Neuroscience Letters 558 (2014) 26–30
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotection by platelet-activating factor acetylhydrolase in a mouse model of transient cerebral ischemia Yijuan Wu a,b , Lijuan Wang b,∗ , Chengbo Dai b , Guixian Ma b , Yuhu Zhang b , Xiong Zhang b,∗ , Zhuohua Wu a a
Department of Neurology, First Affiliated Hospital of Guangzhou Medical University, 510120 Guangzhou, Guangdong, China Department of Neurology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangdong Neuroscience Institute, 510080 Guangzhou, China b
h i g h l i g h t s • It seems that human recombinant PAF-AH (rPAF-AH) may have neuroprotection in middle cerebral artery occlusion mice. • In these mice models we found that rPAF-AH might provide neuroprotection against ischemic injury. • Neuroprotection might be induced not only by decrease in MMP-2 and MMP-9 expression, but also by increased VEGF expression.
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Article history: Received 19 June 2013 Received in revised form 2 September 2013 Accepted 3 September 2013 Keywords: MCAO Matrix metalloproteinase PAF-acetylhydrolase Transient ischemia Treatment VEGF
a b s t r a c t Neuronal damage after transient cerebral ischemia is exacerbated by signaling pathways involving activated platelet-activating factor (PAF) and ameliorated by PAF-acetylhydrolase (PAF-AH); but whether cerebral neurons can be rescued by human recombinant PAF-AH (rPAF-AH) remains unknown. Adult male mice underwent a 60 min middle cerebral artery occlusion (MCAO) and reperfusion for 24 h. Then, the mice received intravenous tail injections with different drugs. Neurological behavioral function was evaluated by Bederson’s test, and cerebral infarction volume was assessed with tetrazolium chloride (TTC) staining. mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2, collagenase1), MMP-9 (gelatinase-B), and vascular endothelial growth factor (VEGF) were determined by quantitative real-time PCR (RT-PCR) and western blot analysis, respectively. Compared with the vehicle group, rPAFAH significantly improved sensorimotor function (42%, P = 0.0001). The volume of non-infarcted brain tissue was increased by the rPAF-AH treatment (16.3 ± 4.6% vs. 46.0 ± 10.3%, respectively). rPAF-AH significantly reduced mRNA and protein levels of MMP-2 and MMP-9, but increased the mRNA (P < 0.001) and protein levels (P < 0.01) of VEGF. These results demonstrate that rPAF-AH provides neuroprotection against ischemic injury. Neuroprotection might be induced not only by decrease in MMP-2 and MMP-9 expression, but also by increased VEGF expression. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-snglycero-3-phosphorylcholine) is a potent lipid mediator[1] that was initially extracted from immunoglobin E-stimulated basophils. It is reported to be involved in a large number of pathological processes, including shock, trauma, allergy, ischemia and inflammation [2,3]. Recent studies have shown that PAF is associated with ischemic injury in the central nervous system (CNS) and plays an important role in the aggravation of neuronal damage in postischemic and posttraumatic brains [4]. It was also reported that
∗ Corresponding authors. E-mail addresses: [email protected], [email protected] (X. Zhang). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.09.005
selective PAF receptor antagonists could prevent ischemia-induced CNS damage in animal models [5]. Collectively, the evidence indicates that the use of PAF receptor antagonists may be a potent treatment for stroke [6]. The acetyl group at the sn-2 position of the glycerol backbone in the PAF molecule is crucial to its biological activity. Deacetylation catalyzed by PAF-acetylhydrolase (PAF-AH) can directly deactivate PAF both in vivo and in vitro. Umemura et al. [7] reported that PAF-AH exerted strong neuroprotective effects against ischemic injuries, but the cellular mechanism underlying this effect is still poorly understood. The concentration of the zinc-dependent endopeptidases, matrix metalloproteinases (MMPs), serves as an indicator of various cellular activities. MMP expression and activity are enhanced in various pathologic conditions, such as Alzheimer’s disease, diabetes, cancer, atherosclerosis, or cerebral ischemia. In acute
Y. Wu et al. / Neuroscience Letters 558 (2014) 26–30
cerebral ischemia, the blood-brain barrier (BBB) disruptionmediated neuronal damage results in upregulated expression of MMP-2 and MMP-9[8]. Recent research has revealed that infarction volumes are reduced by inhibition of MMP-9 in mice [9]. These findings prompted us to hypothesize that modulating MMP expression and activity may be a potential therapeutic option for acute cerebral ischemia [10,11]. Vascular endothelial growth factor (VEGF) is the most significant regulatory element in the vascular system development and differentiation [12]. VEGF has pleiotropic functions in the brain, such as promoting neurogenesis and angiogenesis and mediating neuroprotection [13]. Existing studies on stroke have mainly concentrated on its neuroprotective effects, ignoring the potential ability of VEGF to reorganize ischemic vascular structure [14,15], even though angiogenesis and neurogenesis are known to occur simultaneously [16]. Increasing evidence suggests that vascular remodeling happens after stroke [15,17], and higher blood vessel counts are associated with longer survival in stroke patients [18]. These findings indicate that it is necessary to develop a new drug for stroke and ischemia that targets vascular remodeling in the brain. The objective of the present study was to test the hypothesis that rPAF-AH is neuroprotective in a mouse model of transient cerebral ischemia. We also investigated the roles of MMP-2, MMP-9, and VEGF in cerebral vascular remodeling.
