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Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury
Life Sciences 80 (2007) 530 – 537 www.elsevier.com/locate/lifescie
Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury Lei Zhang, Wei-Ping Zhang, Ke-Da Chen, Xiao-Dong Qian, San-Hua Fang, Er-Qing Wei ⁎ Department of Pharmacology, School of Medicine, Zhejiang University, 388, Yu Hang Tang Road, Hangzhou 310058, People's Republic of China Received 14 June 2006; accepted 29 September 2006
Abstract Traumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects against ischemic brain injuries with anti-oxidant and anti-inflammatory properties, and by scavenging reactive species. However, whether caffeic acid has protective effects against traumatic brain injury is unknown. Therefore, we determined the effect of caffeic acid on the lesion in the early (1 day) and late phases (7 to 28 days) of cryoinjury in mice. We found that caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days, but attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and glial fibrillary acidic protein (GFAP) protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduction of superoxide dismutase activity and the increase in malondialdehyde content in the brain 1 day after cryoinjury. These results indicate that caffeic acid exerts a protective effect in traumatic brain injury, especially on glial scar formation in the late phase, which at least is associated with its anti-oxidant ability. © 2006 Elsevier Inc. All rights reserved. Keywords: Caffeic acid; Traumatic brain injury; Cryoinjury; Neuroprotection; Astrogliosis; Glial scar
Introduction Traumatic brain injury produces sequential consequences that can be divided into early and late (or chronic) phases. In the early phase, the main changes include excitotoxicity, oxidative stress, free radical production, apoptosis, inflammation and degeneration (Bramlett and Dietrich, 2004; Raghupathi, 2004; Sofroniew, 2005). In the late phase, one of the changes is the formation of a glial scar resulting from reactive gliosis (mainly consisting of proliferated astrocytes) (Logan and Berry, 2002), which may be a physical and biochemical barrier for the regeneration of axons (Silver and Miller, 2004). To investigate traumatic brain injury, a number of animal models have been developed, among which cryoinjury (also called as cold injury) is one widely used model (Murakami et al., 1999; Hortobagyi et al., 2000). Cryoinjury can mimic some characteristics of ⁎ Corresponding author. Tel.: +86 571 8820 8224; fax: +86 571 8820 8022. E-mail address: [email protected] (E.-Q. Wei). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.09.039
traumatic brain injuries and the related repair responses, such as apoptosis (Flentjar et al., 2002), inflammation (Sewell et al., 2004; Shin et al., 2005), angiogenesis (Nag, 2002), astrocyte proliferation and glial scar formation (Hermann et al., 2004; Hirano et al., 2004; Tada et al., 2004). Currently, the pharmacological interventions in traumatic brain injury are limited to the early injuries (Gorlach et al., 2001; Turkoglu et al., 2005) while the interventions in the late injuries are clinically more important and need to be investigated. Since we recently found that caffeic acid inhibited the astrocyte proliferation and neuronal injury in the late phase of focal cerebral ischemia in rats (Zhou et al., 2006), we propose that caffeic acid may also have an effect on late changes in cryoinjury. Caffeic acid (3, 4-dihydroxycinnamic acid) is one of the natural phenolic compounds widely distributed in plant materials such as vegetables, fruits, coffee and tea (Sondheimer, 1958). Caffeic acid possesses anti-oxidant properties as it scavenges a number of reactive species, including 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH) (Kikuzaki et al., 2002; Gulcin, 2006), peroxyl
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(Castelluccio et al., 1995) and hydroxyl radicals (Kono et al., 1997), as well as superoxide anion, peroxynitrite and mutagenic compounds such as nitrosamines (Kono et al., 1997; Gulcin, 2006). Caffeic acid also inhibits 5-lipoxygenase (5-LOX) activity (Koshihara et al., 1984), and inhibits protein kinase C (PKC), PKA and nuclear factor-κB (NF-κB) activation induced by ceramides in U937 cells (Nardini et al., 2000, 2001). Pharmacological studies have shown that caffeic acid exerts protective effects on glutamate-induced neurodegeneration in primary cultured cortical neurons (Kim and Kim, 2000) and PrP106-126 neurotoxicity in cerebellar granule neurons (Stewart et al., 2001), and hippocampal excitotoxic injury induced by systemic injection of kainite in vivo (Uz et al., 1998). A derivative of caffeic acid, caffeic acid phenethyl ester (CAPE), attenuates cerebral vasospasm after experimental subarachnoid hemorrhage (Aladag et al., 2006) and neonatal hypoxic– ischemic neuronal death (Wei et al., 2004). However, its effects on early and late injuries after traumatic brain injury are still unknown. Therefore, in the present study we determined the effect of caffeic acid on the injuries in the early phase (1 day) and the late phase (7 to 28 days) of cryoinjury in mice. To clarify the possible mechanism, we also determined the anti-oxidant ability of caffeic acid in the early phase.
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injected intraperitoneally 30 min, 2 and 6 h after cryoinjury on the first day and twice daily on days 2 to 7. For measuring superoxide dismutase (SOD) activity and malondialdehyde
Materials and methods Animals A total of 220 male ICR mice weighing 25–35 g (Experimental Animal Center of Zhejiang Academy of Medical Sciences, Hangzhou, China, Certificate No. 20030001) were used in this study. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of School of Medicine, Zhejiang University. Mice were housed under a controlled temperature (22 ± 1 °C), 12 h light/12 h dark cycle and allowed free access to food and water. Brain cryoinjury and caffeic acid administration Mice were anesthetized with intraperitoneal injection of chloral hydrate (400 mg/kg), and placed on a stereotactic frame (SR-5, Narishige, Tokyo, Japan). Rectal (core) temperature was measured and maintained at 37± 0.5°C with a heating pad and a heating lamp during the surgery. Brain cryoinjury was induced according to the reported method (Sewell et al., 2004) with modifications. Briefly, the scalp was incised on the midline to expose the skull. A metal probe (probe weight: 100 g; tip diameter: 3 mm) cooled in liquid nitrogen was applied to the surface of the intact skull above the right parietal lobe (1.5 mm lateral to the midline; −3.0 mm from bregma) for 30 s. Incisions were sutured after cryoinjury. Mice recovered from anesthesia in a warmed box. Caffeic acid (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in dimethyl sulphoxide (DMSO); the solution was freshly diluted with saline before use. Caffeic acid (10 and 50 mg/kg) or saline containing 20% DMSO (v/v, 10 ml/kg) was
Fig. 1. Effect of caffeic acid on cryoinjury lesion in mice. Gross photographs of whole brains (A) show the brain surface 1 and 7 days after cryoinjury. Coronal sections (B) were used to calculate lesion area (C). Caffeic acid (50 mg/kg) significantly attenuated the lesion over 28 days after cryoinjury (A–C). Values summarized in C are reported as mean ± S.D.; n = 8–12; #P b 0.05, compared with saline control, one-way ANOVA followed by Dunnett's Multiple Comparison test. Bar = 5 mm (A) or 500 μm (B). nd: not detectable. CA: caffeic acid.
