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Experimental Neurology

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Experimental Neurology

Experimental Neurology

    Volume 227, Issue 1, January 2011, Pages 224-231

doi:10.1016/j.expneurol.2010.11.009 | How to Cite or Link Using DOI

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    窗体顶端

    TAT-mediated d

    窗体底端

    TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice Bin Caia, Yi Lina, Xie-Hua Xuea, Ling Fanga, Ning Wanga, , , Zhi-Ying Wub, ,

    a Department of Neurology and Institute of Neurology, First Affiliated Hospital, Center of Neuroscience, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, China

    b Department of Neurology and Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, 12 Wulumuqi Zhong Road, Shanghai 200040, China

    Received 18 September 2010; revised 8 November 2010; Accepted 9 November 2010. Available online 17 November 2010.

Abstract

    Neuroglobin (Ngb), a newly identified globin in vertebrate brain, has been suggested to be able to protect against brain hypoxicischemic injury. However, owing to its large size, the

    brain barrier (BBB) to Ngb limits its application in brain injury. impermeability of the blood

    Recently, the 11-amino-acid human immunodeficiency virus trans-activator of transcription (TAT) protein transduction domain was shown to successfully deliver macromolecules into the brain. This study explored whether the TATNgb fusion protein can cross the BBB and protect the brain

    from cerebral ischemia. The TATNgb fusion protein generated from Escherichia coli BL21 (DE3) was efficiently delivered into mice brain tissues by intravenous injection as demonstrated by immunohistochemistry and Western blotting. Two groups of mice were treated with filamentous middle cerebral artery occlusion (MCAO) for 30 min or 2 h followed by reperfusion. Each group

    Ngb, Ngb, or saline was then divided into sub-groups and was injected intravenously with TAT

    respectively before MCAO or immediately after reperfusion. Compared with the Ngb- and saline-treated group, the group with TATNgb treated 2 h before MCAO showed significantly less

    brain infarct volume and had better neurologic outcomes (p < 0.05). Furthermore, a TATNgb

    injection following a 30-min MCAO treatment significantly increased neuronal survival in the striatum (p < 0.05). Our results demonstrated that the exogenous Ngb fusion protein containing the TAT protein transduction domain could be efficiently transduced into neurons in the mouse and protect the brain from mild or moderate ischemic injury.

    Research Highlights

    ?The exogenous Ngb fusion protein containing TAT-PTD can be efficaciously transduced into the brain tissue. ?The exogenous Ngb fusion protein containing TAT-PTD protects the brain from

    ischemic injury. ?The TAT-mediated protein transduction provides a promising tool to deliver

neuroprotective agents across the bloodbrain barrier.

    Keywords: Ischemia; Stroke; Neuroglobin; Tat-PTD; Neuroprotection

    Article Outline

    Introduction

    Materials and methods

Construction of pETTATNgb and pETNgb expression vectors

    Expression and purification of the TATNgb/Ngb fusion protein

    Animals and experimental groups

    Western blotting

    Double-immunofluorescence staining

    Transient focal cerebral ischemia

    Measurement of cerebral blood flow

    Blood pressure measurement and arterial blood gas analysis

    Neurological deficit scores

    Determination of infarct volume

    Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and cresyl violet staining

    Statistical analyses

    Results

Generation of the fusion TATNgb and Ngb proteins

    TAT cargoes Ngb into the brain in vivo

    Physiological parameters

    TATNgb protects against ischemic injury in the 2-h MCAO mouse

    TATNgb inhibits ischemic neuronal apoptosis in the 30-min MCAO mouse

    Discussion

    Acknowledgments

    References

    Introduction

    Acute stroke is one of the leading causes of death and long-term disability worldwide. In most countries, only thrombolysis with alteplase has been approved for the treatment of acute ischemic stroke, yet few patients have received it (California., 2005). So far, neuroprotection in patients with stroke has been unsuccessful (Shuaib et al., 2007), and there is an urgent need for novel therapeutic approaches.