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of MCAO model preoperative 15 min pretreatment drug injected into the tail vein of mice, reperfusion 2 h after middle cerebral artery occlusion: (1) The sham-operation group in which the mice received equal volumes of saline solution (n = 4, Sham group), (2) the MCAO surgery group in which the mice received equal volumes of saline solution (n = 4, NS group), (3) the MCAO surgery group in which the mice received intravenous administration of 100 mg/kg ginaton (n = 4, Ginaton group), (4) the MCAO surgery group in which the mice received intravenous administration of 5 mg/kg ozagrel sodium (n = 4, OS group), and (5) the MCAO surgery group in which the mice received intravenous administration of 1 mg/kg rPAF-AH (n = 4, rPAF-AH group). 2.4. Behavior evaluation A standardized battery of behavioral tests was used to quantify sensorimotor neurological function 2 h and 24 h after MCAO. The Bederson score was determined according to the following scoring system: 0, no deficit; 1, forelimb flexion; 2, forelimb flexion and decreased resistance to lateral push; 3, unidirectional circling; 4, longitudinal spinning or seizure activity; 5, no movement [20]. The test was conducted by an observer who was blinded to the treatment groups. 2.5. Evaluation of cerebral edema and infarction
2. Materials and methods 2.1. Preparation and administration of rPAF-AH Human recombinant PAF-AH (PeproTech EC Ltd., Rocky Hill, NJ, US) (4 mg/ml) was dissolved in 0.9% saline before it was intravenously injected into the tail vein within 15 min before the reperfusion in a middle cerebral artery occlusion (MCAO) mouse model. Reperfusion after 2 h embolism. 2.2. Ischemic animal model All animals were obtained from the Experimental Animal Center of Sun Yat-sen University. Transient MCAO was performed as previously described [19]. Briefly, male Kunming mice (25–30 g) were temporarily anesthetized with 5% isoflurane in oxygen/nitrogen (N2 O/O2 [30/70]) and a mixture of 2% isoflurane and N2 O/O2 (30/70) during the course of surgery. Rectal temperatures were maintained at 37 ◦ C throughout the surgical procedures with a thermostat-controlled heating pad (Neuroscience, Tokyo, Japan). Changes in regional cerebral blood flow (rCBF) before and after MCAO were measured using laserDoppler flowmetry (FLO-C1; Omegawave, Tokyo, Japan). The tip of the flexible probe was affixed with glue perpendicularly to the ipsilateral skull over the area between the secondary somatosensory cortex and the rostral part of the auditory cortex. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed through a ventral midline incision. An 8.0-monofilament nylon suture with a rounded tip was introduced into the CCA lumen and gently advanced into the ICA until it blocked the bifurcating origin of the middle cerebral artery (MCA). Sixty minutes after occlusion, animals were reperfused by withdrawing the suture from the CCA lumen. All experiments were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publications No. 80–23; revised in 1996).
Brains were dissected, and coronal slices (2 mm in thickness) were acquired from frozen forebrains using a mouse brain matrix (Braintree Scientific, Braintree, MA, USA). Brain slices were then stained with 2,3,5- triphenyltetrazolium chloride (TTC) (2%) (Sigma, St. Louis, MO, USA) at 37 ◦ C for 15 min. The areas in the left hemisphere and those showing infarction were quantitated for each section with the ImageJ version 1.63 software (NIH, Bethesda, MD, USA). The relative infarction volume was expressed as the percentage of the infarct volume corrected to the ipsilateral hemispheric volume. The cerebral infarction volume was measured using an MPLAS-500 multimedia color pathological graphic analysis system. The investigators who performed the image analyses were blinded to the study groups. 2.6. Western blot analysis and enzyme assays After reperfusion, mice were anesthetized and perfused with ice-cold phosphate-buffered saline. Hemispheric tissue was immediately frozen in liquid nitrogen and stored at −80 ◦ C. Tissue was homogenized on ice in lysis buffer containing protease inhibitors, centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. For each sample, 20 g of protein was loaded on 4–15% sodium dodecyl sulfate polyacrylamide gels, separated by electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking for 1 h at room temperature with 10% skim milk (Inner Mongolia Yili Industrial Group Co. Ltd., Inner Mongolia, China), blots were incubated overnight at 4 ◦ C with any one of the following primary antibodies from Abcam (Cambridge, UK): rabbit anti-MMP-2 (2 g/ml), rabbit anti-MMP-9 (1:400), and rabbit anti-VEGF (1 g/ml). Horseradish peroxidase-conjugated anti-rabbit antibody was used as the secondary antibody, and signals were detected using the standard chemical luminescence method (ECL; Thermo Fisher Scientific Inc., Waltham, MA, USA). 2.7. RT-PCR for MMP-2, MMP-9, and VEGF
2.3. Experimental groups Twenty mice were randomly divided into five groups according to different drug treatments after MCAO surgery. In the production
Total RNA was isolated from tissues using TRIzol reagent (Invitrogen) and reverse-transcribed using oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase
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Y. Wu et al. / Neuroscience Letters 558 (2014) 26–30
Fig. 1. Cerebral ischemic area 24 h after MCAO. Treatment with Ginaton or PAF-AH markedly reduced infarct volume, and the PAF-AH group showed a significant reduction in infarct volume compared to the other groups. *P < 0.05, **P < 0.01, n = 6/group.
