Background  The underlying mechanism of early neurobiological impairment after subarachnoid hemorrhage (SAH) is not well understood, but the system of reactive oxygen superoxide (ROS) might be involved. Edaravone (MCI-186), a potent free radical scavenger that prevents apoptosis of neurons, was thus used in this study to see its possible therapeutic effect in early brain injury due to SAH in a rat model.
Methods  One hundred and twenty male Sprague-Dawley rats were randomly assigned to four groups: group 1, control rats receiving sham operation only; group 2, rats with SAH treated by saline; group 3, rats with SAH treated with 1 mg/kg MCI-186 injected intraperitoneally; and group 4, rats with SAH treated with 3 mg/kg MCI-186. Treated with either saline or MCI-186 twice daily for two consecutive days after SAH, the rats were sacrificed for measurements of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) and histological analysis of caspase-3 protein by Western blotting and immunohistochemical staining. In addition, mortality and neurological scores were statistically analyzed by the chi-square test and Dunn′s procedure respectively for each group. One-way analysis of variance followed by the Tukey′s procedure was also used in data analysis.
Results  The rats in group 2 that received saline only showed neurological impairment as well as elevated mortality, and were found to have significantly increased levels of MDA and caspase-3, but reduced SOD activities in brain tissues (P <0.05). When treated with MCI-186 at two different dosages, the rats in groups 3 and 4 had markedly decreased levels of MDA and caspase-3 but increased SOD activities in the brain tissue (P <0.05), along with improved scores of neurological evaluation (P <0.05).
Conclusions  This study sheds some lights on the therapy of SAH-induced early brain injury by providing the promising data indicating that MCI-186, a radical scavenger, can efficiently diminish apoptosis of neurons and thus prevent the function loss of the brain in rats with SAH.

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Subarachnoid hemorrhage (SAH) is associated with a high mortality: 12.4% of the patients have a sudden death¹ and 40%–60% die within the first 48 hours because of initial bleeding,^{2, 3} and the survivors usually suffer from early brain injury. The basic mechanism of this injury after the initial bleeding may be related to overproduction of reactive oxygen species (ROS), which causes severe cell apoptosis and necrosis.^4,5

ROS is involved directly in lipid peroxidation and mitochondrial activating apoptosis cascade of neuron in aggravating brain damage.^6,7 Additionally, ROS, especially hydroxyl radicals, induces vasospasm through contracting blood vessel⁸ or depleting nitric oxide (NO).^9,10

Edaravone (MCI-186), a potent ROS scavenger with a high blood-brain barrier (BBB) permeability, has been intensively studied to be effective and efficient in vivo^11-15/vitro¹⁶ experiments and clinical settings.^17-20 Now it is widely applied in the treatment of acute ischemic cerebrovascular diseases. SAH, a limited bleeding in the subarachnoid cavity, can cause ROS involving neuron impairment and function loss. So far, little research work has been done in the treatment of SAH with MCI-186 in animal models. In this study, MCI-186 was used in rats with experimental SAH and its protective effect on early brain injury after SAH was evaluated.

Experimental groups
One hundred and twenty male Sprague-Dawley rats weighing 300 to 350 g from the Animal Laboratory, Nanjing Medical University, Nanjing, China were randomly assigned to 4 groups, 30 rats for each group: group 1, control rats receiving sham operation only; group 2, rats with SAH treated with saline; group 3, rats with SAH treated with MCI-186 1 mg/kg; and group 4, rats with SAH treated with MCI-186 3 mg/kg. Both saline and MCI-186 were injected intraperitoneally after SAH.

