Neurochemical Research

, Volume 35, Issue 2, pp 348–355 | Cite as

Edaravone Protects Against Apoptotic Neuronal Cell Death and Improves Cerebral Function After Traumatic Brain Injury in Rats

  • Tatsuki Itoh
  • Takao Satou
  • Shozo Nishida
  • Masahiro Tsubaki
  • Motohiro Imano
  • Shigeo Hashimoto
  • Hiroyuki Ito
Original Paper

Abstract

Edaravone is a novel free radical scavenger used clinically in patients with acute cerebral infarction; however, it has not been assessed in traumatic brain injury (TBI). We investigated the effects of edaravone on cerebral function and morphology following TBI. Rats received TBI with a pneumatic controlled injury device. Edaravone (3 mg/kg) or physiological saline was administered intravenously following TBI. Numbers of 8-OHdG-, 4-HNE-, and ssDNA-positive cells around the damaged area after TBI were significantly decreased in the edaravone group compared with the saline group (P < 0.01). There was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the edaravone group compared with the saline group (P < 0.01). Edaravone administration following TBI inhibited free radical-induced neuronal degeneration and apoptotic cell death around the damaged area. In summary, edaravone treatment improved cerebral dysfunction following TBI, suggesting its potential as an effective clinical therapy.

Keywords

Edaravone Traumatic brain injury Neuroprotection Apoptosis Oxidative stress 

Introduction

It is well established that brain ischemia induces excitotoxic cell death in the central nervous system (CNS) [1]. Excitotoxic death cell is induced by the release of glutamate from neurons injured by ischemia, and the subsequent over-activation of glutamate receptors and excessive intracellular Ca2+ influx through these receptors [1]. Increased intracellular Ca2+ leads to the production of free radicals such as super oxide (O2) and hydroxyl radicals (•OH), and ultimately to cell degeneration and death [1].

In experimental focal ischemia in the rat brain, free radical production is increased by facilitation of the arachidonic acid cascade. Free radicals can induce lipid peroxidation in neuronal and glial cell membranes resulting in production of 4-hydroxy-2-nonenal (4-HNE), and can directly cause DNA damage in neuronal and glial cells resulting in production of 8-hydroxy-2′-deoxyguanosine (8-OHdG). These cascades of membrane peroxidation and DNA damage can induce apoptotic neuronal and glial cell death, which can be detected as immunopositive single-stranded DNA (ssDNA), and neuronal dysfunction [2, 3, 4, 5]. Free radical-induced oxidative stress has been shown to contribute to neuronal dysfunction and death in focal brain ischemia models [2, 3]. Thus, 4-HNE and 8-OHdG have been used a pre-apoptotic markers, and ssDNA as an apoptotic marker, after experimental brain injury [4, 5].

Traumatic brain injury (TBI) occurs as a result of a direct mechanical insult to the brain, and induces CNS degeneration and neuronal cell death [6, 7]. Following the initial mechanical insult, secondary effects including blood–brain barrier disruption, excitotoxic damage, and free radical production (such as O2 and •OH) are induced due to the circulatory disturbance caused by ischemia [8, 9, 10]. 3-Methyl-1-phenyl-pyrazolin-5-one (edaravone) is a novel brain-permeable free radical scavenger which interacts biochemically with a wide range of free radicals (O2 and •OH), donates electrons, and eventually transforms itself to the stable chemical, 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB) [11]. Experimentally, edaravone was shown to inhibit O2 and •OH production induced by brain injury and to reduce brain edema in a rat ischemic stroke model [12]. Edaravone also suppressed O2- and •OH-induced delayed neuronal cell death in the hippocampus and cerebral cortex following ischemia in the rat [13, 14], and attenuated ischemic brain injury in adult and immature animal models. Furthermore, several clinical trials have demonstrated neuroprotective effects of edaravone in patients with acute cerebral infarction [15], while edaravone was used to successfully treat a patient with acute cerebral ischemia [16]. However, there are no studies examining the effects of edaravone for treatment of TBI.

In the present study, we hypothesized that edaravone administration would inhibit free radical production and neuronal/glial cell apoptosis induced by TBI, resulting in improved brain dysfunction after TBI. Thus, we used the rat TBI model to investigate the effects of edaravone administration on neural and glial cells around the damaged tissue using morphological and behavioral methods.

