Clinical neuroscience 635
Neuroprotective effects of edaravone after intraventricular hemorrhage in rats Zhi Chen, Jianbo Zhang, Qianwei Chen, Jing Guo, Gang Zhu and Hua Feng Intraventricular hemorrhage (IVH) is a severity factor and treatment target in intracerebral hemorrhage. This study aimed to investigate whether systemic edaravone, a freeradical scavenger, could attenuate the brain injury after IVH in a rat model. Our findings showed that an intraventricular injection of autologous whole blood resulted in acute brain edema, increased malondialdehyde level, and decreased superoxide dismutase enzyme activity. Immediate edaravone treatment after IVH can reduce IVH-induced brain edema and elevated lipid peroxidation. Furthermore, repeated edaravone treatment (immediately, 24 h, and 48 h after IVH) improved the IVH-induced learning and memory damage. These effects suggest that edaravone may be a potential therapeutic agent for IVH,
especially those intracerebral hemorrhage patients with c 2014 ventricular extension. NeuroReport 25:635–640 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
Chongqing, China) weighing 250–350 g were used in this study. The animals were anesthetized with pentobarbital (40 mg/kg intraperitoneally). Body temperature was maintained at 37.51C using a feedback-controlled heating pad. A polyethylene catheter (PE-50) was inserted into the right femoral artery as a source for collecting the arterial blood. Rats were then positioned in a stereotaxic frame and a cranial burr hole (1 mm) was drilled (coordinates: 0.6 mm posterior and 1.6 mm lateral to the bregma). Nonheparinized arterial blood (from the femoral artery catheterization) or saline was collected in a 1 ml syringe with a 26 G needle, which was positioned in the infusion pump and stereotaxically inserted 4.5 ventrally into the right lateral ventricle through the burr hole. Autologous arterial blood (200 ml) or saline was infused at a rate of 14 ml/min using a microinfusion pump. The needle was removed after injection and the skin incision was closed with sutures.
Intracerebral hemorrhage (ICH) is the most severe of the major stroke subtypes. Extension of the hemorrhage into the ventricles after ICH results in intraventricular hemorrhage (IVH), which may occur in up to 50% of patients with ICH [1,2]. Recent studies have confirmed that IVH is an independent predictor of poor outcome after ICH [2–4]. However, the mechanisms of brain injury after IVH are not well understood, and no valid medical treatment is available at present. It is known that brain iron overload is associated with free-radical formation, and contributes toward brain edema, oxidative injury, and brain atrophy after ICH [5,6]. Our recent study has shown that IVH resulted in brain iron accumulation and enlargement of the lateral ventricles in a rat model [7]. However, there is limited information on the role of oxidative stress and neurobehavioral deficits after IVH. The free-radical scavenger edaravone is known to exert antioxidant effects by inhibiting hydroxyl radical-dependent and radical-independent lipid peroxidation. Therefore, the present study investigated whether systemic edaravone treatment could attenuate the IVH-induced acute brain injury and improve behavioral deficits after IVH in a rat model.
Materials and methods Animal preparation and intraventricular infusion
Animal use protocols were approved by the Animal Committee of the Third Military Medical University. A total of 48 male Sprague–Dawley rats (the animal center of the Third Military Medical University, c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
NeuroReport 2014, 25:635–640 Keywords: behavior, cilia, edaravone, intraventricular hemorrhage, oxidative stress Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing, China Correspondence to Hua Feng, MD, PhD, Department of Neurosurgery, Southwest Hospital, Third Military Medical University, No. 30, Gaotanyan Street, Chongqing 400038, China Tel: + 86 23 68765759; fax: + 86 23 68765265; e-mail: [email protected] Received 18 August 2013 accepted 18 September 2013
Experimental groups
These experiments were conducted in two parts. In the first part (short-term study), rats were divided into three groups including intraventricular saline injection (saline control), intraventricular blood injection with vehicle treatment (IVH + Veh), and intraventricular blood injection with edaravone treatment (IVH + Edv). Rats received an injection of 200 ml saline or autologous whole blood into the right lateral ventricle and were treated with either edaravone (6 mg/kg subcutaneously; SigmaAldrich, St Louis, Missouri, USA) or vehicle injection immediately (15 min) after blood injection. This dose of edaravone can reduce ICH-induced brain edema [8]. Rats were euthanized at day 1 for brain water content DOI: 10.1097/WNR.0000000000000050
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
636
NeuroReport 2014, Vol 25 No 9
determination (n = 6 for each group) as well as malondialdehyde (MDA) level and superoxide dismutase (SOD) activity (n = 5 for each group). In the second part (long-term study), rats were divided into three groups as in the first part (n = 5 for each group). The rats with blood injection received either edaravone (6 mg/kg/day subcutaneously) or vehicle treatment every 24 h up to 3 days (immediately, 1 and 2 days after intraventricular injection). Behavioral tests were performed from day 23 after IVH, and these rats were then euthanized for morphological observation at day 28 after injection. Brain water content