Experimental model of SAH
Endovascular perforation was induced in the SAH rat model according to the reports of Bederson et al²¹ and Schwartz et al.²² Briefly, the rats were anesthetized with 1% pentobarbital (50 mg/kg, intraperitoneally), supplied with intubation and respirator apparatus, maintained at (37.0±0.5)°C of rectal temperature with a heating pad. To perform SAH, the right external carotid artery was isolated and severed to leave a stump in length of 3 to 4 mm which was to be reopened after the internal carotid artery and the common carotid artery were clamped. A monofilament nylon suture of size 4.0 was then inserted up through the reopened internal carotid artery for about 20 mm, at which a slight resistance can usually be felt. The suture was then advanced little further to perforate the artery and immediately withdrawn, allowing reperfusion to induce SAH. The duration of the operation was less than 30 seconds. A similar procedure was performed in the sham group but with a blunt suture unable to pierce the arterial wall. After the surgery, the rats were released to their cages with free access to food and water after recovery from anesthesia.

Although we were not sure whether the bleeding was continuing during the recovery, we always found the blood clot around the bifurcation of the middle-anterior cerebral artery on each autopsy of the rat.

Drug administration
One mg/kg, 3 mg/kg MCI-186 (Nanjing Xiansheng Pharmaceutical Company, China) or 0.9% saline in equal volume was intraperitoneally injected into the rats in the proper groups respectively, twice daily for 2 days till all the rats were euthanized for further study. All animal procedures complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised, 1985).

Mortality and neurological function assessment
The Garcia scoring system²³ with a little modification²⁴ was employed to evaluate the animal neurological behavior and function. Briefly, the neurobehavioral examination (scoring scale 5–27) was performed at 6, 24, and 48 hours after SAH or sham operation by an examiner blinded to group assignment. A motor score (0–12) was derived from four aspects: spontaneous activity; symmetry of limb movements; climbing; and balance and coordination (score scale: 0–3 each). A sensory score (5–15) was also derived by examination of body proprioception, vibrissae, visuality, olfactory, and tactile responses to stimulus (score scale: 1–3 each).

Assay of MDA content and SOD activity
Malondialdehyde (MDA)，the well established indicator of lipid peroxidation and superoxide dismutase (SOD) which is an endogenous scavenger of ROS was measured in brain tissue using relative comercial assay kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer′s instructions. The MDA content was assayed in the form of thiobarbituric acid reacting substances (TBARS). One ml of 20% trichloroacetic acid and 1 ml of 1% TBARS reagent were added to 500 µl aliquot of brain tissue, then mixed and incubated at 95°C for 80 minutes. After cooling on ice, samples were centrifuged at 1000 ×g for 20 minutes, and then the absorbance of the supernatant was read at 532 nm (Pharmacia Biotech, Ultrospec 2000, USA). TBARS results were expressed as MDA equivalents using tetraethoxy-propane as a standard. MDA content in the brain tissue was expressed as millimore per milliliter (mmol/ml). SOD activity was determined by inhibition of nitroblue tetrazolium reduction due to superoxide anion generation by a xanthine-xanthine oxidase system. Briefly, 500 µl aliquot of brain tissue, together with 50 µmol/L xanthine and 2.5 µmol/L xanthine oxidase in 50 µmol/L potassium phosphate buffer were mixed and incubated at 37°C for 40 minutes, then nitro blue tetrazolium (NBT) was added. The product (nitrite) produced by the oxidation of oxyamine was measured by monitoring the absorbance at 550 nm (Pharmacia Biotech, Ultrospec 2000). One nitrite unit (NU) of SOD activity was determined as the amount of enzyme to reach an inhibition of 50% NBT reduction rate. The SOD activity in aliquot was expressed as nitrite units per milliliter (NU/ml).

Histology and immunohistochemistry
The rats were sacrificed under deep anesthesia by perfusion through the left ventricle with 200 ml of ice-cold 0.1 mol/L PBS followed by 400 ml of 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS) (pH 7.4). After fixation, their brains were cryopreserved and sectioned coronally for 20 mm thickness using a cryostat (CM3050S, Leica). For immunohistochemistry, the sections were incubated overnight at 4°C with primary antibody against caspase-3 antibody (1: 100; Cell Signaling, Beverly, Mass), further incubated with goat anti-rabbit biotinylated secondary antibody (Santa Cruz Biotechnology) and placed in avidin-biotin-peroxidase complex enzyme. Slides were visualized by incubation with 3, 3-diaminobenzidine (DAB) and hydrogen peroxide. The sections from different groups containing the hippocampus were selected for examination.

