Inhibition of p21-activated kinase 1 by IPA-3 attenuates secondary injury after traumatic brain injury in mice

brain research 1585 (2014) 13–22

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Inhibition of p21-activated kinase 1 by IPA-3 attenuates secondary injury after traumatic brain injury in mice Xinran Ji1, Wei Zhang1, Lihai Zhang, Licheng Zhang, Yiling Zhang, Peifu Tangn The General Hospital of People’s Liberation Army (301 Hospital), 28 Fuxing Road, Wukesong, Beijing 100000, China

art i cle i nfo

ab st rac t

Article history:

The p21-activated kinase 1 (PAK1) is up-regulated in the brain following traumatic brain

Accepted 11 August 2014

injury (TBI). Inhibition of PAK1 has been found to alleviate brain edema in a rat model of

Available online 20 August 2014

subarachnoid hemorrhage. Suppressing PAK1 activity might represent a novel therapeutics of attenuating secondary injury following TBI. Here we confirmed that the mRNA and

Keywords:

protein levels of PAK1 and the protein level of p-PAK1 were significantly increased after

Traumatic brain injury

inducing TBI in mice via M.A. Flierl’s weight-drop model. A single intraperitoneal

PAK1

administration of IPA-3, a specific PAK1 inhibitor, immediately after TBI significantly

IPA-3

reduced the protein level of p-PAK1, cleaved caspase-3 level, the number of apoptotic cells

Brain edema

at the lesion sites of TBI mice. It also reduced brain water content and the blood–brain

BBB disruption

barrier permeability in TBI mice. Furthermore, the administration of IPA-3 significantly

Neurological functions

reduced the neurological severity score and increased the grip test score in TBI mice. Taken together, we demonstrate that PAK1 inhibition by IPA-3 may attenuate the secondary injury following TBI, suggesting it might be a promising neuroprotective strategy for preventing the development of secondary injury after TBI. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Much of the mortality and morbidity in severe traumatic brain injury (TBI) patients is due to secondary injury (Badolia Abbreviations: PAK1, TUNEL,

p21-activated kinase 1; TBI,

et al., 2014). During the hours following the primary injury of TBI, cascades of several secondary processes such as hypoxia, ATP depletion, glutamate excitotoxicity, production of free radicals and apoptosis, take place (Algattas and Huang, 2014;

traumatic brain injury; BBB,

blood–brain barrier; CNS,

central nervous system;

terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; IF, immunofluorescence; PCR,

reaction; SAH, kinase; NIH,

subarachnoid hemorrhage; IP,

intraperitoneal injection; NF-kB, nuclear factor kB; MAPK,

National Institutes of Health; qPCR,

real-time quantitative polymerase chain reaction; NSS,

ROS, reactive oxygen species; AD, Alzheimer’s disease n Corresponding author. E-mail address: [email protected] (P. Tang). 1 Xinran Ji and Wei Zhang are Co-first author. http://dx.doi.org/10.1016/j.brainres.2014.08.026 0006-8993/& 2014 Elsevier B.V. All rights reserved.

polymerase chain

mitogen-activated protein neurological severity score;

14

brain research 1585 (2014) 13–22

Tran, 2014), and greatly increase the secondary injury of the primary lesion. Extensive efforts are being made to prevent neurological deficits after TBI through a variety of techniques including reducing oxidative stress (Campolo et al., 2013; Cornelius et al., 2013), repressing inflammation (Nassar et al., 2012; Wang et al., 2012), neutralization of apoptotic factors (Chen et al., 2014; Leeds et al., 2014), administration of neurotropic factors (Karki et al., 2014; Leeds et al., 2014), and transplantation of stem cells (Arien-Zakay et al., 2014; Liu et al., 2014). Unfortunately, the mechanisms underlying secondary injury are still poorly understood and efficient treatments of the second injury following TBI remain to be discovered. Recently, it has been found that PAK1 expression is upregulated in both mRNA and protein levels after brain injury (Mitsios et al., 2007; Raghavendra Rao et al., 2003). PAK1 is a kind of serine/threonine kinases that are activated by p21 GTPases, GTP-Cdc42 and Rac, and can activate a lot of other signaling pathways (Nikolic, 2008; Zegers, 2008). Activated PAK1 can phosphorylate p47Phox, which is a subunit of NADPH oxidase, and contributes to the respiratory burst (Roepstorff et al., 2008), which could enhance the excessive injurious oxidative stress after TBI. Moreover, activated PAK1 can activate NF-kB (Frost et al., 2000) and P38MAPK (Zhang et al., 1995, 2012), which could promote inflammatory response and result in aggravated secondary injury after TBI. In addition, activated PAK1 can activate caspase-1, which is necessary for the maturation of pro-IL-1β (Basak et al., 2005). These data clearly underscore the important roles of PAK1 after brain injuries. Besides, PAK-1 is a key regulator in the PAR-1-c-Src-PAK1vascular endothelial-cadherin phosphorylation pathway, and the inhibition of PAK1 activity by IPA3 (1,10 -disulfanediyldinaphthalen-2-ol), a specific and noncompetitive Inhibitor of PAK1 (Badolia et al., 2014; Deacon et al., 2008; Wong et al., 2013), attenuates brain edema in a rat model of subarachnoid hemorrhage (Yan et al., 2013). Since brain edema is predominantly responsible for the development of secondary injury after TBI (Singhal and Kandel, 2012), inhibition of PAK1 activation could be a promising strategy for reducing the secondary brain injury after TBI. However, to date the therapeutic potential of PAK1 inhibition in the treatment of TBI has not been explored. The development of a cell permeable, specific and noncompetitive Inhibitor of PAK1, which selectively blocks the phosphorylation and activation of PAK1, represents an important tool of inhibiting PAK1 activity. In this study, we investigated whether the inhibition of PAK1 activity by IPA-3 after TBI was effective in reducing secondary injury and promoting functional recovery in TBI mice.