Materials and Methods

Animals and Surgical Procedures

All experimental protocols conformed to the guidelines of the National Institutes of Health (Guide for the Care and Use of Laboratory Animals 1996), and were approved by the Institutional Animal Experimentation Committee of Kinki University School of Medicine. Ten week old male Wistar rats were used in the present study. Rats were anesthetized by intraperitoneal pentobarbital (50 mg/kg) injection. The scalp was incised on the midline and the skull was exposed. A 2 mm hole was drilled (1 mm posterior + 1 mm right lateral to bregma) in the right parietal calvaria [17, 18]. Brain injury above the dura mater was then induced with a pneumatic controlled injury device [17, 18] at an impact velocity of 4 m/s, with an impact tip diameter of 1 mm and a fixed impact deformation depth of 2 mm from the cerebral surface.

Rats were randomly divided into three groups: (1) TBI plus physiological saline-treated group (saline group, n = 20), (2) TBI plus 3 mg/kg edaravone-treated group (edaravone group, n = 20) [19], and (3) sham TBI (no impact) plus physiological saline-treated group (sham group, n = 10). Sham operation rats were used as controls for the Morris water maze experiments. Edaravone (Mitsubishi Pharma Corporation, Tokyo, Japan) was dissolved in 1 M NaOH and titrated to pH 7.4 with 1 M HCl. This solution was then diluted with physiological saline to a concentration of 3 mg/ml edaravone. Immediately following TBI, rats received single intravenous injections of 3 mg/kg edaravone [19] (edaravone group) or physiological saline (saline group). The experimental protocol is summarized in Fig. 1.
Fig. 1

Experimental design. Top, Timeline of TBI, edaravone injection, animal sacrifice, and Morris water maze examination. TBI traumatic brain injury

Immunohistochemistry

At 1 and 3 days following TBI, and after the last the Morris water maze experiments at 7 days, five rats in each of the saline and edaravone groups were deeply anesthetized by intraperitoneal pentobarbital (150 mg/kg), and then perfused intracardially with 300 ml of 0.1 M phosphate-buffered saline (PBS, pH 7.4–7.5) followed by 300 ml of 4% paraformaldehyde (PFA) in PBS. The brains were removed and stored in PFA for 3 days. The maximum lesion size was sliced into 50 μm thick serial coronal sections using a microslicer (Dousaka EM, Kyoto, Japan). Each section was treated with 3% H2O2 in Tris-buffered saline (TBS; 0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl) for 30 min to block endogenous peroxidase activity. Sections were then washed three times with TBS containing 0.1% Triton X-100 (TBS-T), blocked with 3% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) in TBS-T for 30 min, and incubated overnight at room temperature with the following primary antibodies in blocking solution: rabbit polyclonal anti-ssDNA antibody (apoptosis marker; 1:1000; DAKO, Glostrup, Denmark), monoclonal anti-Hu antibody (neuronal cell marker; 1:10000; Molecular Probes, Inc., Eugene, OR, USA), monoclonal anti-8-OHdG antibody (DNA damage; 1:50; JaICA, Shizuoka, Japan), or monoclonal anti-4-HNE antibody (lipid peroxidation marker; 1:50; JaICA). Following extensive washing, the sections were further incubated with a HISTIFINE Rat-PO (mouse)-kit or HISTIFINE Rat-PO (rabbit)-kit (Nichirei, Osaka, Japan), comprised of peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody, respectively, for 60 min at room temperature. The HISTIFINE Rat-PO kit contained pre-absorbed rat serum and exhibited negligible non-specific binding of rat serum in injured rat tissues. Labeling was visualized using diaminobenzidine (DAB; Vector Peroxidase Substrate Kit; Vector Laboratories, Burlingame, CA, USA) for 5 min, and the sections were counterstained with hematoxylin to quantify the number of Hu-, 8-OHdG-, and 4-HNE-positive cells. Negative control staining was performed with normal mouse serum instead of primary antibodies, following the procedure outlined above.

Quantification of ssDNA-, Hu-, 8-OHdG-, or 4-HNE-Positive Cells

To determine the number of ssDNA-, Hu-, 8-OHdG- and 4-HNE-positive cells, all DAB-labeled cells within 500 μm of the edge of the damaged region (cortex), excluding the white matter, following TBI were counted in three serial sections under a Nikon E 1000 M microscope (Nikon Corporation, Tokyo, Japan) using a 20× objective. The number of DAB-labeled cells in three serial sections was averaged. An image of the measured area was captured at 20× magnification using a CCD camera (ACT-2U; Nikon Corporation), and the measured area and volume in each image was traced and measured by computer. The number of DAB-labeled cells was calculated from the average number of DAB-positive cells and the volume. Numbers of ssDNA-, Hu-, 8-OHdG-, and 4-HNE-positive cells were expressed as the number/100 μm3 [20].