Western blotting analysis
Under the deep anesthesia mentioned above, brain tissues containing the hippocampus of the rats were isolated and kept at –80°C. When an analysis was made, the frozen brain tissues were gently homogenized into whole-cell lysates in 5 ml buffer (20 mmol/L HEPES, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 250 mmol/L sucrose, 0.1 mmol/L phemylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, and proteinase inhibitor cocktail tablets; pH 7.9).

Equal amounts of protein (50 µg) were loaded in each lane of polyacrylamide-sodium dodecyl sulfate gels, which were electrophoresed, followed by a transfer of the protein to a nitrocellulose membrane. The membrane was then blocked with a blocking solution and probed with primary antibodies overnight at 4°C. The primary antibodies used in Western blotting analysis were caspase-3. Immunoblots were next processed with secondary antibodies of horseradish peroxidase conjugated anti-goat IgG (1:2000, Promega Biotechnology) for 1 hour at room temperature. Blotting bands were quantified by densitometry with Image J software (Image J 1.33u, NIH). β-actin (1:2000, Santa Cruz Inc) was blotted on the same membrane as a loading control for the fractions. The quantified values were expressed as a percentage of (100%) β-actin.

Statistical analysis
Data were expressed as mean ± standard deviation (SD). Statistical significance was verified by one-way analysis of variance (ANOVA) followed by Tukey's procedure for multiple comparisons. The significance of differences in neurologic scores was analyzed by the Kruskal-Wallis test followed by multiple comparison procedures by Dunn′s procedure. Differences in mortality among the groups were tested using the chi-square test. The P value of less than 0.05 was considered statistically significant. SPSS 11.0 was used for data analysis.

Mortality and neurological scores
Of the 120 rats, 40 (33%) died in the course of the experiment (Figure 1A). No rats in the sham operation group died. Within the SAH groups, 25% (5 of 20) died either on the table or within the first 3 hours. These rats had not been allocated to a treatment or non-treatment group at that time. From the saline group, 45% (9 of 20) died, whereas only 25%, 30% (5, 6 of 20) of the treated groups died respectively. Treatment with MCI-186 at both dosages decreased the mortality. However, this reduction in mortality after administration of MCI-186 was not significant (χ²= 3.33; P=0.189) (Figure 1A).

In the neurological scores, a poor score was observed for all treated and untreated SAH rats at 6 hours (14±1, 14±1 versus 14±1; Figure 1B). However, by 24 hours, the treated rats had a slightly better score (21±1, 22±1 versus 19±1), and by 48 hours, a significant difference was seen among the groups (24±1, 24±1 versus 21±1; P <0.05) (Figure 1B).

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Figure 1. Mortality and neurological scores. A: The mortality of SD rats increased rapidly 24 hours after SAH, and treatment with MCI-186(M) given at the doses of 1 mg/kg and 3 mg/kg decreased the mortality. *P < 0.01 compared with the sham-operation group. †P >0.05 compared with rats with SAH treated with saline; ‡P >0.05 compared with rats with SAH treated with 1 mg/kg MCI-186. B: All rats developed neurological deficits at 6 hours after SAH (P <0.05 vs the sham-operation group); however, by 24 hours and 48 hours, a significant difference was seen between the two MCI-186 treated groups and the saline group (P <0.05 vs the rats with SAH treated with saline). *P <0.05 compared with the sham-operation group. †P <0.05 compared with the rats with SAH treated with saline and sham-operation; ‡P >0.05 compared with rats with SAH treated with 1 mg/kg MCI-186. §P <0.05 compared with the sham-operation group without treatment.