    Neuroglobin (Ngb) is a newly discovered globin that is widely expressed in the vertebral central (Burmester et al., 2000) and peripheral nervous systems (Reuss et al., 2002), as well as in the retina (Schmidt et al., 2003) and endocrine system (Reuss et al., 2002). Its high affinity for oxygen and preferential expression in cerebral neurons imply a possible role in neuronal protection from neuronal hypoxia and cerebral ischemia. Studies have shown that Ngb can protect neurons from hypoxia in vitro ( [Khan et al., 2008] , [Liu et al., 2009] and [Sun et al., 2001] ) and the brain from ischemic damage in vivo ( [Khan et al., 2006] , [Sun et al., 2003] and [Wang et al., 2008] ). [Sun et al., 2001] and [Sun et al., 2003] reported that neuronal hypoxia and cerebral ischemia upregulated Ngb expression, which could protect neurons from hypoxia in vitro and protect the brain from

    experimentally induced stroke, whereas inhibition of Ngb could aggravate hypoxic-ischemic insults.Khan et al. (2006) and Wang et al. (2008) found that two different kinds of Ngb-overexpressing transgenic mice were more resistant to focal cerebral ischemia compared with wild-type mice. Furthermore, they found that primary cultured neurons from these transgenic mice were also resistant to death from hypoxia ( [Khan et al., 2008] and [Liu et al., 2009] ). These findings have suggested that Ngb plays an important role in neuronal protection following hypoxic and ischemic injury. Therefore, it is reasonable to assume that Ngb could be a promising option for stroke therapy.

    However, due to its large molecular size of 17 kDa, Ngb is unable to freely pass through biological membranes ( [Mendoza et al., 2005] , [Peroni et al., 2007] and [Zhou et al., 2008] ) and the bloodbrain barrier (BBB). As a result, the neuroprotection of Ngb in a stroke has been largely analyzed by viral-vector-mediated overexpression (Sun et al., 2003) and protein overexpression in transgenic mice ( [Khan et al., 2006] and [Wang et al., 2008] ). Although gene therapy is a promising approach used to overexpress proteins, it is not suitable for acute stroke treatment because of its time delay and the possible severe side effects (e.g., autoreactivity). Thus, an efficient and safe method for delivering Ngb across the BBB is needed.

    It has been shown that fusion proteins containing the protein transduction domain (PTD) sequence derived from HIV-1 trans-activator of transcription (TAT) can be transduced into the brain following systemic administration (Schwarze et al., 1999). Thus far, several TAT fusion proteins, such as TAT-FNK ( [Asoh et al., 2002] and [Katsura et al., 2008] ), TAT-Bcl-xL (Cao et al., 2002), TAT-GDNF (Kilic et al., 2003), TAT-XIAP (Li et al., 2006), and Tat-Hsp70 (Nagel et al., 2008), can cross the intact BBB and protect the brain from injury due to ischemia. Recently, it was shown that TATNgb generated by fusing Ngb with the PTD of HIV-TAT can efficiently transduce rat cortical neurons (Zhou et al., 2008) and human islets (Mendoza et al., 2005) in culture, and protect cultures from hypoxia in vitro. The in vitro studies have suggested that the transduction of TATNgb may be a promising approach for stroke treatment. However, until now, the effect of the TATNgb fusion protein has not been demonstrated in an in vivo model.

    In this study, we generated a TATNgb fusion protein in Escherichia coli BL21 (DE3) and

    evaluated the ability of TATNgb to cross the BBB into the mouse brain tissue. Then, we tested the protective effect of TATNgb against brain injury following 30 min or 2 h of transient focal cerebral ischemia with injection of TATNgb into the caudal vein either before or after ischemia.