Double-Immunofluorescence Staining for ssDNA with Hu or Glial Fibrillary Acidic Protein

Sections collected at 3 days post-TBI were washed and blocked with 50 mM glycine in TBS for 2 h at 37°C to reduce non-specific fluorescence. The sections were washed with TBS-T, blocked with 3% BSA in TBS-T, and incubated with the anti-ssDNA antibody (1:300) overnight at room temperature. Following extensive washing, the sections were further incubated with AlexaFluor 488 anti-rabbit IgG (1:300; BD Biosciences Pharmingen, San Diego, CA, USA) for 80 min at room temperature. Next, the sections were washed extensively and incubated with a mouse monoclonal anti-Hu antibody (1:300) or mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:300), an astrocyte marker, overnight at room temperature. Following extensive washing, the sections were further incubated with AlexaFluor 555 anti-mouse IgG (1:300; BD Biosciences Pharmingen) for 80 min at room temperature. The sections were subsequently observed using a confocal laser-scanning microscope (LSM5 PASCAL; Carl Zeiss Jena GmbH, Jena, Germany).

Morris Water Maze Experiments

Morris water maze experiments were performed according to a modification of Elvander’s method [20, 21]. A circular, thermostatically regulated, dark gray PVC-plastic water tank (180 cm wide, 45 cm deep; filled with tap water at 22 ± 1°C), located in the center of the testing room and surrounded by extra-maze cues, was used in the spatial learning task. A constant asymmetrical array of lamps and pictures served as cues for spatial orientation. A circular dark gray platform (15 cm wide) submerged 1 cm below the water surface served as a platform. The platform was placed in the center of the target quadrant of the water maze [20]. Experiments were monitored using a digital TV system connected to a computer [20]. Training took place between 8:00 am and 3:00 pm over 7 consecutive days. Each daily training session consisted of four trials with a 120 s cutoff time, followed by 30 s rest on the platform. Memory was tested in fifteen animals from each of the saline group and the edaravone group, and in ten sham operation group animals, starting at day 1 and continued up to 7 days after TBI/sham operation.

Statistical Analysis

Water maze data are expressed as mean ± SE. Data were analyzed using ANOVA and Fisher’s PLSD-test (Stat View®; SAS Institute Inc., Cary, NC, USA). Other data are expressed as mean ± SD and analyzed using ANOVA. P < 0.05 was considered statistically significant.

Results

Quantification of 8-OHdG-Immunopositive Cells Following TBI

Immunostaining results for 8-OHdG expression around the damaged area after TBI can be seen in Fig. 2. At 1, 3, and 7 days following TBI in the saline group, many 8-OHdG-positive cells were present around the damaged area, and possessed an 8-OHdG-immunopositive cytoplasm and projections morphologically similar to those of large neurons or small astrocytes (Fig. 2a). By contrast, at 1, 3, and 7 days following TBI in the edaravone group, only a few 8-OHdG-immunopositive cells were present around the damaged area (Fig. 2b), with a significant reduction in the number of 8-OHdG-positive cells at 1, 3, and 7 days following TBI in the edaravone group (193.2 ± 43.3, 201 ± 67.2, 237.9 ± 79.1, respectively) compared with the saline group (944.3 ± 119.9, 964.7 ± 67.7, 994.2 ± 87.2, respectively; P < 0.001) (Fig. 2c).
Fig. 2

8-OHdG immunostaining around the damaged cerebral cortex following TBI in the rat. At 3 days after injury there were many 8-OHdG-immunopositive cells in the saline treated group (a), but only a few 8-OHdG-positive cells in the edaravone treated group (b). Scale bars = 50 μm. 8-OHdG-immunopositive cell numbers around the damaged cerebral cortex following TBI (c). Data are mean ± SD. *** P < 0.001, saline treated group vs. edaravone treated group at the same post-TBI day, n = 5