(Takara, Otsu, Japan). Next, 10 ng cDNA was used as a template for quantitative RT-PCR in the presence of a fluorescent dye, SYBR Green (Takara). Relative mRNA abundance was normalized to glyceraldehyde 3-phosphate dehydrogenase mRNA. The primers for MMP2, MMP9, and VEGF were as follows: MMP-2: 5 -GTGCC AAGGTGGAAATCAGAG-3 (reverse); MMP-9: 5 -AGACGACATAGACGGCATCC-3 (forward), 5 GTGGTTCAGTTGTGGTGGTG-3 (reverse); VEGF: 5 -CCAGGAGGACCTTGTGTGAT-3 (forward), 5 -GGGAAGGGAAGATGAGGAAG-3 (reverse). 2.8. Statistical analyses Data are presented as mean ± standard error of mean. Behavioral test has been evaluated by Nonparametric Test-K Independent Samples, Kruskal–Wallis Test. (After 2 h Chi-square = 0.8129, Sig = 0.043; after 24 h Chi-square = 0.8263, Sig = 0.016; and compared with the NS group, *P < 0.05; **P < 0.01.) The other data include the volume of brain section, Western-blot and PCR have been evaluated by one-way ANOVA analysis, LSD was performed to evaluate the differences among the treatment groups and controls. Differences for which P < 0.05 were considered statistically significant.
3. Results 3.1. rPAF-AH treatment decreased infarct volume As shown in Fig. 1, the infarct volumes of all treatment groups were markedly decreased compared with the control group. The rPAF-AH and OS groups were significantly different from the NS group. 3.2. rPAF-AH treatment improved behavior As shown in Fig. 2A, the rPAF-AH group showed significantly improved performance on the Bederson test 2 h after MCAO compared to the NS group. However, the OS and Ginaton groups were not significantly different from the NS group. Fig. 2B indicates that rPAF-AH improved behavioral performance 24 h after ischemic injury compared with the NS group. 3.3. rPAF-AH decreased MMP-2 and MMP-9 expression and increased VEGF expression In mice treated with rPAF-AH, both MMP-2 and MMP-9 expression were found to be decreased on western blots. Compared with other groups, rPAF-AH-treated mice exhibited decreased MMP
Fig. 2. The rPAF-AH group showed the highest behavioral performance scores on the Bederson test. (A) Temporal change in nervous system dysfunction score 2 h after MCAO. *P < 0.05, **P < 0.01. (B) Nervous system dysfunction score 24 h after reperfusion. Ginaton, OS, and rPAF-AH significantly improved neural dysfunction status. *P < 0.05, **P < 0.01, n = 9/group.
Y. Wu et al. / Neuroscience Letters 558 (2014) 26–30
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Fig. 3. Western blots showing decrease in MMP-2 and MMP-9 expression in the rPAF-AH-treated mice; VEGF expression was significantly upregulated. (A) MMP-2 levels in the right hemisphere after MCAO. MMP-2 was significantly increased in the NS group compared to the Sham group. *P < 0.05, **P < 0.01, n = 4/group. (B) MMP-9 levels in the right hemisphere after MCAO. *P < 0.05, **P < 0.01, n = 4/group. (C) VEGF levels in the right hemisphere after MCAO. VEGF was significantly increased in the NS group compared to the Sham group. Ginaton, OS, and rPAF-AH treatment significantly increased VEGF expression. *P < 0.05, **P < 0.01, n = 4/group.
expression (Fig. 3A and B). Western blot analyses demonstrated that VEGF expression was significantly upregulated in the rPAF-AH group compared to the NS group (Fig. 3C). 3.4. rPAF-AH decreased MMP mRNA expression but increased VEGF mRNA expression RT-PCR analysis showed decreased MMP-2 and MMP-9 mRNA levels but increased VEGF mRNA levels in the rPAF-AH group (Fig. 4). 4. Discussion Recently, activation of the PAF-PAF receptor signaling system has been shown to play a crucial part in the appearance and succession of neuronal injuries caused by cerebral ischemia. More selective PAF receptor antagonists perform better in ameliorating brain damage caused by cerebral infarction [21]. However, whether ischemia-damaged neurons can be rescued by human rPAF-AH is still largely unknown. Here, we used behavioral analysis to demonstrate that rPAF-AH provides neuroprotection against ischemic injury following MCAO.