MDA level and SOD activity
MDA content elevated and SOD activity declined in the SAH group as compared with the sham operation group (P <0.05). The data obtained after treatment with 1 mg/kg MCI-186 and 3 mg/kg MCI-186 revealed a significant reduction of MDA level and an elevation of SOD activity versus the group treated with 0.9% saline (P <0.05). A higher dosage of 3 mg/kg MCI-186 did not exhibit stronger protection than that of 1 mg/kg MCI-186 (P <0.05). These results indicate that MCI-186 inhibits lipid peroxidation and restores the activity of SOD through clearing superoxide (Figure 2).

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Figure 2. Content of MDA and activity of SOD. A: The content of MDA increased in all groups even in the sham-operation group, and treatment with MCI-186 at both doses of 1 mg/kg and 3 mg/kg decreased it. *P <0.05 compared with the sham-operation group. †P <0.05 compared with rats with SAH treated with saline. ‡P >0.05 compared with rats with SAH treated with 1 mg/kg MCI-186. B: The activity of SOD decreased significantly in rats with SAH treated with saline , and increased significantly after treatment with MCI-186 at doses of 1 mg/kg and 3 mg/kg; *P <0.05 compared with the sham-operation group. †P <0.05 compared with the rats with SAH treated with saline. ‡P >0.05 compared with the rats with SAH treated with 1 mg/kg MCI-186.

Immunostaining of caspase-3
The hippocampus of the sham-operated rats showed very weak positive caspase-3 immunostaining. The extensive foci of positive caspase-3 staining were present in the hippocampus of rats with SAH treated with saline. Marked attenuation of positive caspase-3 staining was observed in brain sections from rats treated with MCI-186 at dosages of 1 mg/kg and 3 mg/kg (Figure 3).

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Figure 3. Immunostaining of caspase-3. Large amounts of caspase-3 positive cells containing brown granules in nuclei were shown in the hippocampus at 48 hours after SAH. The amounts were decreased after treatment with MCI-186 at doses of 1 mg/kg and 3 mg/kg. A: Sham-operation group. B: rats with SAH treated with saline. C: rats treated with 1 mg/kg MCI-186. D: rats treated with 3 mg/kg MCI-186 (SP, original magnification × 200).

Western blotting
Content of cleaved caspase-3 increased after SAH induction compared with the sham operation group. Both 1 mg/kg MCI-186 and 3 mg/kg MCI-186 treatments reduced the content of cleaved caspase-3 in brain tissue (P <0.05) (Figure 4).

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Figure 4. Western blotting. A: Representative bands of cleaved caspase-3 protein (17 kDa) were shown in brain tissue at 48 hours after SAH. The number of the bands was decreased after treatment with MCI-186 at doses of l mg/kg and 3 mg/kg. 1: Sham-operation group; 2: rats with SAH treated with saline; 3: rats treated with 1 mg/ kg MCI-186; 4: rats treated with 3 mg/kg MCI-186. B: Cleaved caspase-3 protein expression was measured by densitometry analysis. Values were expressed as mean ± standard error with 6 rats per group for 3 independent experiments. *P <0.01 compared with the sham-operation group; †P <0.05 compared with the rats with SAH treated with saline; ‡P >0.05 compared with the rats treated with 1 mg/kg MCI-186.

In the present study, 2 days after SAH induction, MDA levels in the brain tissue were significantly higher than those in the sham operation group, whereas superoxide dismutse (SOD) activity showed the opposite results. The increased level of apoptosis-related caspase-3 protein and the increased expression of caspase-3 in hippocampal neuron were accompanied with deficit of neurological function and high mortality. Treatment with either 1 mg/kg MCI-186 or 3 mg/kg MCI-186 significantly decreased MDA levels and increased SOD activity in the brain tissue, with a subsequent decrease of both protein content and expression of apoptosis-related gene, caspase-3, accompanied with improved neurological outcome. Apoptotic death of cerebral neuron cells induced by ROS might have an important impact on neurological function because of MCI-186 improved neurological outcome at 48 hours with a significant reduction of MDA levels, an increase of SOD activity, and suppression of apoptosis. These data support that MCI-186 may decrease free radicals and suppress apoptosis of neurons, thus mitigating early brain injury and improving neurological function.