Quantification of 4-HNE-Immunopositive Cells Following TBI

Immunostaining results for 4-HNE formation around the damaged area after TBI can be seen in Fig. 3. At 1, 3, and 7 days following TBI in the saline group, many 4-HNE-immunopositive cells were present around the damaged area, and possessed a 4-HNE-immunopositive cytoplasm and projections morphologically similar to those of large neurons or small astrocytes (Fig. 3a). By contrast, at 1, 3, and 7 days following TBI in the edaravone group, only a few 4-HNE-immunopositive cells were present around the damaged area (Fig. 3b), with a significant reduction in the number of 4-HNE-immunopositive cells at 1 and 3 days following TBI in the edaravone group (171.4 ± 37.2, 154.1 ± 16.2, 168.7 ± 17.4, respectively) compared with the saline group (480.6 ± 37.5, 477 ± 58.5, 173.3 ± 28.9, respectively; P < 0.001) (Fig. 3c); there was no difference between the groups at 7 days. The number of 4-HNE-positive cells in the saline group was significantly lower at 3 days compared with 1 day and 7 days after TBI (P < 0.001).
Fig. 3

4-HNE immunostaining around the damaged cerebral cortex following TBI in the rat. At 3 days after injury there were many 4-HNE-immunopositive cells in the saline treated group (a), but only a few 4-HNE-positive cells in the edaravone treated group (b). Scale bars = 50 μm. 4-HNE-immunopositive cell numbers around the damaged cerebral cortex following TBI (c). There was a significant reduction in the number of 4-HNE-immunopositive cells at 1 and 3 days following TBI in the edaravone group compared with the saline group (P < 0.001). However, there was no difference between the groups at 7 days. Data are mean ± SD. *** P < 0.001, saline treated group vs. edaravone treated group at the same post-TBI day, n = 5

Quantification of ssDNA-Immunopositive Cells Following TBI

Immunostaining results for ssDNA expression around the damaged area after TBI can be seen in Fig. 4. At 1, 3, and 7 days following TBI in the saline group, many ssDNA-immunopositive cells were present around the damaged area, and possessed an ssDNA-immunopositive cytoplasm morphologically similar to those of neurons or small astrocytes (Fig. 4a). By contrast, at 1, 3, and 7 days following TBI in the edaravone group, only a few ssDNA-immunopositive cells were present around the damaged area (Fig. 4b), with a significant reduction in the number of ssDNA-immunopositive cells at 1, 3, and 7 days following TBI in the edaravone group (15.6 ± 9.0, 27.4 ± 10.8, 19.6 ± 4.8, respectively) compared with the saline group (68.5 ± 18.0, 290.6 ± 15.3, 56.4 ± 15.7; P < 0.01) (Fig. 4c). The number of ssDNA-immunopositive cells at 3 days after TBI in the saline group was markedly higher than at 1 and 7 days after TBI (P < 0.001).
Fig. 4

ssDNA immunostaining around the damaged cerebral cortex following TBI in the rat. At 3 days after injury, abundant ssDNA immunoreactivity was present mainly in the nuclei in the saline treated group (a). By contrast, there were only a few ssDNA immunoreactive nuclei in the edaravone treated group (b). Scale bars = 50 μm. ssDNA immunopositive cell numbers around the damaged cerebral cortex following TBI (c). Data are mean ± SD. ** P < 0.01, *** P < 0.001, n = 5

Double-Immunofluorescence Staining for ssDNA with Hu or GFAP

There were many Hu-immunopositive cells around the damaged area at 3 days after TBI in both the saline group and the edaravone group (Fig. 5b, e). The majority of Hu-immunopositive cells were also immunopositive for ssDNA in the saline group after TBI (Fig. 5c). By contrast, there were only a few Hu-positive cells colocalized with ssDNA in the edaravone group (Fig. 5f). In addition, although there were GFAP-positive cells and projections in both the saline group (Fig. 5h) and the edaravone group (Fig. 5k) at 3 days after TBI, these cells were smaller in the saline group than in the edaravone group. A subset of GFAP-positive cells were also ssDNA-immunopositive in the saline group (Fig. 5i), but only a few GFAP–ssDNA colocalized cells were observed in the edaravone group (Fig. 5l).
Fig. 5