Animals were decapitated under deep anesthesia 1 day after IVH for brain water content measurement. The brains were removed immediately and the frontal poles (4 mm) were cut off. The remaining brains were divided into three tissue samples: bilateral cerebral hemispheres and the cerebellum. Brain samples were weighted immediately to obtain the wet weight, and were then dried at 1001C for 24 h to obtain the dry weight. Tissue water content was calculated as (wet weight – dry weight)/wet weight. Malondialdehyde assay and superoxide dismutase activities
The rats were reanesthetized and perfused with 0.1 mol/l PBS, and then the brains were removed immediately and the frontal poles (4 mm) were cut off. Then, the whole right cortex and hippocampus were dissected and stored at – 701C to determine the level of MDA and SOD enzyme activities. The level of lipid peroxidation products (MDA) in the right cerebral cortex and hippocampus was measured by the thiobarbituric acid method using the assay kit following the manufacturer’s instruction (Nanjing Jiancheng Biochemistry Co., Nanjing, China) [9]. The protein concentration was determined using Commassie blue assay. The level of MDA was calculated as nmol/mg protein in the sample according to the standard curve derived. Meanwhile, SOD activity was measured using the kit following the manufacturer’s instruction (Nanjing Jiancheng Biochemistry Co.) [9]. It uses a xanthine–xanthine oxidase system to determine the inhibition of nitroblue tetrazolium reduction because of superoxide anion generation. Total nitrite produced by the oxidation of oxyamine was measured by detecting the absorbance at 550 nm. One unit of SOD was defined as the enzyme amount causing 50% inhibition of the nitroblue tetrazolium reduction rate. SOD activity was expressed in U/mg protein. Learning and memory test
The Morris water maze was used to assess learning and memory of the animals [10]. The test was performed in a metal pool (200 cm in diameter, 50 cm in depth) filled with water. The spatial acquisition training was performed at day 23 after IVH. All the rats received
5 days of acquisition training with the platform remaining in a single location. Each training day consisted of four trials from one of four start positions with a 10 min interval, and the latencies and the distances to the goal were averaged across four trials per day. The probe trial to assess reference memory was conducted 1 day after the last acquisition with the platform removed. The time and distance traveled in each of the four quadrants was recorded. The percent time and the percent distance in the target quadrant were calculated. Dissection of lateral ventricles and scanning electron microscopy
Animals were decapitated under deep anesthesia, and the brains were removed immediately. The right lateral ventricles were exposed widely using the whole-mount dissection technique as described previously [11]. The distribution of the yellow-stained area was measured using Image J software (National Institutes of Health, Bethesda, Maryland, USA), and presented as a percentage of the total area of the right lateral ventricle. Brain samples containing the dorsal–lateral wall of the right lateral ventricle were then prepared for scanning electron microscopy (FEI Quanta 450; FEI Company, Hillsboro, Oregon, USA) as described previously [12]. Briefly, the tissues were fixed in Sorensen’s phosphate-buffered glutaraldehyde (4%, pH 7.4) for 48 h. After postfixation in 1% osmium tetroxide, samples were dehydrated through ethanol gradients and dried. After sputter coating with platinum, the mounted samples were ready for examination. Statistical analysis
The data in this study are given as means±SD. Data were analyzed by one-way analysis of variance, followed by Scheffe’s post-hoc test. Differences were considered statistically significant at a P-value of less than 0.05.
Results Edaravone treatment attenuates intraventricular hemorrhage-induced brain edema and free-radical stress
Brain water content was significantly increased in bilateral cerebral hemispheres of the vehicle-treated rats. Treatment with a single dose of edaravone at 6 mg/kg reduced the brain edema in the right hemisphere (78.1±0.3 vs. 78.5±0.2% in the IVH + Veh group, P < 0.01, Fig. 1a). MDA activity and SOD level were measured to evaluate lipid peroxidation and cellular antioxidant potential 1 day after IVH (Fig. 1b and c). IVH led to an increased level of MDA and decreased activity of SOD in the right cortex and hippocampus (P < 0.01, Fig. 1b and c). Edaravone treatment significantly reduced the MDA level in the right cortex (5.2±0.4 vs. 5.8±0.6 nmol/mg protein in the IVH + Veh group, P < 0.05, Fig. 1b) and the hippocampus (5.2±0.6 vs. 5.9±0.5 nmol/mg protein in the IVH + Veh group, P < 0.05, Fig. 1b), and significantly increased SOD
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Protective effects of edaravone after IVH Chen et al. 637
activity only in the right cortex (72.7±2.6 vs. 67.8±3.3 U/mg protein in the IVH + Veh group, P < 0.05, Fig. 1c).