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(MDA) content, mice were treated with three intraperitoneal injections of caffeic acid (10 and 50 mg/kg) or edaravone (3 mg/ kg, Conba Pharmaceutical Co., Hangzhou, China) for 1 day only. Histological examination and immunohistochemistry For histological measurement, sham-operated mice (n = 12) and the cryoinjured mice treated with saline, caffeic acid 10 and 50 mg/kg (n = 8 for each group at each time point) were anesthetized 1, 7, 14, 21 and 28 days after cryoinjury. Then, the mice were perfused transcardially with 4% paraformaldehyde after a pre-wash with saline. Brains were removed, fixed in 4% paraformaldehyde overnight, and then transferred to 30% sucrose for 3–5 days. The whole brains were photographed with a digital camera (FinePix S602 Zoom, Fuji, Japan). Then, 15 continuous 10-μm coronal sections were cut from each brain (from bregma - 2.3 to -2.45 mm) by cryomicrotomy (CM1900, Leica, Wetzlar, Germany), and stained with 1% toluidine blue to measure the lesion area. The lesion area was calculated by ImageTool 2.0 software (University of Texas Health Science Center, San Antonio, TX, USA). To measure the neuron density, the sections were sequentially reacted with a mouse monoclonal antibody against NeuN (1:200, Chemicon, Temecula, CA, USA) overnight at 4 °C and FITC-
conjoined rabbit anti-mouse IgG (1:100, Chemicon) for 2 h. Negative control sections were treated by identical procedure, except that the primary antibodies were omitted. NeuN-positive cells were counted in one 100 μm2 square of the lesion core region and three 100 μm2 squares of the periphery region (neocortex layers III and IV, 1.8–2.0 mm caudal from bregma) under a fluorescence microscope (DP70, Olympus, Tokyo, Japan). The neuron density is reported as NeuN-positive cells per mm2. To measure the astrocyte density and the glial scar wall thickness, the sections were sequentially reacted with a mouse monoclonal antibody against GFAP (1:800, Chemicon) overnight at 4 °C, biotinylated rabbit anti-mouse IgG (1:200), horseradish peroxidase streptavidin (1:200, Zhongshan Biotechnology Co., Beijing, China) for 2 h, and finally visualized using diaminobenzidine (DAB Kit, Zhongshan Biotechnology Co). Negative control sections were treated by identical procedure, except that the primary antibodies were omitted. GFAP-positive cells were counted in three 100 μm2 squares in the periphery of scar wall outer side (neocortex layers III and IV, 1.8–2.0 mm caudal from bregma) under a light microscope. The astrocyte density was reported as GFAP-positive cells per mm2. The glial scar thickness was measured with ImageTool 2.0 software (University of Texas Health Science Center, San Antonio, TX, USA).
Fig. 2. Effect of caffeic acid on neuron loss after cryoinjury in mice. NeuN-positive neurons were lost in the lesion core at 1 day and the periphery at 28 days after cryoinjury, and this loss was attenuated by caffeic acid (CA, 50 mg/kg) (A). Caffeic acid (10 and 50 mg/kg) significantly attenuated the neuron loss in the periphery over 28 days after cryoinjury (A and B). Values in B are reported as mean ± S.D.; n = 8–12; ##P b 0.01, compared with saline control, one-way ANOVA followed Dunnett's Multiple Comparison test. Bar = 20 μm.
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Western blotting For Western blotting analysis of GFAP, brains were rapidly removed from sham-operated mice, cryoinjured mice at 1, 7, 14, 21 and 28 days, or the cryoinjured mice treated with caffeic acid 10 and 50 mg/kg at 14 days after cryoinjury (n = 6 for each group at each time point). Samples from right cortex (100 mg) were homogenized and lysed in Cell and Tissue Protein Extraction Solution, containing 1 mmol/l pepstatin, 2 mmol/ l leupeptin, 80 mmol/l aprotinin, 1 mmol/l phenylmethylsulfonyl fluoride (Kangchen Biotechnology Inc., Shanghai, China). Protein concentrations were determined by the BioRad protein assay (Bio-Rad Lab, Hercules, CA, USA). Protein samples (50 μg) were separated by 10% SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes. The membranes were incubated with mouse monoclonal antibodies against GFAP (1:1000) and glyceraldehyde-3-phosphate dehydrogease (1:5000, GAPDH, Kangchen). After repeated washing, the membranes were incubated with IRDyeTM 800 conjugated affinity purified secondary antibodies (Rockland Immunochem-
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ical Inc., Gilbertsville, PA, USA), and the proteins were detected by Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and analyzed using ImagePro Plus program (Media Cybernetics Inc., Silver Spring, MD, USA). GFAP protein expression was normalized as GFAP/ GAPDH ratio. SOD activity and MDA content measurements For measuring SOD activity and MDA content, shamoperated mice and the cryoinjured mice treated with saline, caffeic acid (10 and 50 mg/kg) and edaravone (an anti-oxidative agent) were sacrificed 1 day after cryoinjury (n = 6 mice for each group). The right cortexes were rapidly removed and homogenized on ice in homogenizing buffer (10 mmol/l Tris–HCl, 0.1 mmol/l EDTA-2Na, 10 mmol/l sucrose, and 0.8% NaCl). The homogenate was collected to assay SOD activity with xanthine oxidase method and MDA content with thiobarbituric acid method according to the instructions of the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The
Fig. 3. Effect of caffeic acid on the number of astrocytes 7 to 28 days after cryoinjury in mice. GFAP-positive astrocytes were stained in the hemispheres from shamoperated, cryoinjured mice with or without caffeic acid treatment 14 days after cryoinjury (A). Few GFAP positive cells were found in sham-operated mice (left in A), and intense proliferation of astrocytes was found in the periphery of the foci (middle upper in A). The amplifications show the hypertrophied astrocytes (arrow, middle lower in A). Caffeic acid attenuated astrocyte proliferation (50 mg/kg, right panels in A), and GFAP-positive cell density (both 10 and 50 mg/kg, B). Values summarized in B are reported as mean ± S.D.; n = 8–12; ##P b 0.01, compared with saline control, one-way ANOVA followed Dunnett's Multiple Comparison test. Bars in A = 500 μm (upper) or 20 μm (lower). CA: caffeic acid.
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results were normalized by the protein concentrations in the homogenates. Statistical analysis Data are reported as means ± S.D. Significance of differences between two groups was analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison test (SPSS 10.0 for Windows, 1999. SPSS Inc., USA). P b 0.05 was considered statistically significant. Results Lesion area Lesion areas are shown in the gross photographs of the brain surface 1 and 7 days after cryoinjury (Fig. 1A). Hemorrhage was obvious in the lesion 1 day and had disappeared 7 days after cryoinjury (left upper panel in Fig. 1A). Caffeic acid reduced the lesion area and the hemorrhage (left lower panel in Fig. 1A). The lesions are shown in the coronal sections stained with toluidine blue 1 and 7 days after cryoinjury (Fig. 1B). The lesion was obviously larger 1 day and gradually decreased to 7 days after cryoinjury (Fig. 1C). Caffeic acid (10 and 50 mg/kg) reduced the lesion area from 1 to 28 days after cryoinjury, but the reduction was significant only at the dose of 50 mg/kg (Fig. 1C).