We compared the effect of rPAF-AH against ginaton and OS. Ginaton, an extract prepared from Ginkgo biloba leaves, was previously reported to act as an antagonist of PAFR. It was hypothesized that inflammation could be inhibited and ischemic injuries be avoided by blocking PAFR signaling [22]. OS is a selective thromboxane A2 synthase inhibitor that suppresses vasospasm and platelet aggregation in animals and humans [23]. In our study, we employed the Bederson score analysis and found less improvement in behavioral disability in the NS group compared to the treatment groups. Mice in the rPAF-AH group showed significant improvement of behavioral disability at both 2 h and 24 h after MCAO. Ginaton or OS monotherapy also showed significant improvements compared with treatment in the NS group, but the rPAF-AH group performed significantly better at 2 h after MCAO. These results are similar to what previous researchers have found. Belayev demonstrated that LAU-0901, a novel platelet-activating factor antagonist, protects against neurological dysfunction and improves neurologic scores in cerebral ischemic animals [24]. Although PAF-AH has been suggested to be beneficial in preventing inflammation, little is known about the exact mechanistic role of PAF-AH. Ko and colleagues [25] hypothesized that the effects are partly mediated by MMP-2 and MMP-9.
Fig. 4. MMP-2, MMP-9, and VEGF mRNA levels in the right hemisphere in MCAO mice were assessed by RT-PCR. (A and B) rPAF-AH significantly downregulated MMP-2 and MMP-9 expression, respectively, # NS group vs. Sham group, P < 0.05; **Gination group, OS group, rPAF-AH group vs. NS group, P < 0.01; no significant difference was found among the Gination group, OS group, and rPAF-AH group. (C) VEGF mRNA levels were higher in the NS group than in the Sham group. rPAF-AH significantly increased VEGF expression (**P < 0.001), but Ginaton and OS did not. ## NS group vs. Sham group, P < 0.01; & Gination group, OS group vs. rPAF-AH group, P < 0.01.
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Indeed, neuronal and vascular injuries may be exacerbated by MMP-2 and MMP-9 upregulation [26]. Melnikova et al. [27] demonstrated that in metastatic melanoma cells, PAF stimulated the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), which resulted in MMP-2 overexpression. PAF injection increases MMP-2 and MMP-9 mRNA expression, protein levels, and activities [26]. These lines of evidence support the hypothesis that rPAF-AH reduces the infarction volume in mice by inhibiting MMPs. VEGF is a potent angiogenic factor that plays prominent roles in the central nervous system, and has neurotrophic and neuroprotective effects on spinal motor neurons [28,29]. One study demonstrated that the combination of intracortically administered VEGF and environmental enrichment enhances brain protection in developing rats [30]. VEGF antisense treatment increases ischemiainduced neuronal damage and results in larger infarct volumes [31]. Furthermore, VEGF overexpression reduces hypoxic–ischemic brain infarct, hypoxic neuron apoptosis, and neurological deficits [32]. VEGF has dual functions in the brain; it mediates both neuroprotection and BBB permeability through VEGF/VEGFR2/Flk1 and PI3K/Akt pathways [33,34]. The results presented here suggest that the elevated VEGF level after rPAF-AH treatment might have neuroprotective effects. In conclusion, infarction volume was sharply reduced and functional performance was significantly improved in a mouse model of cerebral ischemia following 1 mg/kg treatment with rPAF-AH. MMP-2 and MMP-9, which are important mediators of neurovascular injuries after cerebral ischemia, may be down-regulated by rPAF-AH. Elevated VEGF might be an important component in the neuroprotective effect of rPAF-AH via its angiogenic effects. Our data provide a firm basis for further exploring a molecular approach for blocking PAF signaling in stroke pathophysiology. The results described here suggest that rPAF-AH administration is a viable therapy for ischemic stroke. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 30870863 and No. 30801219), the Natural Science Foundation of Guangdong Province (No. 10151008004000030 and No. 8151008004000008), the Science and Technology Planning Project of Guangdong Province (No. 2009B030801251), and the Medical Scientific Research Foundation of Guangdong Province, China (No. A2009038, No. B2010007, and No. A2010029). The authors have no conflicts of interest to declare. References [1] V. Alfaro, Role of histamine and platelet-activating factor in allergic rhinitis, J. Physiol. Biochem. 60 (2004) 101–111. [2] N.K. Liu, X.M. Xu, Phospholipase A2 and its molecular mechanism after spinal cord injury, Mol. Neurobiol. 41 (2010) 197–205. [3] K. Mukai, K. Obata, Y. Tsujimura, H. Karasuyama, New insights into the roles for basophils in acute and chronic allergy, Allergol. Int. 58 (2009) 9–11. [4] A.I. Faden, P.A. Tzendzalian, Platelet-activating factor antagonists limit glycine changes and behavioral deficits after brain trauma, Am. J. Physiol. 263 (1992) R909–R914. [5] R. Farbiszewski, H. Dudek, E. Skrzydlewska, J. Lewko, The role of platelet activating factor (PAF) in physiology and pathology of the central nervous system, Neurol. Neurochir. Pol. 36 (2002) 801–808. [6] S. Uchiyama, T. Nakamura, M. Yamazaki, Y. Kimura, M. Iwata, New modalities and aspects of antiplatelet therapy for stroke prevention, Cerebrovasc. Dis. Suppl. 1N (2006) 7–16. [7] K. Umemura, I. Kato, Y. Hirashima, Y. Ishii, T. Inoue, J. Aoki, N. Kono, T. Oya, N. Hayashi, H. Hamada, S. Endo, M. Oda, H. Arai, H. Kinouchi, K. Hiraga, Neuroprotective role of transgenic PAF-acetylhydrolase II in mouse models of focal cerebral ischemia, Stroke 38 (2007) 1063–1068.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotection by platelet-activating factor acetylhydrolase in a mouse model of transient cerebral ischemia Yijuan Wu a,b , Lijuan Wang b,∗ , Chengbo Dai b , Guixian Ma b , Yuhu Zhang b , Xiong Zhang b,∗ , Zhuohua Wu a a
Department of Neurology, First Affiliated Hospital of Guangzhou Medical University, 510120 Guangzhou, Guangdong, China Department of Neurology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangdong Neuroscience Institute, 510080 Guangzhou, China b
h i g h l i g h t s • It seems that human recombinant PAF-AH (rPAF-AH) may have neuroprotection in middle cerebral artery occlusion mice. • In these mice models we found that rPAF-AH might provide neuroprotection against ischemic injury. • Neuroprotection might be induced not only by decrease in MMP-2 and MMP-9 expression, but also by increased VEGF expression.