Double-immunofluorescence staining of ssDNA with Hu or GFAP around the damaged area at 3 days after TBI. Numerous ssDNA-immunopositive (a, g, green) and Hu-immunopositive (b, red) or GFAP-immunopositive (h, red) cells were observed in the saline treated group. The ssDNA-immunopositive cells were double-immunopositive for Hu (c, yellow) or GFAP (i, yellow). By contrast, only a few ssDNA-immunopositive (d, j, green) and many Hu- (e, red) or GFAP-immunopositive (k, red) cells were observed in the edaravone treated group, while double-labeling of ssDNA-immunopositive cells with Hu (f) or GFAP (l) was not observed. Scale bars = 50 μm. For interpretation of the references to color in this figure legend, the reader is referred to the online version of this article

Quantification of Hu-Immunopositive Cells Following TBI

At 7 days following TBI in the saline group (Fig. 6a) and the edaravone group (Fig. 6b) there were many Hu-positive cells present around the damaged area, which possessed a cytoplasmic and projection staining pattern. There was a significant increase in the number of Hu-positive cells at 7 days following TBI in the edaravone group (348.0 ± 45.5) compared with the saline group (132.9 ± 44.4; P < 0.001) (Fig. 6c).
Fig. 6

Hu expression around the damaged region at 7 days following TBI. A few small Hu-positive cells and a few fibers were observed in the saline treated group (a), while numerous large Hu-positive cells and fibers were observed in the edaravone treated group (b). Scale bar = 50 μm. c Number of Hu-positive cells in each group (n = 5/group). *** P < 0.001, saline treated group vs. edaravone treated group

Water Maze Experiments

The arrival time to platform was significantly increased in the saline group compared with the sham operation group at 7 days after TBI (P < 0.01; Fig. 7). By contrast, the arrival time to platform was significantly decreased in the edaravone group compared with the saline group at 7 days after TBI (P < 0.01), and was not different from the sham operation group.
Fig. 7

The effect of intravenous edaravone administration on cerebral function at 7 days after TBI. Saline (1 ml/kg) or edaravone (3 mg/kg) was administered by intravenous injection following TBI. There was no injury in the sham operation plus saline treated group. The effects of treatment group on arrival time to platform are shown. Values represent mean ± SE (n = 10/group). ** P < 0.01, saline treated group vs. the edaravone treated group vs. sham operation group

Discussion

Edaravone has been previously shown to exhibit neuroprotective effects against ischemic injury by preventing free radical production [13, 14]. TBI injury can also produce free radicals in damaged tissues, resulting in neuronal and glial injury in those regions [2]. However, the effects of free radicals on neuronal and glial cells around the damaged area after TBI are poorly understood, and the role of edaravone in inhibiting free radical release around the damaged area after TBI is unknown.

In the present study, at 7 days after TBI in the saline group the number of 4-HNE and ssDNA-positive cells decreased, but there was no change in number of 8-OHdG-positive cells (Figs. 2 and 3). The early stage change of poly(ADP-ribose) polymerase, a DNA repair-associated enzyme, is similar to that of 8-OHdG [22, 23]. An overexpression of poly(ADP-ribose) polymerase with cleaved casepase-3 appearance has been shown to occur at 24 h after a brain insult, and then end at 72 h [22, 24]. Although in dividing cells, damaged DNA is enzymatically repaired with or without 8-OHdG production, post-mitotic cells such as neurons show low enzyme activity for DNA repair and are quite vulnerable to a brain insult [22, 24]. Such vulnerable neurons died by DNA repair dysfunction, DNA fragmentation, and apoptosis [22, 24]. These data suggest that, in the present study, 8-OHdG production may have accumulated following DNA repair dysfunction, resulting in an increase in the number of 8-OHdG-positve cells at 7 days after TBI.

Following TBI the number of 8-OHdG- and 4-HNE-positive cells in the edaravone group was significantly decreased compared with the saline group, suggesting that edaravone eliminated and absorbed free radicals produced by TBI around the damaged brain region (Figs. 2 and 3). Consistent with these data, edaravone was previously shown to inhibit free radical-mediated DNA peroxidation and decrease numbers of 8-OHdG-positive cells in the brain following neonatal hypoxic–ischemia [22]. Furthermore, edaravone inhibited and absorbed free radical production and decreased numbers of 4-HNE- and 8-OHdG-positive cells in the brain at an early time point after infarction [25] and ischemia [11, 25, 26].