Neuron loss In the lesion core, neuron density was substantially reduced from 3976 ± 472 cells/mm2 to 25 ± 46 cells/mm2 at 1 day (middle upper panel in Fig. 2A), and almost completely disappeared at 7 to 28 days after cryoinjury. Caffeic acid (50 mg/kg) significantly attenuated the neuron loss (263 ± 92 cells/mm2, P b 0.05 compared with cryoinjury control treated with saline) in the lesion core at 1 day after cryoinjury (middle lower panel in Fig. 2A), but did not reverse the neuron loss thereafter (7 to 28 days). In the periphery adjacent to the lesion core, the neuron numbers were significantly reduced by about 55–75% from 1 to 28 days after cryoinjury (right upper panel in Fig. 2A,B). Caffeic acid (10 and 50 mg/kg) significantly attenuated the neuron loss in the periphery 1 to 28 days after cryoinjury (right lower panel in Fig. 2A,B). Astrocyte proliferation and glial scar wall formation One day after cryoinjury (early phase), no obviously proliferated GFAP-positive astrocytes were found around the lesion area. However, 14 days after cryoinjury (late phase), intensely proliferated astrocytes were localized in the periphery of the lesion (middle upper panel in Fig. 3A), which showed a hypertrophic property (middle lower panel in Fig. 3A). The density of GFAP-positive astrocytes was increased 7 days and
Fig. 4. Effect of caffeic acid on glial scar formation from 7 to 28 days after cryoinjury in mice. GFAP-positive glial scar wall (between two arrows) 7, 14, 21 and 28 days after cryoinjury around the lesion (A). Caffeic acid (10 and 50 mg/kg) inhibited the glial scar formation. The glial scar wall thickness was summarized in B; values are reported as mean ± S.D.; n = 8–12. #P b 0.05 and ##P b 0.01, compared with saline control, one-way ANOVA followed Dunnett's Multiple Comparison test. Bars in A = 500 μm. nd: not detectable. CA: caffeic acid.
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Fig. 5. Effect of caffeic acid on the expression of GFAP protein after cryoinjury in mice. GFAP (51 kD) and GAPDH (36 kD) were detected by Western blotting assay (A and B). GFAP protein was up-regulated after 7 to 28 days (A and C). Caffeic acid (50 mg/kg) reduced GFAP expression (B and D). Values summarized in C and D are reported as mean ± S.D.; n = 6 for each group. *P b 0.05, **P b 0.01 and ***P b 0.001, compared with sham-operated mice; ##P b 0.01, compared with saline control (14 days after cryoinjury), one-way ANOVA followed Dunnett's Multiple Comparison test.
remained up to 28 days after cryoinjury (Fig. 3B). Caffeic acid (10 and 50 mg/kg) significantly inhibited astrocyte proliferation and reduced the GFAP-positive astrocyte density (right upper panel in Fig. 3A,B). Moreover, an obvious glial scar was formed around the lesion from 7 to 28 days after cryoinjury (upper panels in Fig. 4A,B). Caffeic acid (10 and 50 mg/kg) significantly attenuated the glial scar wall thickness (lower panels in Fig. 4A, B). Correspondingly, GFAP protein expression abruptly increased from 7 to 28 days after cryoinjury (Fig. 5A and C). Caffeic acid (50 mg/kg) significantly attenuated the up-regulation of GFAP expression 14 days after cryoinjury (Fig. 5B and D). SOD activity and MDA content One day after cryoinjury, SOD activity was decreased and MDA content was increased (P b 0.01 compared with sham operation). Caffeic acid inhibited both changes at 50 mg/kg, but
Table 1 Effect of caffeic acid on SOD and MDA production in mouse brain tissue 1 day after cryoinjury Group
n
Dosage (mg/kg)
SOD (U/mg protein)
MDA (nmol/mg protein)
Sham operation Caffeic acid Cryoinjury 20% DMSO-saline + cryoinjury Edaravone + cryoinjury Caffeic acid + cryoinjury Caffeic acid + cryoinjury
6 6 6 6
– 50 – –
467 ± 134 440 ± 98 260 ± 51⁎⁎ 252 ± 32⁎⁎
5.76 ± 0.84 5.45 ± 1.48 9.12 ± 1.54⁎⁎ 8.97 ± 1.40⁎⁎
6
3
410 ± 78#
5.54 ± 0.56##
6
10
363 ± 47
6.60 ± 1.75#
6
50
399 ± 32#
5.90 ± 0.96##
Values are reported as mean ± S.D.; ⁎⁎P b 0.01, compared with Sham operated mice, #P b 0.05 and ##P b 0.01, compared with saline treated cryoinjured mice, one-way ANOVA followed by Dunnett's Multiple Comparison test.