Fig. 1
(a) 79
∗∗
Brain water content (%)
∗∗
Edaravone treatment improved learning and memory ability after intraventricular hemorrhage
#
78
77
76
Left brain
Right hemisphere
Cerebellum
(b) 8
MDA level (nmol/mg protein)
7
∗∗
6
∗∗ #
#
5 4 3
Morphological findings after intraventricular hemorrhage
2
Hemosiderin deposit in the ventricular wall was an obvious finding in both of the vehicle-treated rats and the edaravone-treated rats at 28 days after IVH (Fig. 3a–c). There was a significant reduction in the yellow-stained area in edaravone-treated rats (17.9±2.7 vs. 12.5±3.4% in vehicle-treated rats, P < 0.01). Scanning electron microscopy of the surface of the dorsal–lateral wall of the lateral ventricle indicated injury of ependymal cilia after IVH (Fig. 3d–f). The ependyma of the salineinjected rats (Fig. 3d) was covered with normal cilia evenly, whereas the ependyma of vehicle-treated rats (Fig. 3e) was largely devoid of cilia. The loss of cilia on ependyma was attenuated in all edaravone-treated rats (Fig. 3f) compared with vehicle-treated rats.
1 0
Cortex
Hippocampus
(c) 100
SOD activity (U/mg protein)
In acquisition training, there was a trend toward a decrease in the latencies and distances to the goal after IVH, suggesting IVH-induced learning deficits (Fig. 2a and b). For latencies, the differences between the saline group and the IVH + Veh group reached significance at 1, 3, 4, and 5 training days (P < 0.05 or 0.01, Fig. 2a), but only reached significance at 2, 3, and 5 training days for distances (P < 0.01, Fig. 2b). Repeated edaravone treatment (6 mg/kg/day 3 days) significantly improved the latencies at 2, 3, and 5 training days (P < 0.05 or 0.01, Fig. 2a), and the distance at 2, 3, 4, and 5 training days (P < 0.05 or 0.01, Fig. 2b). In the probe trial, the vehicle-treated animals spent less percent time and remained less percent distance in the target quadrant than the saline group (P < 0.01, Fig. 2c and d), indicating IVH-induced memory deficits. Repeated edaravone treatment improved the percent time and percent distance in the target quadrant significantly (P < 0.01 and 0.05, Fig. 2c and d). Thus, repeated edaravone treatment improved memory deficits after IVH.
80
∗∗
# ∗∗
60
40
Discussion
20
0
Cortex Saline
Hippocampus IVH+Veh
IVH+Edv
Bar graph showing the effect of edaravone (Edv) treatment on the brain water content (a), MDA assay (b), and SOD activity (c) at 1 day after intraventricular hemorrhage. Values are expressed as the means±SD, n = 5–6; **P < 0.01 versus the saline group; #P < 0.05 versus the IVH + Veh group. IVH, intraventricular hemorrhage; MDA, malondialdehyde; SOD, superoxide dismutase; Veh, vehicle.
The major findings of the current study are as follows: (a) an intraventricular injection of autologous whole blood resulted in brain edema, oxidative stress, and cognitive deficits; (b) systemic edaravone treatment can reduce IVH-induced brain edema and cognitive deficits. It is known that IVH may result in direct brain injury as well as posthemorrhagic hydrocephalus [4]. Most researches on the adult experimental IVH have focused on hydrocephalus. In contrast, IVH-induced acute brain injuries and neurobehavioral deficits have seldom been described [7,13,14]. Iron overload and iron-induced oxidative injury after ICH and other neurological
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
638
NeuroReport 2014, Vol 25 No 9
Fig. 2
(a)
(b) 3000
∗
120
IVH+Veh IVH+Edv
Latency (s)
100 80 #
60
∗∗ ##
40
∗
∗∗ #
4
5
20 0
∗∗
Saline
1
2
3 Training day
(c)
Saline IVH+Veh
2500 Distance (cm)
140
IVH+Edv
2000 ##
1500
∗∗ ## ∗∗
1000 500 0
#
# 1
2
3 Training day
4
5
(d) ##
70 60 50
∗∗
40 30 20 10 0
Saline
IVH+Veh
60 Percent distance in quadrant
Percent time in quadrant
80
∗∗
40 30 20 10 0
IVH+Edv
#
50
Saline
IVH+Veh
IVH+Edv
(e)
Saline
IVH+Veh
IVH+Edv
Effect of edaravone (Edv) treatment on the learning and memory function (water maze) after intraventricular hemorrhage. (a) Escape latency in training trials. (b) Escape distance in training trials. (c) Percent time in the target quadrant. (d) Percent distance in the target quadrant. (e) Representative swim traces in probe trials. Values are expressed as the means±SD, n = 5; *P < 0.01, **P < 0.01 versus the saline group; #P < 0.05, ##P < 0.01 versus the IVH + Veh group. IVH, intraventricular hemorrhage; Veh, vehicle.