Following TBI the number of ssDNA-positive cells in the edaravone group was also decreased compared with the saline group (Fig. 4). Previous studies provide strong evidence that the signaling pathways induced by free radicals can cause cellular damage and even apoptotic cell death during brain injury. For example, activation of mitochondrial pathways [27] and kinase signaling pathways [28], both of which can occur after a variety of injuries including focal cerebral ischemia and TBI, have been demonstrated. Activation of caspase-3 was also increased in hippocampal CA1 neurons at 3 days after an ischemic insult, following which the release of cytochrome c from the mitochondria to the cytosol induces neural and glial apoptotic cell degeneration and death [27]. In the present study, the peak in numbers of apoptotic ssDNA-positive cells at 3 days after TBI in the saline group (Fig. 4) is consistent with these data. Edaravone treatment was also shown to inhibit the release of cytochrome c and block caspase-3 activation in the cortex and hippocampus after an ischemic insult, and to block free radical production, resulting in a reduction in neural and glial apoptotic cell degeneration and death [29]. The mitogen-activated protein kinase (MAPK) family is involved in ischemic brain injury, with an increase in p38 activity observed after the onset of cerebral ischemia [30]. Furthermore, c-Jun N-terminal kinase (JNK) activation is involved in free radical-mediated apoptotic cell death [31], and a spin-trapped α-butylnitrone was shown to protect the hippocampal CA1 region after induction of global ischemia in the gerbil, a protective effect associated with the inhibition of JNK and p38 activity [31]. Edaravone can inhibit JNK [32] and p38 [33] activation and block free radical-mediated apoptotic neural cell degeneration and death after brain injury. Thus, in the present study, it is likely that edaravone absorbed free radicals generated by TBI and/or inhibited their function, thereby preventing free radical-mediated apoptotic neural and glial cell degeneration and death following TBI. In addition, there were less Hu- and GFAP-positive cells co-localized with ssDNA staining in the edaravone group compared with the saline group following TBI (Fig. 5), and many more Hu-positive cells around the damaged area during the chronic phase after TBI in the edaravone group (Fig. 6), providing further support that edaravone administration inhibited free radical-mediated neuronal and glial apoptotic cell death following TBI.

Although the Morris water maze is typically used to evaluate memory and learning, it was recently used to evaluate cortical brain dysfunction after TBI [20, 34]. In the present study, the arrival time to platform was significantly decreased in the edaravone group compared with the saline group after TBI, to levels similar to the sham operation group, suggesting that the neuroprotective effects of edaravone resulted in an improvement in brain dysfunction following TBI (Fig. 7). We were unable to show an acute phase improvement in brain dysfunction following TBI in the edaravone group as we did not examine the Morris water maze at 1 and 3 days after TBI. However, improvement in brain dysfunction during the acute phase following TBI is suggested by the acute prevention of neuronal apoptotic death in the edaravone administration.

In conclusion, edaravone administration following TBI eliminated and/or absorbed free radicals (such as O2 and •OH) induced by brain injury, resulting in inhibition of free radical-mediated neuronal and glial cell degeneration and apoptotic cell death around the damaged area, and improved brain dysfunction. Thus, edaravone administration has potential as an effective therapy following TBI.

Notes

Acknowledgment

The authors thank Mari Machino for technical assistance.