2.

Results

2.1.

The expression of PAK1 and p-PAK1 following TBI

To assess the influence of TBI on PAK1 protein activation and expression, Western blot was performed to detect the temporal expression of p-PAK1 and PAK1. As shown in Fig. 1, the sham group showed a relative low protein expression of p-PAK1 and PAK1. However, both p-PAK1 and PAK1 proteins were

Fig. 1 – The temporal expression of p-PAK1 and PAK1 proteins. (A) The representative Western blot autoradiogram of p-PAK1 and PAK1 protein expression in the ipsilateral cortex after onset of TBI in mice, showing that both p-PAK1 and PAK1 were significantly up-regulated at 12, 24 and 48 h after the onset of TBI. (B) The quantitative analysis of the Western blot results for p-PAK1 and PAK1 protein expression. Protein levels have been normalized with β-actin. Both P-PAK1 and PAK1 protein levels were significantly increased at 12, 24 and 48 h after TBI. Bars represent the mean 7SD. (nPo0.05; ##, nnPo0.01 vs. the sham group, n ¼ 6 for each time point).

Fig. 2 – The mRNA expression of PAK1. The level of PAK1 mRNA in the ipsilateral cortex was significant increased starting at 3 h after the onset of TBI in mice compared with the sham group. β-actin was used as an internal control, and the data were normalized with β-actin and expressed as folds of the sham group. Data were represented as mean7SD. (nPo0.05, nnPo0.01 vs. the sham group, n¼ 6 for each time point).

significantly up-regulated after TBI, peaked at 24 h and showed a significant difference as compared with the sham group (Fig. 1, Po0.01). Subsequently, the expression of these two proteins was gradually reduced. Further, we performed realtime qPCR to test whether the mRNA level of PAK1 was changed following TBI. Fig. 2 showed that the mRNA of PAK1 significantly increased as early as 3 h (Po0.01) after TBI, and reached the peak at 6 h (Po0.01), which was 2 times as that of

brain research 1585 (2014) 13–22

15

Fig. 3 – Representative photomicrographs of double immunofluorescent staining of different neural cell markers (green) and p-PAK1 (red) near the lesion site of mouse brain at 24 h post TBI. p-PAK1 is expressed in neuron, astrocyte and microglia/ macrophage, and is mainly expressed in the cytoplasm. Arrows indicated the colocalization of p-PAK1 with NeuN, GFAP or Iba1. Scale bars¼ 20 μm.

the sham group. Subsequently, the mRNA level of PAK1 was gradually decreased. In addition, we performed double immunostaining of p-PAK1 and NeuN (neuron marker), GFAP

(astrocyte marker), or Iba1 (microglia/macrophage marker) to identify which types of cells express p-PAK1. Double positive neurons, astrocytes, and microglia/macrophagy were observed

16

brain research 1585 (2014) 13–22

upregulated around the lesion site in TBI group at 24 h (Fig. 3), suggesting PAK1 was activated in both neuronal and glial cells following TBI.

2.2. IPA-3 inhibits PAK1 and caspase-3 activation after TBI The ability of IPA-3 to inhibit PAK1 phosphorylation and caspase-3 cleavage was assessed by Western blot assay. The results showed that there was a 4-fold increase of p-PAK1 activation (phosphorylation) 24 h after TBI compared to the unlesioned animals (Fig. 4A and B). IPA-3 treatment reduced p-PAK1 to a level just a little higher than the sham brain (Fig. 4A and B). The result also showed that cleaved caspase-3, one of the key executioners in the apoptotic pathway, exhibited the same trend as p-PAK1 showed. Thus PAK1 inhibition confers neuroprotection via a decreased activation of caspase-3. These results underline that in this experimental context, PAK1 regulates the death cascade associated with caspase-3.

2.3. IPA-3 protects against cell apoptosis in the cortex following TBI To investigate the protective role of PAK1 inhibition in TBI, we studied the effect of IPA-3 treatment on neural apoptosis in the cortex at 24 h post TBI by using the TUNEL staining method to detect the number of apoptotic cells. As shown in

Fig. 5, a very small amount of TUNEL-positive neurons were found in the cortex of sham group mice (a). Compared with the sham group, the number of apoptotic cells significantly increased in the TBI group (b) and vehicle group (c) (Po0.01). IPA-3 treatment remarkably reduced the number of apoptotic cells in the cortex in comparison with the vehicle group (d) (Po0.05). The result showed that the treatment of IPA-3 led to less cell apoptosis in the cortex surrounding the contusive lesion and had neuroprotective effects following TBI.