disorders have been well established [15–17]. Recently, obvious iron accumulation was also observed by T2* MRI sequences and decreased slowly up to 28 days in an adult rat model of IVH [7]. Therefore, we consider that the observed oxidative stress and brain injury may be at least partially related to the iron accumulation after IVH. In the present study, MDA as a product of lipid peroxide decomposition was measured as an indicator of oxidative stress, and SOD as one of the intrinsic antioxidant systems was measured to evaluate the cellular antioxidant
potential after IVH. Our findings showed that IVH led to an increase in the MDA level, as well as decreased activity of SOD in both of the right cortex and the hippocampus at 1 day after injection. Brain edema also developed at 1 day after blood injection. These shortterm findings showed the acute brain injury and the potential role of oxidative stress. To our knowledge, cognitive functions have not been evaluated in an adult IVH model before. This study showed that IVH induced spatial learning and memory deficits in 3–4 weeks after injury. Activities of normal ependymal cilia are considered
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Protective effects of edaravone after IVH Chen et al.
639
Fig. 3
(a–c) Representative gross photograph of the right brain hemisphere from saline-injected rats (a), vehicle-treated rats (b), and edaravone-treated rats (c) at day 28 after injection, showing the obvious hemosiderin-staining in the dorsal–lateral wall of the right lateral ventricle. (d–f) Representative images by scanning electron microscopy from saline-injected rats (d), vehicle-treated rats (e), and edaravone-treated rats (f) at day 28 after injection, showing the presence and density of the ependyma cilia. Scale bar = 10 mm.
to direct cerebrospinal fluid flow, and their absence or abnormal function may cause hydrocephalus. Ependymal surface damage has been described after IVH previously and our study further confirmed the extensive loss of cilia on the ventricular wall at 28 days after IVH. Edaravone has been used for ischemic stroke in some countries and has also gradually been investigated for use in hemorrhagic stroke. In a rat ICH model, a single dose of edaravone (6 mg/kg) administered immediately or 2 h after ICH reduced brain edema, neurologic deficits, and oxidative injury after ICH [8]. In contrast to the recommended clinical regimen of edaravone (30 mg/12 h for 14 days) for the treatment of adult acute cerebral infarction, the periods of edaravone treatment in animal models were mostly shorter. A 3-day period of edaravone treatment has been shown to be neuroprotective in adult rat models of a chronic hypoperfusion model or a traumatic brain injury model [18,19], which was our
treatment period as well. In the present study, edaravone treatment immediately after IVH reduced brain edema and oxidative stress. More importantly, repeated edaravone treatment for 3 days could improve the IVH-induced learning and memory damage as improvement of neurologic deficits is always a key point for stroke therapy. In addition, less yellow staining and loss of cilia on the ventricular surface was observed in the edaravonetreated animals compared with vehicle-treated animals. It is known that ciliated epitheliums are vulnerable to oxidative damage. It has been shown that very shortduration exposure to H2O2 (3%), which was routinely used intraoperatively in neurosurgery as a hemostatic agent, could also cause cessation of ciliary beat frequency and damage of the ependyma [12]. Therefore, we presumed that edaravone might protect the ependymal cilia against oxidative injury after ICH and facilitate the clearance of hemosiderin deposits on the ventricular surface.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
640 NeuroReport 2014, Vol 25 No 9
Conclusion Our results indicate that oxidative injury occurs after IVH, and edaravone treatment reduced IVH-induced brain injury and neurobehavioral deficits. These effects suggest that edaravone may be a potential therapeutic agent for IVH, especially those ICH patients with ventricular extension.
7 8
9
10
Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (81070929, 81271281).
11
12
Conflict of interest
13
There are no conflicts of interest.