    Materials and methods

    Construction of pETTATNgb and pETNgb expression vectors

    To create the expression vector pETTATNgb, the mouse Ngb cDNA (provided by Burmester T,

    University of Hamburg, Hamburg, Germany) was amplified by a standard polymerase chain reaction with the forward primer F1 (5′ATGAATTCGATGGAGCGCC 3′) and reverse primer R (5′CTCGAGCTCCCCATCCCA 3′), which contains XhoI and EcoRI sites, respectively. The

    approximate 456-bp amplified fragment was cut with XhoI and EcoRI (New England Biolabs, Beverly, MA) and was then ligated into a pTATv1 expression vector (provided by S. F. Dowdy, University of California San Diego, CA, USA) digested with XhoI and EcoRI. In addition, another expression vector, pETNgb, was generated with the same procedure and the Ngb fragments were ligated into pET28b vector (Invitrogen, California, USA), resulting in the same Ngb cDNA sequences as pETTATNgb but without the TAT-PTD sequence. The accuracy of the inserted

    genes was confirmed by both restriction enzyme analysis and automated DNA sequencing.

Expression and purification of the TATNgb/Ngb fusion protein

    The plasmid pETTATNgb/pETNgb was transformed into E. coli BL21 (DE3). A selected

    single colony was cultured in 50 ml of LB medium containing 50 mg/l kanamycin (~ 1214 h;

    with rotation at 200 rpm at 37 ?C) and then transferred to 1 l of LB medium. After the OD600 reached about 0.8, fusion protein expression was induced with 0.5 mM IPTG for 6 h at 37 ?C. The bacterial cells were harvested by centrifugation at 10,000×g, resuspended in binding buffer (500 mM NaCl, 20 mM TrisHCl and 5 mM imidazole, pH 7.9) and then disrupted by sonication on ice.

    The bacterial lysates were centrifuged (10,000×g at 4 ?C for 30 min), and the supernatants were added to a Ni-NTA resin column (Novagen, Madison, WI, USA) equilibrated with binding buffer. Following washing with washing buffer (500 mM NaCl, 20 mM TrisHCl and 60 mM imidazole,

    pH 7.9), the proteins were eluted with elution buffer (500 mM NaCl, 20 mM TrisHCl and 1 M

    imidazole, pH 7.9). The eluted proteins were desalted on a HiTrap desalting column (GE Healthcare, Uppsala, Sweden). The purified fusion proteins were verified by SDS/PAGE, Coomassie Brilliant Blue staining and Western blotting analysis with anti-His-tag (1:500, Cell Signaling, Beverly, MA, USA) and anti-Ngb antibodies (1:200, Abcam, Cambridge, UK). The protein concentrations were estimated by the Bradford method (Bio-Rad, Hercules, CA, USA). The purified proteins were stored at ?70 ?C before use.

    Animals and experimental groups

    C57BL/6J mice were purchased from the Shanghai Laboratory Animal Center (SLAC, Shanghai, China). The experimental designs and all procedures were in accordance with the guidelines for the Care and Use of Laboratory Animals approved by Shanghai Experimental Animal Management Committee. All efforts were made to minimize both the suffering and the number of animals used.

    Adult male C57BL/6J mice weighing 2530 g were randomly assigned to the following groups: A,

    mice were treated with intraluminal thread occlusion for 2 h followed by 24 h of reperfusion and with intravenous injection of (1) 0.1 ml normal saline (NS), (2) 10 mg/kg Ngb dissolved in 0.1 ml of NS, (3) 10 mg/kg TATNgb dissolved in 0.1 ml of NS 2 h before occlusion, or (4) 10 mg/kg TATNgb dissolved in 0.1 ml of NS immediately after reperfusion (n = 8 per group). B, mice were treated with intraluminal thread occlusion for 30 min followed by 72 h of reperfusion and with intravenous injection of (1) 0.1 ml NS, (2) 10 mg/kg Ngb dissolved in 0.1 ml of NS, or (3) 10 mg/kg TATNgb dissolved in 0.1 ml of NS immediately after reperfusion (n = 6 per group). C, mice were injected intravenously with 10 mg/kg TATNgb or Ngb dissolved in 0.1 ml of NS and

    sacrificed 1 or 4 h after injection. Western blotting and immunofluorescence staining were, respectively, applied to the left and right sides of the cerebral hemisphere in order to observe the transduction of TATNgb (n = 4 per group). The animal assignment and sampling were

    randomized, and all of the assessments were blinded to the observer.