2.4. Effects of IPA-3 treatment on the brain edema and BBB disruption Vascular changes such as disruption of the blood brain barrier (BBB) occur very early after TBI. To confirm the protective effect of IPA-3 in the macroscopic level, we detected the brain water content and Evans blue extravasation. Fig. 6 showed that, compared with the sham group, TBI and the vehicle groups showed a significant increase of brain water content and Evans blue extravasation. There was no significant difference between TBI and the vehicle groups. While IPA-3-treated group showed a significant decrease in brain water content and Evans blue extravasation compared with vehicle group (Fig. 6) (Po0.05). These results clearly showed that IPA-3 treatment significantly attenuated the brain edema and BBB disruption observed 24 h after TBI.

Fig. 4 – IPA-3 inhibits PAK1 and caspase-3 activation at 24 h after TBI. (A) Representative autoradiogram of p-PAK1 and cleaved caspase-3 protein expression detected by Western blot assay in the sham, TBI, TBIþvehicle and TBIþIPA-3 group mice (B) Quantitative analysis of Western blot results for the expression of p-Pak1. The expression of p-PAK1 was significantly increased in the TBI and TBIþVeh (vehicle) groups compared with that in the sham group. IPA-3 treatment significantly inhibited the TBI-induced up-regulation of p-PAK1. Data are represented as mean7SD. (nnPo0.01 compared with the sham group, #Po0.05 compared with the TBIþVeh group. n ¼ 6 for each group).

brain research 1585 (2014) 13–22

17

Fig. 5 – IPA-3 reduces the TUNEL-positive cells in the pericontusive cortex at 24 h after TBI in mice. (A) Representative photomicrographs of TUNEL staining. A very small amount of TUNEL-positive cells was observed in the cortex of the sham group (a), while numerous TUNEL-positive cells with intense brown nuclear staining were evident in the TBI group (b) and the TBIþVeh group (c). Fewer TUNEL-positive cells were present in the mouse cortex of the TBIþIPA-3 group. (B) The apoptotic index was calculated according to the TUNEL staining results. The administration of IPA-3 significantly decreased the TUNELpositive cells at 24 h following TBI in the mice. Data are represented as mean7SD. (nnPo0.01 versus the sham group; #Po0.05 versus TBIþVeh group. n ¼ 6 for each group). Scale bar¼ 20 m.)

Fig. 6 – IPA-3 reduces brain water content and Evans blue extravasation in TBI mice. Both Brain water content (A) and Evans blue extravasation (B) were increased significantly in the ipsilateral cortex 24 h after TBI. IPA-3 treatment significantly attenuated both brain water content and Evans blue extravasation. Data are represented as mean7SD. (nnPo0.01 compared with the sham group; #Po0.05 compared with the TBIþVeh group. n ¼ 6 for each group.)

2.5.

IPA-3 rescues neurological function 24 h post TBI

To assess whether PAK1 inhibition by IPA-3 has a positive effect on neurological functions, both NSS (Fig. 7A) and grip test (Fig. 7B) were performed 24 h and 72 h after injury on vehicle and IPA-3 treated animals. Fig. 7A showed that 24 h post TBI, the NSS of IPA-3 treated group was significantly lower than that of the vehicle group. But there was no statistical significance in NSS at 72 h after TBI. Fig. 7B showed that IPA-3 treated group had a significantly higher grip test score at both 24 h and 72 h post TBI, compared with the vehicle group. Obviously, the neurological deficits in TBI mice were significantly attenuated by the treatment of IPA-3.

3.

Discussion

The present study underlined the expression and activation of PAK1 after TBI, and the therapeutic potential of PAK inhibitory treatment by IPA-3 after TBI in mice. Here we reported that PAK1 inhibition by IPA-3 led to a significant improvement of the neurological function assessed with two different tests, the NSS and the grip test at 24 h and 72 h after TBI in mice.

To gain insight into the neuroprotective mechanism(s) of PAK1 inhibition by IPA-3, we analyzed the changes of apoptotic targets induced by IPA-3 in TBI mice. We found that the treatment of IPA-3 significantly inhibited the phosphorylation of PAK1 and the cleavage of caspase-3, reducing TUNEL-positive cells in the ipsilateral cortex 24 h after TBI. This indicates PAK1 inhibition by IPA-3 might protect the brain from secondary injury after TBI by blocking the caspase-3-dependent apoptotic pathway. Among the different processes triggered by the primary mechanical injury, oxidative stress and inflammatory response play an important role in neuronal and glial cell death (Rodriguez-Rodriguez et al., 2014; Tran, 2014). Roepstorff et al. (2008) reported that activated PAK1 can phosphorylate p47Phox, which is a subunit of NADPH oxidase, and contributes to the respiratory burst. And NADPH oxidase is reported to be activated and contributes to reactive oxygen species (ROS) generation after TBI (Ferreira et al., 2013; Lu et al., 2014). The PAK1 inhibition by IPA-3 might reduce NADPH oxidase activation, and therefore reduce ROS production and attenuate secondary injury after TBI. Moreover, activated PAK1 can also activate NF-kB (Foryst-Ludwig and Naumann, 2000; Frost et al., 2000) and P38MAPK (Zhang et al., 1995, 2012), which could induce the inflammatory response cascades. In addition, activated PAK1

18

brain research 1585 (2014) 13–22

Fig. 7 – IPA-3 rescues neurological function post TBI. The mouse neurological behaviour was evaluated by neurological severity score (NSS) and grip test. (A) The IPA-3 treated group showed a significant reduction of NSS 24 h post-TBI. Both groups showed a decrease in NSS as time passed after TBI. (B) The IPA-3 treated group showed a significant increase in grip test score 24 h and 72 h after TBI. Both groups showed an increase in grip test score as time passed after TBI. Two-way ANOVA followed by Bonferroni post-hoc test was used in the test. Data are represented as Mean7SD. (nPo0.05 versus the TBIþVeh group, n ¼ 6 for each group.)