References

  1. 1.
    Kontos HA, George E (1985) Brown memorial lecture. Oxygen radicals in cerebral vascular injury. Circ Res 57:508–516PubMedGoogle Scholar
  2. 2.
    Clausen F, Lundqvist H, Ekmark S, Lewen A, Ebendal T, Hillered L (2004) Oxygen free radical-dependent activation of extracellular signal-regulated kinase mediates apoptosis-like cell death after traumatic brain injury. J Neurotrauma 21:1168–1182CrossRefPubMedGoogle Scholar
  3. 3.
    Hall ED, Braughler JM (1989) Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Radic Biol Med 6:303–313CrossRefPubMedGoogle Scholar
  4. 4.
    Chan PH, Fishman RA, Longar S, Chen S, Yu A (1985) Cellular and molecular effects of polyunsaturated fatty acids in brain ischemia and injury. Prog Brain Res 63:227–235CrossRefPubMedGoogle Scholar
  5. 5.
    Lee EJ, Lee MY, Chen HY, Hsu YS, Wu TS, Chen ST, Chang GL (2005) Melatonin attenuates gray and white matter damage in a mouse model of transient focal cerebral ischemia. J Pineal Res 38:42–52CrossRefPubMedGoogle Scholar
  6. 6.
    Chirumamilla S, Sun D, Bullock MR, Colello RJ (2002) Traumatic brain injury induced cell proliferation in the adult mammalian central nervous system. J Neurotrauma 19:693–703CrossRefPubMedGoogle Scholar
  7. 7.
    Rice AC, Khaldi A, Harvey HB, Salman NJ, White F, Fillmore H, Bullock MR (2003) Proliferation and neuronal differentiation of mitotically active cells following traumatic brain injury. Exp Neurol 183:406–417CrossRefPubMedGoogle Scholar
  8. 8.
    Azbill RD, Mu X, Bruce-Keller AJ, Mattson MP, Springer JE (1997) Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury. Brain Res 765:283–290CrossRefPubMedGoogle Scholar
  9. 9.
    Kawamata T, Katayama Y, Hovda DA, Yoshino A, Becker DP (1995) Lactate accumulation following concussive brain injury: the role of ionic fluxes induced by excitatory amino acids. Brain Res 674:196–204CrossRefPubMedGoogle Scholar
  10. 10.
    Xiong Y, Gu Q, Peterson PL, Muizelaar JP, Lee CP (1997) Mitochondrial dysfunction and calcium perturbation induced by traumatic brain injury. J Neurotrauma 14:23–34CrossRefPubMedGoogle Scholar
  11. 11.
    Arai T, Nonogawa M, Makino K, Endo N, Mori H, Miyoshi T, Yamashita K, Sasada M, Kakuyama M, Fukuda K (2008) The radical scavenger edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) reacts with a pterin derivative and produces a cytotoxic substance that induces intracellular reactive oxygen species generation and cell death. J Pharmacol Exp Ther 324:529–538CrossRefPubMedGoogle Scholar
  12. 12.
    Nishi H, Watanabe T, Sakurai H, Yuki S, Ishibashi A (1989) Effect of MCI-186 on brain edema in rats. Stroke 20:1236–1240PubMedGoogle Scholar
  13. 13.
    Yamamoto T, Yuki S, Watanabe T, Mitsuka M, Saito KI, Kogure K (1997) Delayed neuronal death prevented by inhibition of increased hydroxyl radical formation in a transient cerebral ischemia. Brain Res 762:240–242CrossRefPubMedGoogle Scholar
  14. 14.
    Mizuno A, Umemura K, Nakashima M (1998) Inhibitory effect of MCI-186, a free radical scavenger, on cerebral ischemia following rat middle cerebral artery occlusion. Gen Pharmacol 30:575–578CrossRefPubMedGoogle Scholar
  15. 15.
    Wu T, Ding XS, Wang W, Wu J (2006) MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) attenuated simulated ischemia/reperfusion injury in cultured rat hippocampal cells. Biol Pharm Bull 29:1613–1617CrossRefPubMedGoogle Scholar
  16. 16.
    Dohi K, Satoh K, Mihara Y, Nakamura S, Miyake Y, Ohtaki H, Nakamachi T, Yoshikawa T, Shioda S, Aruga T (2006) Alkoxyl radical-scavenging activity of edaravone in patients with traumatic brain injury. J Neurotrauma 23:1591–1599CrossRefPubMedGoogle Scholar
  17. 17.
    Itoh T, Satou T, Hashimoto S, Ito H (2005) Isolation of neural stem cells from damaged rat cerebral cortex after TBI. Neuroreport 16:1687–1691CrossRefPubMedGoogle Scholar
  18. 18.
    Itoh T, Satou T, Hashimoto S, Ito H (2007) Immature and mature neurons coexist among glial scars after rat traumatic brain injury. Neurol Res 29:734–742CrossRefPubMedGoogle Scholar
  19. 19.
    Watanabe T, Yuki S, Egawa M, Nishi H (1994) Protective effects of MCI-186 on cerebral ischemia: possible involvement of free radical scavenging and antioxidant actions. J Pharmacol Exp Ther 268:1597–1604PubMedGoogle Scholar