    Western blotting

    Four hours after injection, the left cerebral hemisphere was dissected and homogenized in a lysis buffer. Protein concentrations were estimated by the Bradford method (Bio-Rad), equal amounts of protein (80 μg/lane) were diluted in 6 × sample buffer, boiled, and loaded onto 12% SDS-PAGE

    gels and transferred to a nitrocellulose membrane (Hybond-C, Amersham Biosciences, USA) for immunoblotting. The membrane was then incubated in 5% milk/TBS blocking solution, followed by incubation in a mouse anti-Ngb antibody (1:200, Abcam) or mouse anti-β-actin antibody

    (1:1000, Santa Cruz Biotechnology, CA, USA) overnight at 4 ?C. Horseradish peroxidase-labeled anti-mouse secondary antibody (1:3000, Sigma, St Louis, MO, USA) was used to detect the immunoreactivity. Lastly, the membrane was incubated in Lumi-LightPLUS Western blotting substrate (Roche, Mannheim, Germany) and exposed to Kodak film (Kodak, Tokyo, Japan). Quantitative analysis of the relative intensities of the bands was performed using ImageJ 1.42q (http://rsb.info.nih.gov/ij/download/).

    Double-immunofluorescence staining

    One or four hours after injection, the right brain coronal sections were cut into 10 μm at the level

    of the bregma and were incubated with the mouse anti-Ngb antibody (1:100, Abcam) and rabbit anti-neuron-specific enolase (NSE) antibody (1:100, Thermo Fischer Scientific, Fremont, CA, USA) or rabbit anti-GFAP antibody (1:200, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) at 4 ?C for 12 h, followed by an incubation for 2 h at room temperature with goat anti-mouse TRITC (1:200, Santa Cruz) and anti-rabbit FITC immunoconjugate (1:200, Santa Cruz). The sections were washed four times in PBS, mounted with a coverslip in aqueous mounting medium. The sections were examined by a Zeiss LSM 510 confocal microscope (Zeiss, Jena, Germany) with the AxioVision software (Zeiss).

    Transient focal cerebral ischemia

    The mice were subjected to transient focal cerebral ischemia (30 min or 2 h) by MCAO with a modification of intraluminal filament technique as previously described ( [Ji et al., 2009] and [Longa et al., 1989] ). Briefly, the mice were anesthetized with 11.5% isoflurane (Abbott.

    Pharmaceutical Co., Ltd., Shanghai, China) in 30% oxygen (O2) and 70% nitrous oxide (N2O) via a facemask, and the rectal temperature was kept at 37 ? 0.5 ?C using a heating pad connected to feedback-controlled heating system (temperature controller 69000; RWD Life Science, Co. Ltd., Shenzhen, China). Under an Olympus SZ40 stereo zoom microscope (Olympus, Co. Ltd., Tokyo, Japan), a 6-0 nylon monofilament (Ethicon, Somerville, NJ) coated with flexible silicone (Sanchen, Co. Ltd., Beijing, China) was inserted through the ECA stump and gently advanced (8 to 9 mm distal to the carotid bifurcation) to occlude the origin of the middle cerebral artery (MCA); correct placement was confirmed by a rapid decrease in laser-Doppler signal. At 30 min or 2 h after the onset of ischemia, reperfusion of the MCA was achieved by withdrawal of the filament, and the ECA was permanently coagulated. Sham-operated mice underwent the same experimental procedures, but the nylon monofilament was not advanced beyond the common carotid artery. After the surgery and cerebral blood flow recordings were completed, anesthesia was discontinued, and the mice were returned to their home cages.