can activate caspase-1 (Basak et al., 2005), which is necessary for maturation of pro-IL-1β. Thus, PAK1 participates in inflammatory responses, and PAK1 inhibition could alleviate inflammation induced by secondary injury after TBI. Since PAK1 activation was observed in neurons, astrocyte and microglia/ macrophage in our study, and in endothelial cells in Yan’s observation (Yan et al., 2013), IPA-3 inhibition could have a protective effect on these types of cells. Microvascular damage such as the increase of brain water content and BBB permeability occurs following TBI is considered both as a direct consequence of the primary lesion of TBI and as an effect of the up-regulated oxidative stress and inflammatory responses on the endothelial cells following TBI. Interestingly, we showed that IPA-3 treatment reduces the brain water content and Evans blue extravasation in TBI mice. Thus, IPA-3 protects BBB from dysfunction after TBI. This effect was described by Yan’s study in a rat model of subarachnoid hemorrhage, that PAK1 activity inhibition can preserve microvascular integrity and provide neurobehavioral protection via the suppression of VE-cadherin endocytosis. Moreover, PAK1 is required in TNF-α induced expression of MMP-9 (Zhou et al., 2009), which is a key factor results in BBB disruption (Jang et al., 2014; Li et al., 2013; Zheng et al., 2014). Therefore, we demonstrate that PAK inhibition by IPA-3 reduces TBI-induced secondary injury. Our data bring the proof of concept that PAK1 inhibition might be interesting in the treatment of TBI. However, some data show that PAK1 inactivation may impair recognition (Arsenault et al., 2013). Decreases in cytosolic PAK concentrations are found in the cortex and hippocampus of persons who died with advanced Alzheimer’s disease (AD) (Nguyen et al., 2008), and reduction of PAK catalytic activity in vivo induces a decrease in the spine density within cortical neurons (Hayashi et al., 2002, 2007, 2004). Since PAK1 is a common and universal expressing cytokine, PAK1 activation is essential for the proper and ordinary functions of the brain. Hyper-activation of PAK1 induced by disturbances in the brain, however, would result in a cascade expression and activation of cytokines, such as NADPH oxidase, NF-kB, P38, and caspase-1, which would aggravate the oxidative stress and inflammatory responses. Moreover, PAK1 may have dual effects after brain injury as NF-kB’s detrimental effect on increased inflammation in early stage after brain injury and protective effect on tissue repair in

the later stage (Hang et al., 2006; Hu et al., 2014; You et al., 2012, 2013). Furthermore, early inhibition of PAK1 may result in increased expression by feedback, which would be protective in the later stage after brain injury. Thus, PAK1 inhibition acts upon different complementary players in TBI scenario to reduce secondary injury and promote function recovery. Overall, we demonstrate that Inhibition of PAK1 by IPA-3 attenuates secondary injury after TBI in mice. We strongly highlight the potential therapeutic interest of PAK1 inhibition after TBI. And further studies are necessary to completely clarify the mechanisms and the time window of PAK1 inhibition after TBI.

4.

Experimental procedures

4.1.

Animals

ICR mice (200 male, weight: 28–32 g) (The Laboratory animal center of Academy of Military Medical Sciences, Beijing, China) were used in this study. Experiment protocols were approved by the Animal Care and Use Committee of Medical School of Chinese People’s Liberation Army and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH), China. The mice were housed on a 12-h light/dark cycle circumstance with free access to food and water.

4.2.

Experimental design

All mice were randomly distributed into the following groups: Sham, TBI, TBIþvehicle (DMSO) and TBIþIPA-3 groups. In a previous study, a single intraperitoneal injection of IPA-3 (3.5 mg/kg) was found to be enough to inhibit PAK1 activation (Yan et al., 2013). Thus, in this present study, IPA-3 (Sigma– Aldrich Co., St. Louis, MO) at the dose of 3.5 mg/kg was administrated intraperitoneally immediately after TBI surgery. The same volume of 1% DMSO (Sigma–Aldrich Co., St. Louis, MO) in 0.01 M PBS was used as a vehicle control.

brain research 1585 (2014) 13–22

4.3.

Mouse model of TBI

The M.A Flierl’s weight-drop model with some modifications (Flierl et al., 2009) was used to induce TBI in mice in the present study. Briefly, mouse was anesthetized with an intraperitoneal injection of chloral hydrate (4 ml/kg), and a 1.5-cm midline longitudinal scalp incision was made to expose the skull. Then mouse was placed onto the platform directly under the weight-drop device. After identification of the left anterior frontal area (1.5 mm lateral to the midline on the mid-coronal plane) as impact area, the weight was released and dropped onto the skull. Based on the protocol (Kuzelova et al., 2014), the weight was 200 g and the height of the weight-drop was 2.5 cm. All the procedures were processed in sham-injured mice except for weight drop injury. After onset of TBI, the scalp wound was sutured, and the mice were returned to their cages. The mortality occurred about 8% after TBI operation.

4.4.

