Edaravone Protects Against Apoptotic Neuronal Cell Death and Improves Cerebral Function After Traumatic Brain Injury in Rats
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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.
KeywordsEdaravone Traumatic brain injury Neuroprotection Apoptosis Oxidative stress
It is well established that brain ischemia induces excitotoxic cell death in the central nervous system (CNS) . 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 . 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 .
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) . 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 . 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 , while edaravone was used to successfully treat a patient with acute cerebral ischemia . 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.
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 .
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 . Experiments were monitored using a digital TV system connected to a computer . 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.
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.
Quantification of 8-OHdG-Immunopositive Cells Following TBI
Quantification of 4-HNE-Immunopositive Cells Following TBI
Quantification of ssDNA-Immunopositive Cells Following TBI
Double-Immunofluorescence Staining for ssDNA with Hu or GFAP
Quantification of Hu-Immunopositive Cells Following TBI
Water Maze Experiments
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 . 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 . 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  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  and kinase signaling pathways , 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 . 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 . 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 . Furthermore, c-Jun N-terminal kinase (JNK) activation is involved in free radical-mediated apoptotic cell death , 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 . Edaravone can inhibit JNK  and p38  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.
The authors thank Mari Machino for technical assistance.
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