Early brain injury may be attributed to elevated intracranial pressure (ICP), toxicity of blood components, ischemia,²⁵ reperfusion (reduction and restoration of cerebral blood flow (CBF)), acute vasospasm,^26,27 and ROS.⁷ After initial bleeding, the main cause of neuron death is apoptosis. The hippocampus is the most sensitive region to apoptosis in the brain.⁸ In this experiment we concentrated on brain injury in the hippocampus caused by the pathway of apoptosis induced by ROS.

After SAH, Fe²⁺ is released from hematoma. The subsequent Fenton reaction overproduces ROS, especially hydroxyl radicals.²⁸ The endogenous protective antioxidant SOD is consumed by the burst of ROS, which leads to a significant reduction of SOD activity. ROS causes cell injury by compromising the integrity of cell membrane via the oxidation of membrane phospholipids (lipid peroxidation) and integral proteins and by cleaving the DNA.²⁹ More importantly, ROS may attack mitochondria to induce the creation of the apoptosome, which cleaves procaspase-3 to form caspase-3 and activates the apoptosis cascade.³⁰ Caspase-3 has been shown to be the end product for both external and internal apoptotic cascades.³¹ The caspase-3 protein level reflects the extent of apoptosis. ROS can induce contraction of the cerebral vessels directly⁹ and react with endothelium-derived NO to form peroxynitrite (ONOO¯). NO is directly involved in the relaxation of the vascular wall.¹⁰ The ROS-induced NO depletion may lead to acute vasospasm and aggravate early neuron apoptosis through the mechanisms mentioned above. By correlating with the changes of MDA and SOD, Western blotting results in the present study showed caspase-3 protein increased in brain tissue and caspase-3 expressed broadly in the hippocampus two days after SAH. Meanwhile, treatment of MCI-186 significantly reduced the content of caspase-3 and the expression of caspase-3 in hippocampal neurons with a corresponding decrease in MDA level and an increase in endogenous SOD activity.

Many studies have been undertaken to reduce brain injury through clearance of ROS, such as Mn-SOD, SOD mimetic TBAP, and apocynin.^32-34 They are all effective in animal models. However, low permeability through blood brain barrier (BBB), low affinity to tissues, and shorter half-life in vivo prevent their clinical use.

MCI-186 has been effective in eliminating hydroxyl radicals that have a higher reactivity to trigger lipid peroxidation and neuron apoptosis, thus inhibiting the initiation and propagation of lipid peroxidation and apoptosis in neurons.³⁵ MCI-186 can interact with both peroxyl and hydroxyl radicals, and then form a stable oxidation product OPB (2-oxo-3-(phenylhydrazono) -butanoic acid) through a radical intermediate.³⁶ In addition, MCI-186 is effective in attenuating vasospasm after SAH in rats.³⁷ It has been used to treat ischemic cerebral vascular diseases^17-20 and to improve ischemia neurologic deficits in patients after SAH. However, the mechanism is not elucidated. This study demonstrated that MCI-186 protects neurons through ROS clearance and anti-apoptosis in SAH.

A contradicting issue is the remarkable effect of MCI-186 on ROS clearance and anti-apoptosis but weak effect on mortality. This paradox may indicate that scavenging of ROS and anti-apoptosis are important but other factors such as intracranial hypertension or hemoglobin toxicity are also contributive; and it may also illustrate that the mechanism of early brain injury after SAH is multi-factorial.

In conclusion, MCI-186 is a potent neuroprotective agent against early brain injury in SAH. We found in this study that MCI-186 protects neuron cells by scavenging free radicals, suppressing pathological apoptosis in a SAH model. Furthermore, MCI-186 did not result in complete attenuation of apoptosis, suggesting that multiple interventions may be necessary to have a full recovery of early brain injury.

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