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Article history: Received 19 June 2013 Received in revised form 2 September 2013 Accepted 3 September 2013 Keywords: MCAO Matrix metalloproteinase PAF-acetylhydrolase Transient ischemia Treatment VEGF
a b s t r a c t Neuronal damage after transient cerebral ischemia is exacerbated by signaling pathways involving activated platelet-activating factor (PAF) and ameliorated by PAF-acetylhydrolase (PAF-AH); but whether cerebral neurons can be rescued by human recombinant PAF-AH (rPAF-AH) remains unknown. Adult male mice underwent a 60 min middle cerebral artery occlusion (MCAO) and reperfusion for 24 h. Then, the mice received intravenous tail injections with different drugs. Neurological behavioral function was evaluated by Bederson’s test, and cerebral infarction volume was assessed with tetrazolium chloride (TTC) staining. mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2, collagenase1), MMP-9 (gelatinase-B), and vascular endothelial growth factor (VEGF) were determined by quantitative real-time PCR (RT-PCR) and western blot analysis, respectively. Compared with the vehicle group, rPAFAH significantly improved sensorimotor function (42%, P = 0.0001). The volume of non-infarcted brain tissue was increased by the rPAF-AH treatment (16.3 ± 4.6% vs. 46.0 ± 10.3%, respectively). rPAF-AH significantly reduced mRNA and protein levels of MMP-2 and MMP-9, but increased the mRNA (P < 0.001) and protein levels (P < 0.01) of VEGF. These results demonstrate that rPAF-AH provides neuroprotection against ischemic injury. Neuroprotection might be induced not only by decrease in MMP-2 and MMP-9 expression, but also by increased VEGF expression. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-snglycero-3-phosphorylcholine) is a potent lipid mediator[1] that was initially extracted from immunoglobin E-stimulated basophils. It is reported to be involved in a large number of pathological processes, including shock, trauma, allergy, ischemia and inflammation [2,3]. Recent studies have shown that PAF is associated with ischemic injury in the central nervous system (CNS) and plays an important role in the aggravation of neuronal damage in postischemic and posttraumatic brains [4]. It was also reported that
∗ Corresponding authors. E-mail addresses: [email protected], [email protected] (X. Zhang). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.09.005
selective PAF receptor antagonists could prevent ischemia-induced CNS damage in animal models [5]. Collectively, the evidence indicates that the use of PAF receptor antagonists may be a potent treatment for stroke [6]. The acetyl group at the sn-2 position of the glycerol backbone in the PAF molecule is crucial to its biological activity. Deacetylation catalyzed by PAF-acetylhydrolase (PAF-AH) can directly deactivate PAF both in vivo and in vitro. Umemura et al. [7] reported that PAF-AH exerted strong neuroprotective effects against ischemic injuries, but the cellular mechanism underlying this effect is still poorly understood. The concentration of the zinc-dependent endopeptidases, matrix metalloproteinases (MMPs), serves as an indicator of various cellular activities. MMP expression and activity are enhanced in various pathologic conditions, such as Alzheimer’s disease, diabetes, cancer, atherosclerosis, or cerebral ischemia. In acute
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cerebral ischemia, the blood-brain barrier (BBB) disruptionmediated neuronal damage results in upregulated expression of MMP-2 and MMP-9[8]. Recent research has revealed that infarction volumes are reduced by inhibition of MMP-9 in mice [9]. These findings prompted us to hypothesize that modulating MMP expression and activity may be a potential therapeutic option for acute cerebral ischemia [10,11]. Vascular endothelial growth factor (VEGF) is the most significant regulatory element in the vascular system development and differentiation [12]. VEGF has pleiotropic functions in the brain, such as promoting neurogenesis and angiogenesis and mediating neuroprotection [13]. Existing studies on stroke have mainly concentrated on its neuroprotective effects, ignoring the potential ability of VEGF to reorganize ischemic vascular structure [14,15], even though angiogenesis and neurogenesis are known to occur simultaneously [16]. Increasing evidence suggests that vascular remodeling happens after stroke [15,17], and higher blood vessel counts are associated with longer survival in stroke patients [18]. These findings indicate that it is necessary to develop a new drug for stroke and ischemia that targets vascular remodeling in the brain. The objective of the present study was to test the hypothesis that rPAF-AH is neuroprotective in a mouse model of transient cerebral ischemia. We also investigated the roles of MMP-2, MMP-9, and VEGF in cerebral vascular remodeling.