only inhibited the increase in MDA content at 10 mg/kg. Edaravone (3 mg/kg), an anti-oxidant control, also inhibited both changes. Whereas caffeic acid (50 mg/kg) alone did not affect SOD activity and MDA content; the saline containing 20% DMSO did not inhibit cryoinjury-induced changes (Table 1). Discussion In the present study, we confirm the temporal pattern of brain cryoinjury; i.e. the neuronal loss in the lesion core and periphery as well as a hemorrhage in the early phase (1 day), and the astrocyte proliferation and glial scar formation in the periphery in the chronic phase (7 to 28 days). On this basis, we found that caffeic acid not only decreases the lesion area and attenuates neuron loss in the periphery in the early phase, but also inhibits astrocyte proliferation and glial scar formation as well as neuron loss in the periphery in the chronic phase. The most important finding of this study is that caffeic acid inhibited astrocyte proliferation (astrogliosis) and glial scar formation in the late phase of cryoinjury. This finding is consistent with a line of reports that reactive astrocytes (that are responsible for glial scar formation) are increased (Penkowa et al., 2003; Hermann et al., 2004; Hirano et al., 2004) and glial scar is formed (Di Giovanni et al., 2005) after cryoinjury in rats and mice. Moreover, the caffeic acid derivative, CAPE, inhibits cell proliferation of human astrocytoma cells (Kim et al., 1998), and vascular smooth muscle cell proliferation induced by angiotensin II in stroke-prone spontaneously hypertensive rats (Li et al., 2005). To our knowledge, pharmacological interventions for late or chronic changes have been investigated very little although interventions for the early injury have been well investigated (Murakami et al., 1999; Hortobagyi et al., 2000; Gorlach et al., 2001; Flentjar et al., 2002; Sewell et al., 2004; Shin et al., 2005; Turkoglu et al., 2005). Because the glial scar formation in the late phase of brain injury may be a physical and biochemical barrier for the regeneration of axons (Silver and
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Miller, 2004), the effect of caffeic acid may be of benefit for the treatment of brain injury. Another finding is that caffeic acid attenuated other types of damage after cryoinjury. This was shown by the observations that caffeic acid dose-dependently reduced cryoinjury-induced lesion area, attenuated neuron loss in the periphery of the lesion over 28 days, as well as hemorrhage in the early phase. Recently, it has been reported that CAPE prevents neonatal hypoxic–ischemic brain injury in rats (Wei et al., 2004), attenuates lipopolysaccharide (LPS)-induced inflammatory stress in rat hippocampal slice cultures (Montpied et al., 2003), and suppresses ischemia–reperfusion-induced cerebral lipid peroxidation (Irmak et al., 2003) and cerebral ischemic injury in rats (Tsai et al., 2006). The present study further shows the neuroprotective effect of caffeic acid on cryoinjury in mice. The mechanisms of the protective effects of caffeic acid are not fully understood, but the effects can be explained at least in two ways. One is its anti-oxidant and anti-inflammatory ability as shown in lots of studies. Like CAPE, caffeic acid as an antioxidant can scavenge a number of reactive species, such as DPPH radicals (Kikuzaki et al., 2002; Gulcin, 2006), peroxyl (Castelluccio et al., 1995) and hydroxyl (Kono et al., 1997) radicals, as well as superoxide anion, peroxynitrite and mutagenic compounds such as nitrosamines (Kono et al., 1997; Gulcin, 2006). In the present study, we confirmed the anti-oxidant ability of caffeic acid 1 day after cryoinjury, a peak time-point of oxidative stress after cryoinjury (Murakami et al., 1999; Turkoglu et al., 2005). We found that cryoinjury reduces the activity of SOD (an eliminator of free radicals) and increases the amount of MDA (an indicator of lipid peroxidation) in the brain; these changes can be inhibited by caffeic acid, similar as by edaravone, an anti-oxidant agent with neuroprotective effects (Yasuoka et al., 2004; Yoshida et al., 2006). It has been reported that reactive oxygen species activate astrocytes (Keller et al., 1999; Hazell, 2002; Steiner et al., 2002; PerezOrtiz et al., 2004), which can be inhibited by anti-oxidant agents like sesaminol glucosides (Lee et al., 2006), suggesting that oxidative stress after cryoinjury may contribute to astrocyte activation and the resultant formation of a glial scar. Thus, the anti-oxidant ability of caffeic acid might be responsible for the effects on the late astrocyte proliferation and glial scar formation in addition to the early neuronal death. Another explanation for the protective effects of caffeic acid is the inhibition of 5-lipoxygenase (5-LOX) activity (Koshihara et al., 1984). Our recent studies show that 5-LOX is activated after in vitro ischemic-like injury in the cultured rat cortical neurons and PC12 cells; 5-LOX activation is related to cell injury and can be inhibited by caffeic acid (Song et al., 2004; Ge et al., 2006). These findings indicate that the effects of caffeic acid on neuron loss after cryoinjury might be partially due to inhibition of 5-LOX activation. On the other hand, 5-LOX might be involved in astrocyte proliferation and glial scar formation since it is required for cellular proliferation and oncogenesis. Recently, we found that 5-LOX is moderately expressed in grade II astrocytoma and highly expressed in grade III/IV astrocytoma (Zhang et al., 2006). We also found that 5LOX enzymatic activity was increased and its expression was
up-regulated in the proliferated astrocytes in the late phase of focal cerebral ischemia, and the increased 5-LOX enzymatic activity was inhibited by caffeic acid (Zhou et al., 2006). Therefore, caffeic acid might attenuate astrocyte proliferation and glial scar formation via inhibiting 5-LOX activity. Conclusion Caffeic acid attenuates the lesion and neuron loss after cryoinjury, and especially blocks the activation of astrocytes resulting in attenuation of their proliferation and glial scar formation in the late phase of cryoinjury in mice, which at least is associated with its anti-oxidant ability. These findings suggest that caffeic acid may represent a new prototype compound of potential neuroprotective agents in the treatment of early and late traumatic brain injuries. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 30371637) and the Scientific Foundation of Education Ministry of China (20050335105). References Aladag, M.A., Turkoz, Y., Ozcan, C., Sahna, E., Parlakpinar, H., Akpolat, N., Cigremis, Y., 2006. Caffeic acid phenethyl ester (CAPE) attenuates cerebral vasospasm after experimental subarachnoidal haemorrhage by increasing brain nitric oxide levels. International Journal of Developmental Neuroscience 24 (1), 9–14. Bramlett, H.M., Dietrich, W.D., 2004. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. Journal of Cerebral Blood Flow and Metabolism 24 (2), 133–150. Castelluccio, C., Paganga, G., Melikian, N., Bolwell, G.P., Pridham, J., Sampson, J., Rice-Evans, C., 1995. Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBS Letters 368 (1), 188–192. Di Giovanni, S., Movsesyan, V., Ahmed, F., Cernak, I., Schinelli, S., Stoica, B., Faden, A.I., 2005. Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury. Proceedings of the National Academy of Sciences of the United States of America 102 (23), 8333–8338. Flentjar, N.J., Crack, P.J., Boyd, R., Malin, M., de Haan, J.B., Hertzog, P., Kola, I., Iannello, R., 2002. Mice lacking glutathione peroxidase-1 activity show increased TUNEL staining and an accelerated inflammatory response in brain following a cold-induced injury. Experimental Neurology 177 (1), 9–20. Ge, Q.F., Wei, E.Q., Zhang, W.P., Hu, X., Huang, X.J., Zhang, L., Song, Y., Ma, Z.Q., Chen, Z., Luo, J.H., 2006. Activation of 5-lipoxygenase after oxygen– glucose deprivation is partly mediated via NMDA receptor in rat cortical neurons. Journal of Neurochemistry 97 (4), 992–1004. Gorlach, C., Hortobagyi, T., Hortobagyi, S., Benyo, Z., Relton, J., Whalley, E.T., Wahl, M., 2001. Bradykinin B2, but not B1, receptor antagonism has a neuroprotective effect after brain injury. Journal of Neurotrauma 18 (8), 833–838. Gulcin, I., 2006. Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 217 (2–3), 213–220. Hazell, A.S., 2002. Astrocytes and manganese neurotoxicity. Neurochemistry International 41 (4), 271–277. Hermann, D.M., Hossmann, K.A., Mies, G., 2004. Expression of c-jun, mitogen-activated protein kinase phosphatase-1, caspase-3 and glial fibrillary acidic protein following cortical cold injury in rats: relationship to metabolic disturbances and delayed cell death. Neuroscience 123 (2), 371–379. Hirano, S., Yonezawa, T., Hasegawa, H., Hattori, S., Greenhill, N.S., Davis, P.F., Sage, E.H., Ninomiya, Y., 2004. Astrocytes express type VIII collagen