Brain tissue preparation

Animals were deeply anesthetized with solution of chloral hydrate and perfused intracardially with 60 ml cold (4 1C) heparinized 0.9% saline at scheduled time interval after TBI surgery. For Western blot and PCR analysis, brain tissue near the contusion of the left cerebral cortex was collected and stored in liquid nitrogen immediately until use. For TUNEL staining, animals were deeply anesthetized, sacrificed, and perfused intracardially with 60 ml cold heparinized 0.9% saline, then followed by 50 ml of cold 4% paraformaldehyde. The whole brain was removed and immersed in 4% paraformaldehyde overnight. For double immunofluorescence (IF), the fixed brain was immersed in 20% sucrose solution for 24 h, and then immersed in 30% sucrose solution for about 24 h. Sham injured animals underwent the same procedures as the TBI mice.

4.5.

Western blot analysis

Dissected brain tissue was homogenized in 1% Triton X-100 lysis buffer supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich Co, St Louis, MO) and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich Co, St Louis, MO) inhibitors and used for Western blot analysis. Protein concentrations were quantified using Bradford Assay (Bio-Rad Protein Assay 5000006, Munchen, Germany) and 30 μg of brain tissue homogenate was separated by 10% SDS polyacrylamide gel and then transferred to PVDF membranes. The membranes were blocked with 5% no fat milk in TBST (Tris-buffered saline and 0.1% Tween20) for 1 h at room temperature. Primary antibodies (rabbit anti-phosphorylated-PAK1, PAK1, cleaved caspase-3 or β-actin antibody) (1:500; all antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in the same buffer and incubated overnight at 4 1C. Western blots were quantified by densitometry using ImageJ 1.48 (National Institutes of Health).

19

4.6. Real-time quantitative polymerase chain reaction (qPCR) Total RNA was extracted from the ipsilateral cortex using RNAiso Plus (TaKaRa Bio, Dalian, China). The concentration and purity of total RNA were determined with a spectrophotometer (OD260/280¼ 1.8–2.0). RNA was reverse transcripted to cDNA using PrimeScript™ RT reagent Kit (TaKaRa Bio, Dalian, China) and stored at  80 1C. The primers were designed according to PubMed GenBank, and synthesized by Invitrogen Life Technologies (Shanghai, China). The primers sequences were as follows: PAK1: F: 50 -ATGACAAGGTAGGCAATGG-30 , R: 50 -CTGAGTAAGAGAAGTGTGAGA-30 ; β-actin: F: 50 -AGTGTGACGTTGACATCCGTA-30 , R: 50 -GCCAGAGCAGTAATCTCCTTCT-30 . The qPCR analysis adopted the real-time SYBR Green PCR technology and was performed by using the Mxpro3000P System (Stratagene, USA). PCR amplification program consisted of an initial denaturation step of 95 1C for 30 s, followed by 40 cycles of 95 1C for 5 s, a 30 s annealing and elongation step at 60 1C. All samples were analyzed in triplicate. The relative change in PAK1 mRNA expression following TBI was determined by the equation: fold change¼ 2  [ΔΔCt], ΔΔCt ¼(CtPAK1 Ctβ-actin)TBI (CtPAK1  Ctβ-actin)Sham. Ct value is the cycle number at which fluorescence signal crosses the threshold.

4.7.

Immunofluorescence

For IF detection, 8-μm thick cryostat frozen sections mounted on gelatin coated slides were prepared. Before IF staining, slides were warmed up at room temperature for 15 min followed by three washes with PBS for 5 min each. Slides were incubated in blocking buffer (10% normal goat serum in PBS) for 1 h, followed by overnight incubation at 4 1C with primary antibodies: p-PAK1 (1:100; Santa Cruz, USA); Iba-1 (1:100; Abcam, USA); NeuN (1:100; Millipore, USA); or GFAP (1:200; CST, USA). On the next day, the slides were washed with PBS three times for 5 min each, followed by incubation with donkey anti-rabbit IgG-R, donkey anti-goat IgG-FITC or donkey anti-mouse IgG-FITC (1:200; Santa Cruz, CA) antibodies for 1 h at room temperature. Following three washes with PBS slides were counterstained with DAPI for 2 min and rinsed with PBS, and then sections were cover-slipped with mounting medium for imaging analysis. Fluorescence microscopy imaging was performed using Olympus IX71 inverted microscope system.

4.8. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) The 10-μm sections cross the lesion site of the mice brain were deparaffinized, rehydrated and stained by an in situ cell death detection kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s protocol. The positive cells were identified, counted, and analyzed under the light microscope by an invited pathologist and an investigator who were blind to the grouping. The extent of brain damage was evaluated by the apoptotic index, which was defined as the average percentage of TUNEL-positive cells in each section counted in 6 cortical microscopic fields at 400  magnification. A total

20

brain research 1585 (2014) 13–22

Table 1 – Characterization of neurological severity score (NSS). Task

NSS Failure/success

Exit a 30-cm diameter circle within 3 min Absence of mono- or hemiparesis Able to walk straight Presence of startle reflex Presence of seeking behaviour Able to walk on a 3-cm wide beam Able to walk on a 2-cm wide beam Able to walk on a 1-cm wide beam Able to balance on a 1-cm wide beam at least 10 s Able to balance on a round stick (0.5 cm wide) at least 10 s Maximum total

of five sections with the interval of 100 μm from each animal were used for quantification. The final average percentage of TUNEL-positive cells in five sections was regarded as the data for each sample.