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of MCAO model preoperative 15 min pretreatment drug injected into the tail vein of mice, reperfusion 2 h after middle cerebral artery occlusion: (1) The sham-operation group in which the mice received equal volumes of saline solution (n = 4, Sham group), (2) the MCAO surgery group in which the mice received equal volumes of saline solution (n = 4, NS group), (3) the MCAO surgery group in which the mice received intravenous administration of 100 mg/kg ginaton (n = 4, Ginaton group), (4) the MCAO surgery group in which the mice received intravenous administration of 5 mg/kg ozagrel sodium (n = 4, OS group), and (5) the MCAO surgery group in which the mice received intravenous administration of 1 mg/kg rPAF-AH (n = 4, rPAF-AH group). 2.4. Behavior evaluation A standardized battery of behavioral tests was used to quantify sensorimotor neurological function 2 h and 24 h after MCAO. The Bederson score was determined according to the following scoring system: 0, no deficit; 1, forelimb flexion; 2, forelimb flexion and decreased resistance to lateral push; 3, unidirectional circling; 4, longitudinal spinning or seizure activity; 5, no movement [20]. The test was conducted by an observer who was blinded to the treatment groups. 2.5. Evaluation of cerebral edema and infarction
2. Materials and methods 2.1. Preparation and administration of rPAF-AH Human recombinant PAF-AH (PeproTech EC Ltd., Rocky Hill, NJ, US) (4 mg/ml) was dissolved in 0.9% saline before it was intravenously injected into the tail vein within 15 min before the reperfusion in a middle cerebral artery occlusion (MCAO) mouse model. Reperfusion after 2 h embolism. 2.2. Ischemic animal model All animals were obtained from the Experimental Animal Center of Sun Yat-sen University. Transient MCAO was performed as previously described [19]. Briefly, male Kunming mice (25–30 g) were temporarily anesthetized with 5% isoflurane in oxygen/nitrogen (N2 O/O2 [30/70]) and a mixture of 2% isoflurane and N2 O/O2 (30/70) during the course of surgery. Rectal temperatures were maintained at 37 ◦ C throughout the surgical procedures with a thermostat-controlled heating pad (Neuroscience, Tokyo, Japan). Changes in regional cerebral blood flow (rCBF) before and after MCAO were measured using laserDoppler flowmetry (FLO-C1; Omegawave, Tokyo, Japan). The tip of the flexible probe was affixed with glue perpendicularly to the ipsilateral skull over the area between the secondary somatosensory cortex and the rostral part of the auditory cortex. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed through a ventral midline incision. An 8.0-monofilament nylon suture with a rounded tip was introduced into the CCA lumen and gently advanced into the ICA until it blocked the bifurcating origin of the middle cerebral artery (MCA). Sixty minutes after occlusion, animals were reperfused by withdrawing the suture from the CCA lumen. All experiments were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publications No. 80–23; revised in 1996).
Brains were dissected, and coronal slices (2 mm in thickness) were acquired from frozen forebrains using a mouse brain matrix (Braintree Scientific, Braintree, MA, USA). Brain slices were then stained with 2,3,5- triphenyltetrazolium chloride (TTC) (2%) (Sigma, St. Louis, MO, USA) at 37 ◦ C for 15 min. The areas in the left hemisphere and those showing infarction were quantitated for each section with the ImageJ version 1.63 software (NIH, Bethesda, MD, USA). The relative infarction volume was expressed as the percentage of the infarct volume corrected to the ipsilateral hemispheric volume. The cerebral infarction volume was measured using an MPLAS-500 multimedia color pathological graphic analysis system. The investigators who performed the image analyses were blinded to the study groups. 2.6. Western blot analysis and enzyme assays After reperfusion, mice were anesthetized and perfused with ice-cold phosphate-buffered saline. Hemispheric tissue was immediately frozen in liquid nitrogen and stored at −80 ◦ C. Tissue was homogenized on ice in lysis buffer containing protease inhibitors, centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. For each sample, 20 g of protein was loaded on 4–15% sodium dodecyl sulfate polyacrylamide gels, separated by electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking for 1 h at room temperature with 10% skim milk (Inner Mongolia Yili Industrial Group Co. Ltd., Inner Mongolia, China), blots were incubated overnight at 4 ◦ C with any one of the following primary antibodies from Abcam (Cambridge, UK): rabbit anti-MMP-2 (2 g/ml), rabbit anti-MMP-9 (1:400), and rabbit anti-VEGF (1 g/ml). Horseradish peroxidase-conjugated anti-rabbit antibody was used as the secondary antibody, and signals were detected using the standard chemical luminescence method (ECL; Thermo Fisher Scientific Inc., Waltham, MA, USA). 2.7. RT-PCR for MMP-2, MMP-9, and VEGF
2.3. Experimental groups Twenty mice were randomly divided into five groups according to different drug treatments after MCAO surgery. In the production
Total RNA was isolated from tissues using TRIzol reagent (Invitrogen) and reverse-transcribed using oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase
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Fig. 1. Cerebral ischemic area 24 h after MCAO. Treatment with Ginaton or PAF-AH markedly reduced infarct volume, and the PAF-AH group showed a significant reduction in infarct volume compared to the other groups. *P < 0.05, **P < 0.01, n = 6/group.