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Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury Lei Zhang, Wei-Ping Zhang, Ke-Da Chen, Xiao-Dong Qian, San-Hua Fang, Er-Qing Wei ⁎ Department of Pharmacology, School of Medicine, Zhejiang University, 388, Yu Hang Tang Road, Hangzhou 310058, People's Republic of China Received 14 June 2006; accepted 29 September 2006
Abstract Traumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects against ischemic brain injuries with anti-oxidant and anti-inflammatory properties, and by scavenging reactive species. However, whether caffeic acid has protective effects against traumatic brain injury is unknown. Therefore, we determined the effect of caffeic acid on the lesion in the early (1 day) and late phases (7 to 28 days) of cryoinjury in mice. We found that caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days, but attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and glial fibrillary acidic protein (GFAP) protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduction of superoxide dismutase activity and the increase in malondialdehyde content in the brain 1 day after cryoinjury. These results indicate that caffeic acid exerts a protective effect in traumatic brain injury, especially on glial scar formation in the late phase, which at least is associated with its anti-oxidant ability. © 2006 Elsevier Inc. All rights reserved. Keywords: Caffeic acid; Traumatic brain injury; Cryoinjury; Neuroprotection; Astrogliosis; Glial scar
Introduction Traumatic brain injury produces sequential consequences that can be divided into early and late (or chronic) phases. In the early phase, the main changes include excitotoxicity, oxidative stress, free radical production, apoptosis, inflammation and degeneration (Bramlett and Dietrich, 2004; Raghupathi, 2004; Sofroniew, 2005). In the late phase, one of the changes is the formation of a glial scar resulting from reactive gliosis (mainly consisting of proliferated astrocytes) (Logan and Berry, 2002), which may be a physical and biochemical barrier for the regeneration of axons (Silver and Miller, 2004). To investigate traumatic brain injury, a number of animal models have been developed, among which cryoinjury (also called as cold injury) is one widely used model (Murakami et al., 1999; Hortobagyi et al., 2000). Cryoinjury can mimic some characteristics of ⁎ Corresponding author. Tel.: +86 571 8820 8224; fax: +86 571 8820 8022. E-mail address: [email protected] (E.-Q. Wei). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.09.039
traumatic brain injuries and the related repair responses, such as apoptosis (Flentjar et al., 2002), inflammation (Sewell et al., 2004; Shin et al., 2005), angiogenesis (Nag, 2002), astrocyte proliferation and glial scar formation (Hermann et al., 2004; Hirano et al., 2004; Tada et al., 2004). Currently, the pharmacological interventions in traumatic brain injury are limited to the early injuries (Gorlach et al., 2001; Turkoglu et al., 2005) while the interventions in the late injuries are clinically more important and need to be investigated. Since we recently found that caffeic acid inhibited the astrocyte proliferation and neuronal injury in the late phase of focal cerebral ischemia in rats (Zhou et al., 2006), we propose that caffeic acid may also have an effect on late changes in cryoinjury. Caffeic acid (3, 4-dihydroxycinnamic acid) is one of the natural phenolic compounds widely distributed in plant materials such as vegetables, fruits, coffee and tea (Sondheimer, 1958). Caffeic acid possesses anti-oxidant properties as it scavenges a number of reactive species, including 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH) (Kikuzaki et al., 2002; Gulcin, 2006), peroxyl
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(Castelluccio et al., 1995) and hydroxyl radicals (Kono et al., 1997), as well as superoxide anion, peroxynitrite and mutagenic compounds such as nitrosamines (Kono et al., 1997; Gulcin, 2006). Caffeic acid also inhibits 5-lipoxygenase (5-LOX) activity (Koshihara et al., 1984), and inhibits protein kinase C (PKC), PKA and nuclear factor-κB (NF-κB) activation induced by ceramides in U937 cells (Nardini et al., 2000, 2001). Pharmacological studies have shown that caffeic acid exerts protective effects on glutamate-induced neurodegeneration in primary cultured cortical neurons (Kim and Kim, 2000) and PrP106-126 neurotoxicity in cerebellar granule neurons (Stewart et al., 2001), and hippocampal excitotoxic injury induced by systemic injection of kainite in vivo (Uz et al., 1998). A derivative of caffeic acid, caffeic acid phenethyl ester (CAPE), attenuates cerebral vasospasm after experimental subarachnoid hemorrhage (Aladag et al., 2006) and neonatal hypoxic– ischemic neuronal death (Wei et al., 2004). However, its effects on early and late injuries after traumatic brain injury are still unknown. Therefore, in the present study we determined the effect of caffeic acid on the injuries in the early phase (1 day) and the late phase (7 to 28 days) of cryoinjury in mice. To clarify the possible mechanism, we also determined the anti-oxidant ability of caffeic acid in the early phase.
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injected intraperitoneally 30 min, 2 and 6 h after cryoinjury on the first day and twice daily on days 2 to 7. For measuring superoxide dismutase (SOD) activity and malondialdehyde
Materials and methods Animals A total of 220 male ICR mice weighing 25–35 g (Experimental Animal Center of Zhejiang Academy of Medical Sciences, Hangzhou, China, Certificate No. 20030001) were used in this study. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of School of Medicine, Zhejiang University. Mice were housed under a controlled temperature (22 ± 1 °C), 12 h light/12 h dark cycle and allowed free access to food and water. Brain cryoinjury and caffeic acid administration Mice were anesthetized with intraperitoneal injection of chloral hydrate (400 mg/kg), and placed on a stereotactic frame (SR-5, Narishige, Tokyo, Japan). Rectal (core) temperature was measured and maintained at 37± 0.5°C with a heating pad and a heating lamp during the surgery. Brain cryoinjury was induced according to the reported method (Sewell et al., 2004) with modifications. Briefly, the scalp was incised on the midline to expose the skull. A metal probe (probe weight: 100 g; tip diameter: 3 mm) cooled in liquid nitrogen was applied to the surface of the intact skull above the right parietal lobe (1.5 mm lateral to the midline; −3.0 mm from bregma) for 30 s. Incisions were sutured after cryoinjury. Mice recovered from anesthesia in a warmed box. Caffeic acid (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in dimethyl sulphoxide (DMSO); the solution was freshly diluted with saline before use. Caffeic acid (10 and 50 mg/kg) or saline containing 20% DMSO (v/v, 10 ml/kg) was
Fig. 1. Effect of caffeic acid on cryoinjury lesion in mice. Gross photographs of whole brains (A) show the brain surface 1 and 7 days after cryoinjury. Coronal sections (B) were used to calculate lesion area (C). Caffeic acid (50 mg/kg) significantly attenuated the lesion over 28 days after cryoinjury (A–C). Values summarized in C are reported as mean ± S.D.; n = 8–12; #P b 0.05, compared with saline control, one-way ANOVA followed by Dunnett's Multiple Comparison test. Bar = 5 mm (A) or 500 μm (B). nd: not detectable. CA: caffeic acid.