4.9.

Brain water content

For brain water content analysis, after perfusion with ice-cold 0.9% saline with a constant pressure, mouse forebrain was immediately removed and the injured left cerebral hemisphere was quickly isolated and weighed (wet weight). Brain specimen was then dried in an oven at 100 1C for 72 h till the brain water evaporated completely to get the dry weight. The percentage of water content was calculated as (wet weightdry weight)/wet weight.

4.10.

BBB disruption

The integrity of the BBB was investigated using Evans blue extravasation as described by Manaenko et al. (2011). Briefly, 21 h after TBI operation, Evans blue dye (2%, 4 ml/kg of body weight) was injected intraperitoneally, and allowed to absorb for 3 h (Wong et al., 2013). Then mice were killed by intracardial perfusion with saline, and brain samples were collected, weighed, homogenized in PBS, and centrifuged at 15,000g for 30 min. 0.5 ml of the resultant supernatant was added to an equal volume of 50% trichloroacetic acid. After incubation for overnight and centrifugation at 15,000g for 30 min at 4 1C, the supernatant was used for spectrophotometric quantification at 610 nm. Evans blue content was expressed as μg per gram of protein.

4.11.

Behavior tests

To evaluate functional disability after TBI, all animals were subjected to 2 neurological tests: the neurological severity score (NSS) (Flierl et al., 2009) and the grip test score (Bermpohl et al., 2006). NSS is based on the ability of the mouse to perform 10 different tasks that evaluate the motor ability, balance, and alertness of the mouse (Table 1). One point is given for failing to perform each of the tasks; whereas 0 point is given for any success. Thus, a normal and uninjured mouse scores 0; while the maximum score is 10.

1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 10

Table 2 – Characterization of grip test. Task

Score

Unable to grasp wire for 30 s Gripping wire for 60 s with 1 or 2 paws Jump up and grasp wire with 4 paws Grasp wire with 4 paws and wrap tail around Crawl along the wire for at least 5 cm Crawl along wire to terminal and dismount Maximum total

0 1 2 3 4 5 5

The severity of injury is defined by the initial NSS, evaluated 1 h post TBI, and is a reliable predictor for the late outcome of TBI. Grip test was developed to test gross vestibular-motor function. The test consists of placing the mouse on a thin and horizontal wire (45 cm long) suspended (45 cm above a foam pad) between two poles, and grading the ability of the mouse to grip, attach and move as described in Table 2. The grip test was performed in triplicate and a total value was calculated for each mouse. NSS and grip test were performed the day before the surgery to evaluate normal behavior of the mice, and were further performed 24 h and 72 h after TBI by 2 observers who were blind to mice groups.

4.12.

Statistics

Data are reported as mean7SD (standard deviation), and SPSS16.0 (SPSS Inc., Chicago) was used for the statistical analysis. Two-way ANOVA followed by Bonferroni post-hoc test was used for the behavior tests analysis, while one-way ANOVA followed by Bonferroni post-hoc test for other data analysis. P valueo0.05 was deemed statistically significant.

r e f e r e n c e s

Algattas, H., Huang, J.H., 2014. Traumatic brain injury pathophysiology and treatments: early, intermediate, and late phases post-injury. Int. J. Mol. Sci. 15, 309–341. Arien-Zakay, H., Gincberg, G., Nagler, A., Cohen, G., LirazZaltsman, S., Trembovler, V., Alexandrovich, A.G., Matok, I., Galski, H., Elchalal, U., Lelkes, P.I., Lazarovici, P., Shohami, E., 2014. Neurotherapeutic effect of cord blood derived