(Takara, Otsu, Japan). Next, 10 ng cDNA was used as a template for quantitative RT-PCR in the presence of a fluorescent dye, SYBR Green (Takara). Relative mRNA abundance was normalized to glyceraldehyde 3-phosphate dehydrogenase mRNA. The primers for MMP2, MMP9, and VEGF were as follows: MMP-2: 5 -GTGCC AAGGTGGAAATCAGAG-3 (reverse); MMP-9: 5 -AGACGACATAGACGGCATCC-3 (forward), 5 GTGGTTCAGTTGTGGTGGTG-3 (reverse); VEGF: 5 -CCAGGAGGACCTTGTGTGAT-3 (forward), 5 -GGGAAGGGAAGATGAGGAAG-3 (reverse). 2.8. Statistical analyses Data are presented as mean ± standard error of mean. Behavioral test has been evaluated by Nonparametric Test-K Independent Samples, Kruskal–Wallis Test. (After 2 h Chi-square = 0.8129, Sig = 0.043; after 24 h Chi-square = 0.8263, Sig = 0.016; and compared with the NS group, *P < 0.05; **P < 0.01.) The other data include the volume of brain section, Western-blot and PCR have been evaluated by one-way ANOVA analysis, LSD was performed to evaluate the differences among the treatment groups and controls. Differences for which P < 0.05 were considered statistically significant.
3. Results 3.1. rPAF-AH treatment decreased infarct volume As shown in Fig. 1, the infarct volumes of all treatment groups were markedly decreased compared with the control group. The rPAF-AH and OS groups were significantly different from the NS group. 3.2. rPAF-AH treatment improved behavior As shown in Fig. 2A, the rPAF-AH group showed significantly improved performance on the Bederson test 2 h after MCAO compared to the NS group. However, the OS and Ginaton groups were not significantly different from the NS group. Fig. 2B indicates that rPAF-AH improved behavioral performance 24 h after ischemic injury compared with the NS group. 3.3. rPAF-AH decreased MMP-2 and MMP-9 expression and increased VEGF expression In mice treated with rPAF-AH, both MMP-2 and MMP-9 expression were found to be decreased on western blots. Compared with other groups, rPAF-AH-treated mice exhibited decreased MMP
Fig. 2. The rPAF-AH group showed the highest behavioral performance scores on the Bederson test. (A) Temporal change in nervous system dysfunction score 2 h after MCAO. *P < 0.05, **P < 0.01. (B) Nervous system dysfunction score 24 h after reperfusion. Ginaton, OS, and rPAF-AH significantly improved neural dysfunction status. *P < 0.05, **P < 0.01, n = 9/group.
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Fig. 3. Western blots showing decrease in MMP-2 and MMP-9 expression in the rPAF-AH-treated mice; VEGF expression was significantly upregulated. (A) MMP-2 levels in the right hemisphere after MCAO. MMP-2 was significantly increased in the NS group compared to the Sham group. *P < 0.05, **P < 0.01, n = 4/group. (B) MMP-9 levels in the right hemisphere after MCAO. *P < 0.05, **P < 0.01, n = 4/group. (C) VEGF levels in the right hemisphere after MCAO. VEGF was significantly increased in the NS group compared to the Sham group. Ginaton, OS, and rPAF-AH treatment significantly increased VEGF expression. *P < 0.05, **P < 0.01, n = 4/group.
expression (Fig. 3A and B). Western blot analyses demonstrated that VEGF expression was significantly upregulated in the rPAF-AH group compared to the NS group (Fig. 3C). 3.4. rPAF-AH decreased MMP mRNA expression but increased VEGF mRNA expression RT-PCR analysis showed decreased MMP-2 and MMP-9 mRNA levels but increased VEGF mRNA levels in the rPAF-AH group (Fig. 4). 4. Discussion Recently, activation of the PAF-PAF receptor signaling system has been shown to play a crucial part in the appearance and succession of neuronal injuries caused by cerebral ischemia. More selective PAF receptor antagonists perform better in ameliorating brain damage caused by cerebral infarction [21]. However, whether ischemia-damaged neurons can be rescued by human rPAF-AH is still largely unknown. Here, we used behavioral analysis to demonstrate that rPAF-AH provides neuroprotection against ischemic injury following MCAO.
We compared the effect of rPAF-AH against ginaton and OS. Ginaton, an extract prepared from Ginkgo biloba leaves, was previously reported to act as an antagonist of PAFR. It was hypothesized that inflammation could be inhibited and ischemic injuries be avoided by blocking PAFR signaling [22]. OS is a selective thromboxane A2 synthase inhibitor that suppresses vasospasm and platelet aggregation in animals and humans [23]. In our study, we employed the Bederson score analysis and found less improvement in behavioral disability in the NS group compared to the treatment groups. Mice in the rPAF-AH group showed significant improvement of behavioral disability at both 2 h and 24 h after MCAO. Ginaton or OS monotherapy also showed significant improvements compared with treatment in the NS group, but the rPAF-AH group performed significantly better at 2 h after MCAO. These results are similar to what previous researchers have found. Belayev demonstrated that LAU-0901, a novel platelet-activating factor antagonist, protects against neurological dysfunction and improves neurologic scores in cerebral ischemic animals [24]. Although PAF-AH has been suggested to be beneficial in preventing inflammation, little is known about the exact mechanistic role of PAF-AH. Ko and colleagues [25] hypothesized that the effects are partly mediated by MMP-2 and MMP-9.