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(MDA) content, mice were treated with three intraperitoneal injections of caffeic acid (10 and 50 mg/kg) or edaravone (3 mg/ kg, Conba Pharmaceutical Co., Hangzhou, China) for 1 day only. Histological examination and immunohistochemistry For histological measurement, sham-operated mice (n = 12) and the cryoinjured mice treated with saline, caffeic acid 10 and 50 mg/kg (n = 8 for each group at each time point) were anesthetized 1, 7, 14, 21 and 28 days after cryoinjury. Then, the mice were perfused transcardially with 4% paraformaldehyde after a pre-wash with saline. Brains were removed, fixed in 4% paraformaldehyde overnight, and then transferred to 30% sucrose for 3–5 days. The whole brains were photographed with a digital camera (FinePix S602 Zoom, Fuji, Japan). Then, 15 continuous 10-μm coronal sections were cut from each brain (from bregma - 2.3 to -2.45 mm) by cryomicrotomy (CM1900, Leica, Wetzlar, Germany), and stained with 1% toluidine blue to measure the lesion area. The lesion area was calculated by ImageTool 2.0 software (University of Texas Health Science Center, San Antonio, TX, USA). To measure the neuron density, the sections were sequentially reacted with a mouse monoclonal antibody against NeuN (1:200, Chemicon, Temecula, CA, USA) overnight at 4 °C and FITC-
conjoined rabbit anti-mouse IgG (1:100, Chemicon) for 2 h. Negative control sections were treated by identical procedure, except that the primary antibodies were omitted. NeuN-positive cells were counted in one 100 μm2 square of the lesion core region and three 100 μm2 squares of the periphery region (neocortex layers III and IV, 1.8–2.0 mm caudal from bregma) under a fluorescence microscope (DP70, Olympus, Tokyo, Japan). The neuron density is reported as NeuN-positive cells per mm2. To measure the astrocyte density and the glial scar wall thickness, the sections were sequentially reacted with a mouse monoclonal antibody against GFAP (1:800, Chemicon) overnight at 4 °C, biotinylated rabbit anti-mouse IgG (1:200), horseradish peroxidase streptavidin (1:200, Zhongshan Biotechnology Co., Beijing, China) for 2 h, and finally visualized using diaminobenzidine (DAB Kit, Zhongshan Biotechnology Co). Negative control sections were treated by identical procedure, except that the primary antibodies were omitted. GFAP-positive cells were counted in three 100 μm2 squares in the periphery of scar wall outer side (neocortex layers III and IV, 1.8–2.0 mm caudal from bregma) under a light microscope. The astrocyte density was reported as GFAP-positive cells per mm2. The glial scar thickness was measured with ImageTool 2.0 software (University of Texas Health Science Center, San Antonio, TX, USA).
Fig. 2. Effect of caffeic acid on neuron loss after cryoinjury in mice. NeuN-positive neurons were lost in the lesion core at 1 day and the periphery at 28 days after cryoinjury, and this loss was attenuated by caffeic acid (CA, 50 mg/kg) (A). Caffeic acid (10 and 50 mg/kg) significantly attenuated the neuron loss in the periphery over 28 days after cryoinjury (A and B). Values in B are reported as mean ± S.D.; n = 8–12; ##P b 0.01, compared with saline control, one-way ANOVA followed Dunnett's Multiple Comparison test. Bar = 20 μm.
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Western blotting For Western blotting analysis of GFAP, brains were rapidly removed from sham-operated mice, cryoinjured mice at 1, 7, 14, 21 and 28 days, or the cryoinjured mice treated with caffeic acid 10 and 50 mg/kg at 14 days after cryoinjury (n = 6 for each group at each time point). Samples from right cortex (100 mg) were homogenized and lysed in Cell and Tissue Protein Extraction Solution, containing 1 mmol/l pepstatin, 2 mmol/ l leupeptin, 80 mmol/l aprotinin, 1 mmol/l phenylmethylsulfonyl fluoride (Kangchen Biotechnology Inc., Shanghai, China). Protein concentrations were determined by the BioRad protein assay (Bio-Rad Lab, Hercules, CA, USA). Protein samples (50 μg) were separated by 10% SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes. The membranes were incubated with mouse monoclonal antibodies against GFAP (1:1000) and glyceraldehyde-3-phosphate dehydrogease (1:5000, GAPDH, Kangchen). After repeated washing, the membranes were incubated with IRDyeTM 800 conjugated affinity purified secondary antibodies (Rockland Immunochem-
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ical Inc., Gilbertsville, PA, USA), and the proteins were detected by Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and analyzed using ImagePro Plus program (Media Cybernetics Inc., Silver Spring, MD, USA). GFAP protein expression was normalized as GFAP/ GAPDH ratio. SOD activity and MDA content measurements For measuring SOD activity and MDA content, shamoperated mice and the cryoinjured mice treated with saline, caffeic acid (10 and 50 mg/kg) and edaravone (an anti-oxidative agent) were sacrificed 1 day after cryoinjury (n = 6 mice for each group). The right cortexes were rapidly removed and homogenized on ice in homogenizing buffer (10 mmol/l Tris–HCl, 0.1 mmol/l EDTA-2Na, 10 mmol/l sucrose, and 0.8% NaCl). The homogenate was collected to assay SOD activity with xanthine oxidase method and MDA content with thiobarbituric acid method according to the instructions of the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The
Fig. 3. Effect of caffeic acid on the number of astrocytes 7 to 28 days after cryoinjury in mice. GFAP-positive astrocytes were stained in the hemispheres from shamoperated, cryoinjured mice with or without caffeic acid treatment 14 days after cryoinjury (A). Few GFAP positive cells were found in sham-operated mice (left in A), and intense proliferation of astrocytes was found in the periphery of the foci (middle upper in A). The amplifications show the hypertrophied astrocytes (arrow, middle lower in A). Caffeic acid attenuated astrocyte proliferation (50 mg/kg, right panels in A), and GFAP-positive cell density (both 10 and 50 mg/kg, B). Values summarized in B are reported as mean ± S.D.; n = 8–12; ##P b 0.01, compared with saline control, one-way ANOVA followed Dunnett's Multiple Comparison test. Bars in A = 500 μm (upper) or 20 μm (lower). CA: caffeic acid.
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results were normalized by the protein concentrations in the homogenates. Statistical analysis Data are reported as means ± S.D. Significance of differences between two groups was analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison test (SPSS 10.0 for Windows, 1999. SPSS Inc., USA). P b 0.05 was considered statistically significant. Results Lesion area Lesion areas are shown in the gross photographs of the brain surface 1 and 7 days after cryoinjury (Fig. 1A). Hemorrhage was obvious in the lesion 1 day and had disappeared 7 days after cryoinjury (left upper panel in Fig. 1A). Caffeic acid reduced the lesion area and the hemorrhage (left lower panel in Fig. 1A). The lesions are shown in the coronal sections stained with toluidine blue 1 and 7 days after cryoinjury (Fig. 1B). The lesion was obviously larger 1 day and gradually decreased to 7 days after cryoinjury (Fig. 1C). Caffeic acid (10 and 50 mg/kg) reduced the lesion area from 1 to 28 days after cryoinjury, but the reduction was significant only at the dose of 50 mg/kg (Fig. 1C).