brain research 1585 (2014) 13–22

CD45þhematopoietic cells in mice after traumatic brain injury. J. Neurotrauma. Arsenault, D., Dal-Pan, A., Tremblay, C., Bennett, D.A., Guitton, M. J., De Koninck, Y., Tonegawa, S., Calon, F., 2013. PAK inactivation impairs social recognition in 3  Tg-AD Mice without increasing brain deposition of tau and Abeta. J. Neurosci. 33, 10729–10740. Badolia, R., Manne, B.K., Dangelmaier, C., Kunapuli, S.P., 2014. IPA3 non-specifically enhances phosphorylation of several proteins in human platelets. Platelets. Basak, C., Pathak, S.K., Bhattacharyya, A., Mandal, D., Pathak, S., Kundu, M., 2005. NF-kappaB- and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharidestimulated macrophages. J. Biol. Chem. 280, 4279–4288. Bermpohl, D., You, Z., Korsmeyer, S.J., Moskowitz, M.A., Whalen, M.J., 2006. Traumatic brain injury in mice deficient in Bid: effects on histopathology and functional outcome. J. Cereb. Blood Flow Metab. 26, 625–633. Campolo, M., Ahmad, A., Crupi, R., Impellizzeri, D., Morabito, R., Esposito, E., Cuzzocrea, S., 2013. Combination therapy with melatonin and dexamethasone in a mouse model of traumatic brain injury. J. Endocrinol. 217, 291–301. Chen, Q., Xu, J., Li, L., Li, H., Mao, S., Zhang, F., Zen, K., Zhang, C.Y., Zhang, Q., 2014. MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis. Cell Death Dis 5, e1132. Cornelius, C., Crupi, R., Calabrese, V., Graziano, A., Milone, P., Pennisi, G., Radak, Z., Calabrese, E.J., Cuzzocrea, S., 2013. Traumatic brain injury: oxidative stress and neuroprotection. Antioxid. Redox Signal. Deacon, S.W., Beeser, A., Fukui, J.A., Rennefahrt, U.E., Myers, C., Chernoff, J., Peterson, J.R., 2008. An isoform-selective, smallmolecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem. Biol. 15, 322–331. Ferreira, A.P., Rodrigues, F.S., Della-Pace, I.D., Mota, B.C., Oliveira, S.M., Velho Gewehr, C.D., Bobinski, F., de Oliveira, C. V., Brum, J.S., Oliveira, M.S., Furian, A.F., de Barros, C.S., Ferreira, J., Santos, A.R., Fighera, M.R., Royes, L.F., 2013. The effect of NADPH-oxidase inhibitor apocynin on cognitive impairment induced by moderate lateral fluid percussion injury: role of inflammatory and oxidative brain damage. Neurochem. Int. 63, 583–593. Flierl, M.A., Stahel, P.F., Beauchamp, K.M., Morgan, S.J., Smith, W.R., Shohami, E., 2009. Mouse closed head injury model induced by a weight-drop device. Nat. Protoc. 4, 1328–1337. Foryst-Ludwig, A., Naumann, M., 2000. p21-Activated kinase 1 activates the nuclear factor kappa B (NF-kappa B)-inducing kinase-Ikappa B kinases NF-kappa B pathway and proinflammatory cytokines in Helicobacter pylori infection. J. Biol. Chem. 275, 39779–39785. Frost, J.A., Swantek, J.L., Stippec, S., Yin, M.J., Gaynor, R., Cobb, M.H., 2000. Stimulation of NFkappa B activity by multiple signaling pathways requires PAK1. J. Biol. Chem. 275, 19693–19699. Hang, C.H., Chen, G., Shi, J.X., Zhang, X., Li, J.S., 2006. Cortical expression of nuclear factor kappaB after human brain contusion. Brain Res. 1109, 14–21. Hayashi, K., Ohshima, T., Mikoshiba, K., 2002. Pak1 is involved in dendrite initiation as a downstream effector of Rac1 in cortical neurons. Mol. Cell Neurosci. 20, 579–594. Hayashi, K., Ohshima, T., Hashimoto, M., Mikoshiba, K., 2007. Pak1 regulates dendritic branching and spine formation. Dev. Neurobiol. 67, 655–669. Hayashi, M.L., Choi, S.Y., Rao, B.S., Jung, H.Y., Lee, H.K., Zhang, D., Chattarji, S., Kirkwood, A., Tonegawa, S., 2004. Altered cortical synaptic morphology and impaired memory consolidation in

21

forebrain- specific dominant-negative PAK transgenic mice. Neuron 42, 773–787. Hu, Y.C., Sun, Q., Li, W., Zhang, D.D., Ma, B., Li, S., Li, W.D., Zhou, M.L., Hang, C.H., 2014. Biphasic activation of nuclear factor kappa B and expression of p65 and c-Rel after traumatic brain injury in rats. Inflamm. Res. 63, 109–115. Jang, J.W., Lee, J.K., Hur, H., Kim, T.W., Joo, S.P., Piao, M.S., 2014. Rutin improves functional outcome via reducing the elevated matrix metalloproteinase-9 level in a photothrombotic focal ischemic model of rats. J. Neurol. Sci. 339, 75–80. Karki, P., Smith, K., Johnson Jr., J., Lee, E., 2014. Astrocyte-derived growth factors and estrogen neuroprotection: role of transforming growth factor-alpha in estrogen-induced upregulation of glutamate transporters in astrocytes. Mol. Cell Endocrinol. Kuzelova, K., Grebenova, D., Holoubek, A., Roselova, P., Obr, A., 2014. Group I PAK inhibitor IPA-3 induces cell death and affects cell adhesivity to fibronectin in human hematopoietic cells. PLoS One 9, e92560. Leeds, P.R., Yu, F., Wang, Z., Chiu, C.T., Zhang, Y., Leng, Y., Linares, G.R., Chuang, D.M., 2014. A new avenue for lithium: intervention in traumatic brain injury. ACS Chem. Neurosci. Li, Y.J., Wang, Z.H., Zhang, B., Zhe, X., Wang, M.J., Shi, S.T., Bai, J., Lin, T., Guo, C.J., Zhang, S.J., Kong, X.L., Zuo, X., Zhao, H., 2013. Disruption of the blood–brain barrier after generalized tonicclonic seizures correlates with cerebrospinal fluid MMP-9 levels. J. Neuroinflamm 10, 80. Liu, S.J., Zou, Y., Belegu, V., Lv, L.Y., Lin, N., Wang, T.Y., McDonald, J.W., Zhou, X., Xia, Q.J., Wang, T.H., 2014. Co-grafting of neural stem cells with olfactory en sheathing cells promotes neuronal restoration in traumatic brain injury with an antiinflammatory mechanism. J. Neuroinflamm 11, 66. Lu, X.Y., Wang, H.D., Xu, J.G., Ding, K., Li, T., 2014. NADPH oxidase inhibition improves neurological outcome in experimental traumatic brain injury. Neurochem. Int. 69, 14–19. Manaenko, A., Chen, H., Kammer, J., Zhang, J.H., Tang, J., 2011. Comparison Evans blue injection routes: intravenous versus intraperitoneal, for measurement of blood–brain barrier in a mice hemorrhage model. J. Neurosci. Methods 195, 206–210. Mitsios, N., Saka, M., Krupinski, J., Pennucci, R., Sanfeliu, C., Wang, Q., Rubio, F., Gaffney, J., Kumar, P., Kumar, S., Sullivan, M., Slevin, M., 2007. A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion. BMC Neurosci 8, 93. Nassar, N.N., Abdelsalam, R.M., Abdel-Rahman, A.A., Abdallah, D. M., 2012. Possible involvement of oxidative stress and inflammatory mediators in the protective effects of the early preconditioning window against transient global ischemia in rats. Neurochem. Res. 37, 614–621. Nguyen, T.V., Galvan, V., Huang, W., Banwait, S., Tang, H., Zhang, J., Bredesen, D.E., 2008. Signal transduction in Alzheimer disease: p21-activated kinase signaling requires C-terminal cleavage of APP at Asp664. J. Neurochem. 104, 1065–1080. Nikolic, M., 2008. The Pak1 kinase: an important regulator of neuronal morphology and function in the developing forebrain. Mol. Neurobiol. 37, 187–202. Raghavendra Rao, V.L., Dhodda, V.K., Song, G., Bowen, K.K., Dempsey, R.J., 2003. Traumatic brain injury-induced acute gene expression changes in rat cerebral cortex identified by GeneChip analysis. J. Neurosci. Res. 71, 208–219. Rodriguez-Rodriguez, A., Egea-Guerrero, J.J., Murillo-Cabezas, F., Carrillo-Vico, A., 2014. Oxidative stress in traumatic brain injury. Curr. Med. Chem. 21, 1201–1211. Roepstorff, K., Rasmussen, I., Sawada, M., Cudre-Maroux, C., Salmon, P., Bokoch, G., van Deurs, B., Vilhardt, F., 2008. Stimulus-dependent regulation of the phagocyte NADPH oxidase by a VAV1, Rac1, and PAK1 signaling axis. J. Biol. Chem. 283, 7983–7993.