    Measurement of cerebral blood flow

    A flexible 0.5-mm fiberoptic probe (Probe 418-1; Perimed, Stockholm, Sweden) was attached to the intact skull overlying the core region of the MCA territory (2 mm posteri and 6 mm lateral to bregma) with Loctite 4161 instant adhesive (Loctite, Hartford, CT, USA) and Insta-Set CA accelerator (Bob Smith Ind, CA, USA). Then, regional cerebral blood flow was assessed by laser-Doppler flowmetry (PF 5010 LDPM Unit; Perimed, Jarfalla, Sweden) and recorded by using a computer-based data acquisition system (Perisoft).

    Blood pressure measurement and arterial blood gas analysis

    The left femoral artery was cannulated for monitoring of mean arterial blood pressure by a pressure transducer and RM6240C physiological signal acquisition system (Chengdu Instrument, Chengdu, China) and for blood sampling. The arterial blood samples were analyzed for PaO2,

    PaCO2, and pH by the i-STAT portable clinical analyzer (i-STAT Corporation, East Windsor, NJ, USA) 15 min before and after MCAO.

    Neurological deficit scores

    At 24 or 72 h after MCAO, the mice were examined by a blind observer for neurological deficits using a modified five-point scale as previously reported (0, no neurological deficit; 1, failure to fully extend the right forepaw; 2, circling to the right; 3, falling to the right at rest; 4, unable to walk spontaneously and having a depressed level of consciousness) ( [Bederson et al., 1986] and [Longa et al., 1989] ).

    Determination of infarct volume

    At 24 h after the 2-h MCAO experiment, the mice were anesthetized deeply with chloral hydrate (400 mg/kg, i.p.) and decapitated. The brains were removed and placed in ice-cold normal saline for 10 min and sectioned into five 2-mm-thick coronal slices. The brain slices were stained with 2% 2,3,5,-triphenyltetrazolium chloride monohydrate (TTC) (Sigma) at 37 ?C for 20 min, fixed in 10% formalin, and then photographed by digital cameras (Sony, Japan). The areas of the infarction (unstained) and hemisphere were traced and measured by a blind observer using ImageJ 1.42q. The area was numerically integrated across the thickness of the slice to obtain an estimate of the infarct volume and hemisphere volume. To eliminate the effect of brain edema, a formula was applied as follows: the corrected volume of the infarcted hemisphere = volume of contralateral hemisphere ? (volume of ipsilateral hemisphere ? volume of infarct) (Yildirim et al., 2008).

    Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and cresyl violet staining

    At 72 h after 30 min of MCAO, the mice were deeply anesthetized with chloral hydrate (400 mg/kg, i.p.) and were perfused transcardially with cold NS followed by 10% buffered formalin phosphate. The brains were removed and fixed in 10% buffered formalin phosphate for 24 to 48 h for paraffin embedding. The brain coronal sections were cut in 10 μm at the level of the bregma. Apoptosis in the sections was assessed by TUNEL staining using the in situ Apoptosis Detection Kit (KeyGEN Biotech Co. Ltd., Nanjing, China) according to the manufacturer's protocol. The sections were incubated with streptavidinhorseradish peroxidase and visualized with

    3,3′-Diaminobenzidine (DAB), which stained the TUNEL-positive cells brown. The sections were counterstained with Harris' hematoxylin.

    For evaluation of the degree of tissue damage, other brain sections were used for cresyl violet (BDH, Poole, England) staining according to a standardized histologic protocol. TUNEL-positive cells and viable (cresyl violet) neurons in the 6 striatal regions of interest were quantified by a blind observer using light microscopy and ImageJ 1.42q (Hermann et al., 2001). Neuronal viability was expressed as the percentage of viable neurons compared to the total number of neurons counted.

    Statistical analyses

    Statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL). Differences between groups were compared by one-way analysis of variance, followed by Student's t-test or Dunnett's test. All values were given as mean ? SD. Values of p < 0.05 were considered statistically significant.