  20. 20.
    Itoh T, Satou T, Nishida S, Tsubaki M, Hashimoto S, Ito H (2009) Improvement of cerebral function by anti-amyloid precursor protein antibody infusion after traumatic brain injury in rats. Mol Cell Biochem 324:191–199CrossRefPubMedGoogle Scholar
  21. 21.
    Elvander E, Schott PA, Sandin J, Bjelke B, Kehr J, Yoshitake T, Ogren SO (2004) Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition. Neuroscience 126:541–557CrossRefPubMedGoogle Scholar
  22. 22.
    Takizawa Y, Miyazawa T, Nonoyama S, Goto Y, Itoh M (2009) Edaravone inhibits DNA peroxidation and neuronal cell death in neonatal hypoxic–ischemic encephalopathy model rat. Pediatr Res 65:636–641CrossRefPubMedGoogle Scholar
  23. 23.
    Wang X, Karlsson JO, Zhu C, Bahr BA, Hagberg H, Blomgren K (2001) Caspase-3 activation after neonatal rat cerebral hypoxia–ischemia. Biol Neonate 79:172–179CrossRefPubMedGoogle Scholar
  24. 24.
    Weissman L, de Souza-Pinto NC, Stevnsner T, Bohr VA (2007) DNA repair, mitochondria, and neurodegeneration. Neuroscience 145:1318–1329CrossRefPubMedGoogle Scholar
  25. 25.
    Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T (2005) Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 36:2220–2225CrossRefPubMedGoogle Scholar
  26. 26.
    Han N, Ding SJ, Wu T, Zhu YL (2008) Correlation of free radical level and apoptosis after intracerebral hemorrhage in rats. Neurosci Bull 24:351–358CrossRefPubMedGoogle Scholar
  27. 27.
    Sugawara T, Noshita N, Lewen A, Gasche Y, Ferrand-Drake M, Fujimura M, Morita-Fujimura Y, Chan PH (2002) Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation. J Neurosci 22:209–217PubMedGoogle Scholar
  28. 28.
    Irving EA, Bamford M (2002) Role of mitogen- and stress-activated kinases in ischemic injury. J Cereb Blood Flow Metab 22:631–647CrossRefPubMedGoogle Scholar
  29. 29.
    Yasuoka N, Nakajima W, Ishida A, Takada G (2004) Neuroprotection of edaravone on hypoxic–ischemic brain injury in neonatal rats. Brain Res Dev Brain Res 151:129–139CrossRefPubMedGoogle Scholar
  30. 30.
    Walton KM, DiRocco R, Bartlett BA, Koury E, Marcy VR, Jarvis B, Schaefer EM, Bhat RV (1998) Activation of p38MAPK in microglia after ischemia. J Neurochem 70:1764–1767PubMedCrossRefGoogle Scholar
  31. 31.
    Tsuji M, Inanami O, Kuwabara M (2000) Neuroprotective effect of alpha-phenyl-N-tert--butylnitrone in gerbil hippocampus is mediated by the mitogen-activated protein kinase pathway and heat shock proteins. Neurosci Lett 282:41–44CrossRefPubMedGoogle Scholar
  32. 32.
    Wen J, Watanabe K, Ma M, Yamaguchi K, Tachikawa H, Kodama M, Aizawa Y (2006) Edaravone inhibits JNK-c-Jun pathway and restores anti-oxidative defense after ischemia-reperfusion injury in aged rats. Biol Pharm Bull 29:713–718CrossRefPubMedGoogle Scholar
  33. 33.
    Niyaz M, Numakawa T, Matsuki Y, Kumamaru E, Adachi N, Kitazawa H, Kunugi H, Kudo M (2007) MCI-186 prevents brain tissue from neuronal damage in cerebral infarction through the activation of intracellular signaling. J Neurosci Res 85:2933–2942CrossRefPubMedGoogle Scholar
  34. 34.
    Xiong Y, Mahmood A, Lu D, Qu C, Kazmi H, Goussev A, Zhang ZG, Noguchi CT, Schallert T, Chopp M (2008) Histological and functional outcomes after traumatic brain injury in mice null for the erythropoietin receptor in the central nervous system. Brain Res 1230:247–257CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Tatsuki Itoh
    • 1
  • Takao Satou
    • 1
    • 2
    • 3
  • Shozo Nishida
    • 4
  • Masahiro Tsubaki
    • 4
  • Motohiro Imano
    • 5
  • Shigeo Hashimoto
    • 6
  • Hiroyuki Ito
    • 1
  1. 1.Department of PathologyKinki University School of MedicineOsakasayamaJapan
  2. 2.Division of Hospital PathologyHospital of Kinki University School of MedicineOsakaJapan
  3. 3.Division of Sports Medicine, Institute of Life ScienceKinki UniversityOsakaJapan
  4. 4.Kinki University School of Pharmaceutical SciencesOsakaJapan
  5. 5.Department of SurgeryKinki University School of MedicineOsakasayamaJapan
  6. 6.Department of PathologyPL HospitalOsakaJapan

Personalised recommendations