Neuron loss In the lesion core, neuron density was substantially reduced from 3976 ± 472 cells/mm2 to 25 ± 46 cells/mm2 at 1 day (middle upper panel in Fig. 2A), and almost completely disappeared at 7 to 28 days after cryoinjury. Caffeic acid (50 mg/kg) significantly attenuated the neuron loss (263 ± 92 cells/mm2, P b 0.05 compared with cryoinjury control treated with saline) in the lesion core at 1 day after cryoinjury (middle lower panel in Fig. 2A), but did not reverse the neuron loss thereafter (7 to 28 days). In the periphery adjacent to the lesion core, the neuron numbers were significantly reduced by about 55–75% from 1 to 28 days after cryoinjury (right upper panel in Fig. 2A,B). Caffeic acid (10 and 50 mg/kg) significantly attenuated the neuron loss in the periphery 1 to 28 days after cryoinjury (right lower panel in Fig. 2A,B). Astrocyte proliferation and glial scar wall formation One day after cryoinjury (early phase), no obviously proliferated GFAP-positive astrocytes were found around the lesion area. However, 14 days after cryoinjury (late phase), intensely proliferated astrocytes were localized in the periphery of the lesion (middle upper panel in Fig. 3A), which showed a hypertrophic property (middle lower panel in Fig. 3A). The density of GFAP-positive astrocytes was increased 7 days and
Fig. 4. Effect of caffeic acid on glial scar formation from 7 to 28 days after cryoinjury in mice. GFAP-positive glial scar wall (between two arrows) 7, 14, 21 and 28 days after cryoinjury around the lesion (A). Caffeic acid (10 and 50 mg/kg) inhibited the glial scar formation. The glial scar wall thickness was summarized in B; values are reported as mean ± S.D.; n = 8–12. #P b 0.05 and ##P b 0.01, compared with saline control, one-way ANOVA followed Dunnett's Multiple Comparison test. Bars in A = 500 μm. nd: not detectable. CA: caffeic acid.
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Fig. 5. Effect of caffeic acid on the expression of GFAP protein after cryoinjury in mice. GFAP (51 kD) and GAPDH (36 kD) were detected by Western blotting assay (A and B). GFAP protein was up-regulated after 7 to 28 days (A and C). Caffeic acid (50 mg/kg) reduced GFAP expression (B and D). Values summarized in C and D are reported as mean ± S.D.; n = 6 for each group. *P b 0.05, **P b 0.01 and ***P b 0.001, compared with sham-operated mice; ##P b 0.01, compared with saline control (14 days after cryoinjury), one-way ANOVA followed Dunnett's Multiple Comparison test.
remained up to 28 days after cryoinjury (Fig. 3B). Caffeic acid (10 and 50 mg/kg) significantly inhibited astrocyte proliferation and reduced the GFAP-positive astrocyte density (right upper panel in Fig. 3A,B). Moreover, an obvious glial scar was formed around the lesion from 7 to 28 days after cryoinjury (upper panels in Fig. 4A,B). Caffeic acid (10 and 50 mg/kg) significantly attenuated the glial scar wall thickness (lower panels in Fig. 4A, B). Correspondingly, GFAP protein expression abruptly increased from 7 to 28 days after cryoinjury (Fig. 5A and C). Caffeic acid (50 mg/kg) significantly attenuated the up-regulation of GFAP expression 14 days after cryoinjury (Fig. 5B and D). SOD activity and MDA content One day after cryoinjury, SOD activity was decreased and MDA content was increased (P b 0.01 compared with sham operation). Caffeic acid inhibited both changes at 50 mg/kg, but
Table 1 Effect of caffeic acid on SOD and MDA production in mouse brain tissue 1 day after cryoinjury Group
n
Dosage (mg/kg)
SOD (U/mg protein)
MDA (nmol/mg protein)
Sham operation Caffeic acid Cryoinjury 20% DMSO-saline + cryoinjury Edaravone + cryoinjury Caffeic acid + cryoinjury Caffeic acid + cryoinjury
6 6 6 6
– 50 – –
467 ± 134 440 ± 98 260 ± 51⁎⁎ 252 ± 32⁎⁎
5.76 ± 0.84 5.45 ± 1.48 9.12 ± 1.54⁎⁎ 8.97 ± 1.40⁎⁎
6
3
410 ± 78#
5.54 ± 0.56##
6
10
363 ± 47
6.60 ± 1.75#
6
50
399 ± 32#
5.90 ± 0.96##
Values are reported as mean ± S.D.; ⁎⁎P b 0.01, compared with Sham operated mice, #P b 0.05 and ##P b 0.01, compared with saline treated cryoinjured mice, one-way ANOVA followed by Dunnett's Multiple Comparison test.