    Results

    Generation of the fusion TATNgb and Ngb proteins

    The fusion proteins created in this study are shown in Fig. 1A. The recombinant plasmids

    pETTATNgb and pETNgb were verified by restriction enzyme analysis and had the correct insert size of 456 bp (Fig. 1B). The automated DNA sequencing showed that the fragment matched exactly with mouse Ngb (Gene ID: 64242) and the TAT-PTD sequence. The results confirmed the authenticity of the TATNgb/Ngb coding region. The TATNgb and Ngb proteins

    were expressed in E. coli BL21 (DE3) mostly in the soluble form and then purified to near homogeneity (Fig. 1C). The purified fusion proteins were identified using Western blotting analysis with anti-His-tag and anti-Ngb antibodies. Our results showed that TATNgb was 22 kDa

    and Ngb was 21 kDa (Fig. 1D).

    Full-size image (45K)

    Fig. 1.

    Generation of the fusion proteins TATNgb and Ngb. (A) Diagram of TATNgb and control Ngb

    fusion proteins. (B) Recombinant plasmids pETTATNgb and pETNgb digested by restriction

    enzymes. Lane M: 2000-bp molecular-mass marker; lane 1: pETTATNgb; lane 2:

    pETTATNgb digested by XhoI/EcoRI; lane 3: pETNgb; lane 4: pETNgb digested by

    XhoI/EcoRI. (C) TATNgb expression in Escherichia coli BL21(DE3) induced by 0.5 mmol/l IPTG for 6 h. Lane M: molecular markers; lane 1: uninduced Escherichia coli BL21(DE3)/pETTATNgb; lane 2: induced Escherichia coli BL21(DE3)/pETTATNgb; lane 3:

    pellet of the induced cell lysates; lane 4: supernatant of the induced cell lysates, lane 5: the purified TATNgb fusion protein. (D) Verification of purified TATNgb and Ngb fusion protein by

    Western blotting with anti-His-tag antibody and anti-Ngb antibody.

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TAT cargoes Ngb into the brain in vivo

    To determine whether the systemic injection of TATNgb or Ngb could pass through the BBB, we

    initially performed Western blotting to detect TATNgb in brain tissues at 4 h after injection of the

    protein. In the TATNgb-treated mice, both the exogenous TATNgb proteins (22 kDa) and

    endogenous Ngb proteins (17 kDa) were detectable in the brain (Fig. 2), and the quantitative analysis of the relative intensities of the bands indicated that the exogenous Ngb level was about 10 times higher than the endogenous level. However, in the Ngb-treated mice, only the endogenous Ngb proteins were detectable in the brain (Fig. 2). All of these results indicated that TATNgb could achieve efficient transduction in the brain 4 h after systemic injection, but Ngb without TAT could not achieve protein transduction.

    Full-size image (17K)

    Fig. 2.

    In vivo protein transduction of TATNgb in the mouse brain. In the TATNgb-treated mice, both

    the exogenous TATNgb proteins (22 kDa, as indicated by the arrowhead) and endogenous Ngb proteins (17 kDa, as indicated by the arrow) were detectable in the brain (Fig. 2). In the Ngb-treated mice, only the endogenous Ngb proteins were detectable in the brain (Fig. 2). TATNgb fusion proteins served as a positive control.

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The cell location of the transduced TATNgb in the brain was evaluated by double

    immunofluorescence staining. In the Ngb-treated mice, weak fluorescence could be seen in the brain parenchyma 4 h after intravenous delivery (Fig. 3A). However, in the TATNgb-treated mice,

    a strong, red fluorescence was observed in the microvessel walls 1 h after TATNgb infusion

    (Fig. 3D), and a strong, red fluorescence was observed in the cells inside the brain parenchyma at 4 h (Figs. 3G, J). Double immunofluorescent staining with anti-Ngb and anti-NSE antibody confirmed that most of the transduced cells were neurons (Fig. 3I). Double immunofluorescent staining with anti-Ngb and anti-GFAP antibody confirmed that the GFAP-positive-astrocytes have not been transduced (Fig. 3JL). These results indicated that TATNgb could efficiently cross the

    BBB and enter neurons.