22

brain research 1585 (2014) 13–22

Singhal, R., Kandel, E.S., 2012. The response to PAK1 inhibitor IPA3 distinguishes between cancer cells with mutations in BRAF and Ras oncogenes. Oncotarget 3, 700–708. Tran, L.V., 2014. Understanding the pathophysiology of traumatic brain injury and the mechanisms of action of neuroprotective interventions. J. Trauma Nurs 21, 30–35. Wang, J., Liu, Y.M., Cao, W., Yao, K.W., Liu, Z.Q., Guo, J.Y., 2012. Anti-inflammation and antioxidant effect of Cordymin, a peptide purified from the medicinal mushroom Cordyceps sinensis, in middle cerebral artery occlusion-induced focal cerebral ischemia in rats. Metab. Brain Dis. 27, 159–165. Wong, L.L., Lam, I.P., Wong, T.Y., Lai, W.L., Liu, H.F., Yeung, L.L., Ching, Y.P., 2013. IPA-3 inhibits the growth of liver cancer cells by suppressing PAK1 and NF-kappaB activation. PLoS One 8, e68843. Yan, J., Manaenko, A., Chen, S., Klebe, D., Ma, Q., Caner, B., Fujii, M., Zhou, C., Zhang, J.H., 2013. Role of SCH79797 in maintaining vascular integrity in rat model of subarachnoid hemorrhage. Stroke. You, W.C., Li, W., Zhuang, Z., Tang, Y., Lu, H.C., Ji, X.J., Shen, W., Shi, J.X., Zhou, M.L., 2012. Biphasic activation of nuclear factor-kappa B in experimental models of subarachnoid hemorrhage in vivo and in vitro. Mediators Inflamm. 2012, 786242.

You, W.C., Wang, C.X., Pan, Y.X., Zhang, X., Zhou, X.M., Zhang, X.S., Shi, J.X., Zhou, M.L., 2013. Activation of nuclear factor-kappaB in the brain after experimental subarachnoid hemorrhage and its potential role in delayed brain injury. PLoS One 8, e60290. Zegers, M., 2008. Roles of p21-activated kinases and associated proteins in epithelial wound healing. Int. Rev. Cell Mol. Biol. 267, 253 (K.W. Jeon, ed.\widehateds). Zhang, S., Han, J., Sells, M.A., Chernoff, J., Knaus, U.G., Ulevitch, R.J., Bokoch, G.M., 1995. Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J. Biol. Chem. 270, 23934–23936. Zhang, T., Lu, X., Arnold, P., Liu, Y., Baliga, R., Huang, H., Bauer, J.A., Liu, Y., Feng, Q., 2012. Mitogen-activated protein kinase phosphatase-1 inhibits myocardial TNF-alpha expression and improves cardiac function during endotoxemia. Cardiovasc. Res. 93, 471–479. Zheng, M., Wei, J., Tang, Y., Yang, C., Wei, Y., Yin, X., Liu, Q., 2014. ApoE-deficient promotes blood–brain barrier disruption in experimental autoimmune encephalomyelitis via alteration of MMP-9. J. Mol. Neurosci.. Zhou, L., Yan, C., Gieling, R.G., Kida, Y., Garner, W., Li, W., Han, Y.P., 2009. Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21activated kinase-1. BMC Immunol 10, 15.