only inhibited the increase in MDA content at 10 mg/kg. Edaravone (3 mg/kg), an anti-oxidant control, also inhibited both changes. Whereas caffeic acid (50 mg/kg) alone did not affect SOD activity and MDA content; the saline containing 20% DMSO did not inhibit cryoinjury-induced changes (Table 1). Discussion In the present study, we confirm the temporal pattern of brain cryoinjury; i.e. the neuronal loss in the lesion core and periphery as well as a hemorrhage in the early phase (1 day), and the astrocyte proliferation and glial scar formation in the periphery in the chronic phase (7 to 28 days). On this basis, we found that caffeic acid not only decreases the lesion area and attenuates neuron loss in the periphery in the early phase, but also inhibits astrocyte proliferation and glial scar formation as well as neuron loss in the periphery in the chronic phase. The most important finding of this study is that caffeic acid inhibited astrocyte proliferation (astrogliosis) and glial scar formation in the late phase of cryoinjury. This finding is consistent with a line of reports that reactive astrocytes (that are responsible for glial scar formation) are increased (Penkowa et al., 2003; Hermann et al., 2004; Hirano et al., 2004) and glial scar is formed (Di Giovanni et al., 2005) after cryoinjury in rats and mice. Moreover, the caffeic acid derivative, CAPE, inhibits cell proliferation of human astrocytoma cells (Kim et al., 1998), and vascular smooth muscle cell proliferation induced by angiotensin II in stroke-prone spontaneously hypertensive rats (Li et al., 2005). To our knowledge, pharmacological interventions for late or chronic changes have been investigated very little although interventions for the early injury have been well investigated (Murakami et al., 1999; Hortobagyi et al., 2000; Gorlach et al., 2001; Flentjar et al., 2002; Sewell et al., 2004; Shin et al., 2005; Turkoglu et al., 2005). Because the glial scar formation in the late phase of brain injury may be a physical and biochemical barrier for the regeneration of axons (Silver and
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Miller, 2004), the effect of caffeic acid may be of benefit for the treatment of brain injury. Another finding is that caffeic acid attenuated other types of damage after cryoinjury. This was shown by the observations that caffeic acid dose-dependently reduced cryoinjury-induced lesion area, attenuated neuron loss in the periphery of the lesion over 28 days, as well as hemorrhage in the early phase. Recently, it has been reported that CAPE prevents neonatal hypoxic–ischemic brain injury in rats (Wei et al., 2004), attenuates lipopolysaccharide (LPS)-induced inflammatory stress in rat hippocampal slice cultures (Montpied et al., 2003), and suppresses ischemia–reperfusion-induced cerebral lipid peroxidation (Irmak et al., 2003) and cerebral ischemic injury in rats (Tsai et al., 2006). The present study further shows the neuroprotective effect of caffeic acid on cryoinjury in mice. The mechanisms of the protective effects of caffeic acid are not fully understood, but the effects can be explained at least in two ways. One is its anti-oxidant and anti-inflammatory ability as shown in lots of studies. Like CAPE, caffeic acid as an antioxidant can scavenge a number of reactive species, such as DPPH radicals (Kikuzaki et al., 2002; Gulcin, 2006), peroxyl (Castelluccio et al., 1995) and hydroxyl (Kono et al., 1997) radicals, as well as superoxide anion, peroxynitrite and mutagenic compounds such as nitrosamines (Kono et al., 1997; Gulcin, 2006). In the present study, we confirmed the anti-oxidant ability of caffeic acid 1 day after cryoinjury, a peak time-point of oxidative stress after cryoinjury (Murakami et al., 1999; Turkoglu et al., 2005). We found that cryoinjury reduces the activity of SOD (an eliminator of free radicals) and increases the amount of MDA (an indicator of lipid peroxidation) in the brain; these changes can be inhibited by caffeic acid, similar as by edaravone, an anti-oxidant agent with neuroprotective effects (Yasuoka et al., 2004; Yoshida et al., 2006). It has been reported that reactive oxygen species activate astrocytes (Keller et al., 1999; Hazell, 2002; Steiner et al., 2002; PerezOrtiz et al., 2004), which can be inhibited by anti-oxidant agents like sesaminol glucosides (Lee et al., 2006), suggesting that oxidative stress after cryoinjury may contribute to astrocyte activation and the resultant formation of a glial scar. Thus, the anti-oxidant ability of caffeic acid might be responsible for the effects on the late astrocyte proliferation and glial scar formation in addition to the early neuronal death. Another explanation for the protective effects of caffeic acid is the inhibition of 5-lipoxygenase (5-LOX) activity (Koshihara et al., 1984). Our recent studies show that 5-LOX is activated after in vitro ischemic-like injury in the cultured rat cortical neurons and PC12 cells; 5-LOX activation is related to cell injury and can be inhibited by caffeic acid (Song et al., 2004; Ge et al., 2006). These findings indicate that the effects of caffeic acid on neuron loss after cryoinjury might be partially due to inhibition of 5-LOX activation. On the other hand, 5-LOX might be involved in astrocyte proliferation and glial scar formation since it is required for cellular proliferation and oncogenesis. Recently, we found that 5-LOX is moderately expressed in grade II astrocytoma and highly expressed in grade III/IV astrocytoma (Zhang et al., 2006). We also found that 5LOX enzymatic activity was increased and its expression was
up-regulated in the proliferated astrocytes in the late phase of focal cerebral ischemia, and the increased 5-LOX enzymatic activity was inhibited by caffeic acid (Zhou et al., 2006). Therefore, caffeic acid might attenuate astrocyte proliferation and glial scar formation via inhibiting 5-LOX activity. Conclusion Caffeic acid attenuates the lesion and neuron loss after cryoinjury, and especially blocks the activation of astrocytes resulting in attenuation of their proliferation and glial scar formation in the late phase of cryoinjury in mice, which at least is associated with its anti-oxidant ability. These findings suggest that caffeic acid may represent a new prototype compound of potential neuroprotective agents in the treatment of early and late traumatic brain injuries. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 30371637) and the Scientific Foundation of Education Ministry of China (20050335105). References Aladag, M.A., Turkoz, Y., Ozcan, C., Sahna, E., Parlakpinar, H., Akpolat, N., Cigremis, Y., 2006. Caffeic acid phenethyl ester (CAPE) attenuates cerebral vasospasm after experimental subarachnoidal haemorrhage by increasing brain nitric oxide levels. International Journal of Developmental Neuroscience 24 (1), 9–14. Bramlett, H.M., Dietrich, W.D., 2004. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. Journal of Cerebral Blood Flow and Metabolism 24 (2), 133–150. Castelluccio, C., Paganga, G., Melikian, N., Bolwell, G.P., Pridham, J., Sampson, J., Rice-Evans, C., 1995. Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBS Letters 368 (1), 188–192. Di Giovanni, S., Movsesyan, V., Ahmed, F., Cernak, I., Schinelli, S., Stoica, B., Faden, A.I., 2005. Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury. Proceedings of the National Academy of Sciences of the United States of America 102 (23), 8333–8338. Flentjar, N.J., Crack, P.J., Boyd, R., Malin, M., de Haan, J.B., Hertzog, P., Kola, I., Iannello, R., 2002. Mice lacking glutathione peroxidase-1 activity show increased TUNEL staining and an accelerated inflammatory response in brain following a cold-induced injury. Experimental Neurology 177 (1), 9–20. Ge, Q.F., Wei, E.Q., Zhang, W.P., Hu, X., Huang, X.J., Zhang, L., Song, Y., Ma, Z.Q., Chen, Z., Luo, J.H., 2006. Activation of 5-lipoxygenase after oxygen– glucose deprivation is partly mediated via NMDA receptor in rat cortical neurons. Journal of Neurochemistry 97 (4), 992–1004. Gorlach, C., Hortobagyi, T., Hortobagyi, S., Benyo, Z., Relton, J., Whalley, E.T., Wahl, M., 2001. Bradykinin B2, but not B1, receptor antagonism has a neuroprotective effect after brain injury. Journal of Neurotrauma 18 (8), 833–838. Gulcin, I., 2006. Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 217 (2–3), 213–220. Hazell, A.S., 2002. Astrocytes and manganese neurotoxicity. Neurochemistry International 41 (4), 271–277. Hermann, D.M., Hossmann, K.A., Mies, G., 2004. Expression of c-jun, mitogen-activated protein kinase phosphatase-1, caspase-3 and glial fibrillary acidic protein following cortical cold injury in rats: relationship to metabolic disturbances and delayed cell death. Neuroscience 123 (2), 371–379. Hirano, S., Yonezawa, T., Hasegawa, H., Hattori, S., Greenhill, N.S., Davis, P.F., Sage, E.H., Ninomiya, Y., 2004. Astrocytes express type VIII collagen
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