    Full-size image (144K)

    Fig. 3.

    TATNgb crosses the bloodbrain barrier and enters neurons but astrocytes in mice. The weak

    Ngb immunoreactivity is observed in the Ngb-treated control brain (A), and the strong Ngb immunoreactivity is observed in TATNgb-treated brains 1 h (D) or 4 h (G) after injection. At 1 h after TATNgb injection, immunoreactivity is detected mainly in the microvessel walls (D). At 4 h after TATNgb injection, immunoreactivity is detected in a large number of cells (G, J). Double immunofluorescent staining with anti-Ngb and anti-NSE antibody confirmed that most of the transduced cells were neurons (I). Double immunofluorescent staining with anti-Ngb and anti-GFAP antibody indicated that the astrocytes could not be transduced by TATNgb (L). C, F, I

    and l are paired superimposed images of photomicrographs a and B, D and E, G and H, and J and K, respectively.

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Physiological parameters

    There were no differences (p > 0.05) in mean arterial blood pressure, PaO2, PaCO2, pH, rectal temperature, or body weight between the different animal groups (data not shown). The regional cerebral blood flow was reduced to approximately 1020% of the baseline value immediately after

    MCAO and recovered to near baseline with reperfusion.

    TATNgb protects against ischemic injury in the 2-h MCAO mouse

    At 24 h after the 2-h MCAO treatment, the mice developed reproducible infarcts involving the cerebral cortex and the striatum in saline-treated group. TATNgb injected before MCAO

    significantly reduced infarct volume and neurological deficits. Furthermore, in the group treated with TATNgb after reperfusion, there was also a trend towards a reduction in infarct volume and neurological deficits, but it did not reach statistical significance. On the other hand, there were no significant changes in infarct volume and neurological deficits in the Ngb-treated group (Fig. 4AC).

    Full-size image (64K)

    Fig. 4.

    Effect of TATNgb on infarct volumes and neurological deficit scores at 24 h after 2-h MCAO treatment of mice. (A) Representative TTC-stained brain coronal sections of mice treated with saline, Ngb, TATNgb 2 h before ischemia and TATNgb immediately after reperfusion. (B)

    Quantitative analysis of cerebral infarct volumes of mice treated with saline, Ngb, TATNgb 2 h

    before ischemia and immediately after reperfusion (n = 8 per group). (C) Neurological deficit scores of mice treated with saline, Ngb, and TATNgb 2 h before ischemia and immediately after

reperfusion (n = 8 per group). Note that TATNgb treatment 2 h before ischemia significantly

    reduced infarct volume and ameliorated neurological performance. Values given are mean ? SD. #p < 0.05 vs. mice treated with TATNgb immediately after reperfusion. *p < 0.01 vs. mice

    treated with others.

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TATNgb inhibits ischemic neuronal apoptosis in the 30-min MCAO mouse

    Using TUNEL staining and cresyl violet staining, we found that selective neuronal injury only occurred in the striatum at 72 h after 30 min of MCAO. The TUNEL-positive cells with shrunken cell bodies and condensed nuclei were distributed only in the striatum (Fig. 5A). Compared with the saline-treated group, the number of TUNEL-positive cells was significantly (p < 0.01) reduced in the TATNgb-treated group but not in the Ngb-treated group (Fig. 5B). Conversely, neuronal viability was significantly (p < 0.01) increased in the TATNgb-treated group but not in the

    Ngb-treated group compared with the saline-treated group, as shown by cresyl violet staining (Fig. 6A and B).

    Full-size image (26K)

    Fig. 5.

    Effect of TATNgb on TUNEL staining at 72 h after 30-min MCAO treatment of mice. (A) Representative microphotographs showed TUNEL staining cells in the striatum of mouse treated with saline, Ngb or TATNgb. (B) Quantitative analysis showed that TATNgb significantly

    reduced the number of TUNEL-positive cells as compared with saline or Ngb (n = 6 per group). *p < 0.01.

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