Fig. 4. MMP-2, MMP-9, and VEGF mRNA levels in the right hemisphere in MCAO mice were assessed by RT-PCR. (A and B) rPAF-AH significantly downregulated MMP-2 and MMP-9 expression, respectively, # NS group vs. Sham group, P < 0.05; **Gination group, OS group, rPAF-AH group vs. NS group, P < 0.01; no significant difference was found among the Gination group, OS group, and rPAF-AH group. (C) VEGF mRNA levels were higher in the NS group than in the Sham group. rPAF-AH significantly increased VEGF expression (**P < 0.001), but Ginaton and OS did not. ## NS group vs. Sham group, P < 0.01; & Gination group, OS group vs. rPAF-AH group, P < 0.01.
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Indeed, neuronal and vascular injuries may be exacerbated by MMP-2 and MMP-9 upregulation [26]. Melnikova et al. [27] demonstrated that in metastatic melanoma cells, PAF stimulated the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), which resulted in MMP-2 overexpression. PAF injection increases MMP-2 and MMP-9 mRNA expression, protein levels, and activities [26]. These lines of evidence support the hypothesis that rPAF-AH reduces the infarction volume in mice by inhibiting MMPs. VEGF is a potent angiogenic factor that plays prominent roles in the central nervous system, and has neurotrophic and neuroprotective effects on spinal motor neurons [28,29]. One study demonstrated that the combination of intracortically administered VEGF and environmental enrichment enhances brain protection in developing rats [30]. VEGF antisense treatment increases ischemiainduced neuronal damage and results in larger infarct volumes [31]. Furthermore, VEGF overexpression reduces hypoxic–ischemic brain infarct, hypoxic neuron apoptosis, and neurological deficits [32]. VEGF has dual functions in the brain; it mediates both neuroprotection and BBB permeability through VEGF/VEGFR2/Flk1 and PI3K/Akt pathways [33,34]. The results presented here suggest that the elevated VEGF level after rPAF-AH treatment might have neuroprotective effects. In conclusion, infarction volume was sharply reduced and functional performance was significantly improved in a mouse model of cerebral ischemia following 1 mg/kg treatment with rPAF-AH. MMP-2 and MMP-9, which are important mediators of neurovascular injuries after cerebral ischemia, may be down-regulated by rPAF-AH. Elevated VEGF might be an important component in the neuroprotective effect of rPAF-AH via its angiogenic effects. Our data provide a firm basis for further exploring a molecular approach for blocking PAF signaling in stroke pathophysiology. The results described here suggest that rPAF-AH administration is a viable therapy for ischemic stroke. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 30870863 and No. 30801219), the Natural Science Foundation of Guangdong Province (No. 10151008004000030 and No. 8151008004000008), the Science and Technology Planning Project of Guangdong Province (No. 2009B030801251), and the Medical Scientific Research Foundation of Guangdong Province, China (No. A2009038, No. B2010007, and No. A2010029). The authors have no conflicts of interest to declare. References [1] V. Alfaro, Role of histamine and platelet-activating factor in allergic rhinitis, J. Physiol. Biochem. 60 (2004) 101–111. [2] N.K. Liu, X.M. Xu, Phospholipase A2 and its molecular mechanism after spinal cord injury, Mol. Neurobiol. 41 (2010) 197–205. [3] K. Mukai, K. Obata, Y. Tsujimura, H. Karasuyama, New insights into the roles for basophils in acute and chronic allergy, Allergol. Int. 58 (2009) 9–11. [4] A.I. Faden, P.A. Tzendzalian, Platelet-activating factor antagonists limit glycine changes and behavioral deficits after brain trauma, Am. J. Physiol. 263 (1992) R909–R914. [5] R. Farbiszewski, H. Dudek, E. Skrzydlewska, J. Lewko, The role of platelet activating factor (PAF) in physiology and pathology of the central nervous system, Neurol. Neurochir. Pol. 36 (2002) 801–808. [6] S. Uchiyama, T. Nakamura, M. Yamazaki, Y. Kimura, M. Iwata, New modalities and aspects of antiplatelet therapy for stroke prevention, Cerebrovasc. Dis. Suppl. 1N (2006) 7–16. [7] K. Umemura, I. Kato, Y. Hirashima, Y. Ishii, T. Inoue, J. Aoki, N. Kono, T. Oya, N. Hayashi, H. Hamada, S. Endo, M. Oda, H. Arai, H. Kinouchi, K. Hiraga, Neuroprotective role of transgenic PAF-acetylhydrolase II in mouse models of focal cerebral ischemia, Stroke 38 (2007) 1063–1068.
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