14
References 1 2 3
4
5 6
Keep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol 2012; 11:720–731. Hanley DF. Intraventricular hemorrhage: severity factor and treatment target in spontaneous intracerebral hemorrhage. Stroke 2009; 40:1533–1538. Morgan T, Awad I, Keyl P, Lane K, Hanley D. Preliminary report of the clot lysis evaluating accelerated resolution of intraventricular hemorrhage (CLEAR-IVH) clinical trial. Acta Neurochir Suppl 2008; 105:217–220. Strahle J, Garton HJL, Maher CO, Muraszko KM, Keep RF, Xi GH. Mechanisms of hydrocephalus after neonatal and adult intraventricular hemorrhage. Transl Stroke Res 2012; 3:S25–S38. Xi G, Keep RF, Hoff JT. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 2006; 5:53–63. Hua Y, Nakamura T, Keep RF, Wu J, Schallert T, Hoff JT, Xi G. Long-term effects of experimental intracerebral hemorrhage: the role of iron. J Neurosurg 2006; 104:305–312.
15
16
17
18
19
Chen Z, Gao C, Hua Y, Keep RF, Muraszko K, Xi G. Role of iron in brain injury after intraventricular hemorrhage. Stroke 2011; 42:465–470. Nakamura T, Kuroda Y, Yamashita S, Zhang X, Miyamoto O, Tamiya T, et al. Edaravone attenuates brain edema and neurologic deficits in a rat model of acute intracerebral hemorrhage. Stroke 2008; 39:463–469. Hu J, Luo CX, Chu WH, Shan YA, Qian ZM, Zhu G, et al. 20Hydroxyecdysone protects against oxidative stress-induced neuronal injury by scavenging free radicals and modulating NF-kappaB and JNK pathways. PLoS One 2012; 7:e50764. Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006; 1:848–858. Mirzadeh Z, Doetsch F, Sawamoto K, Wichterle H, Alvarez-Buylla A. The subventricular zone en-face: wholemount staining and ependymal flow. J Vis Exp 2010; 39:e1938. Hirst RA, Rutman A, O’Callaghan C. Hydrogen peroxide at a concentration used during neurosurgery disrupts ciliary function and causes extensive damage to the ciliated ependyma of the brain. Childs Nerv Syst 2009; 25:559–561. Lodhia KR, Shakui P, Keep RF. Hydrocephalus in a rat model of intraventricular hemorrhage. Acta Neurochir Suppl 2006; 96:207–211. Mayfrank L, Kissler J, Raoofi R, Delsing P, Weis J, Kuker W, Gilsbach JM. Ventricular dilatation in experimental intraventricular hemorrhage in pigs. Characterization of cerebrospinal fluid dynamics and the effects of fibrinolytic treatment. Stroke 1997; 28:141–148. Nakamura T, Keep RF, Hua Y, Nagao S, Hoff JT, Xi G. Iron-induced oxidative brain injury after experimental intracerebral hemorrhage. Acta Neurochir Suppl 2006; 96:194–198. Wu J, Hua Y, Keep RF, Schallert T, Hoff JT, Xi G. Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. Brain Res 2002; 953:45–52. Kruck TP, Percy ME, Lukiw WJ. Metal sulfate-mediated induction of pathogenic genes and repression by phenyl butyl nitrone and Feralex-G. Neuroreport 2008; 19:245–249. Ueno Y, Zhang N, Miyamoto N, Tanaka R, Hattori N, Urabe T. Edaravone attenuates white matter lesions through endothelial protection in a rat chronic hypoperfusion model. Neuroscience 2009; 162:317–327. Wang GH, Jiang ZL, Li YC, Li X, Shi H, Gao YQ, et al. Free-radical scavenger edaravone treatment confers neuroprotection against traumatic brain injury in rats. J Neurotrauma 2011; 28:2123–2134.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Neuroprotective effects of edaravone after intraventricular hemorrhage in rats Zhi Chen, Jianbo Zhang, Qianwei Chen, Jing Guo, Gang Zhu and Hua Feng Intraventricular hemorrhage (IVH) is a severity factor and treatment target in intracerebral hemorrhage. This study aimed to investigate whether systemic edaravone, a freeradical scavenger, could attenuate the brain injury after IVH in a rat model. Our findings showed that an intraventricular injection of autologous whole blood resulted in acute brain edema, increased malondialdehyde level, and decreased superoxide dismutase enzyme activity. Immediate edaravone treatment after IVH can reduce IVH-induced brain edema and elevated lipid peroxidation. Furthermore, repeated edaravone treatment (immediately, 24 h, and 48 h after IVH) improved the IVH-induced learning and memory damage. These effects suggest that edaravone may be a potential therapeutic agent for IVH,
especially those intracerebral hemorrhage patients with c 2014 ventricular extension. NeuroReport 25:635–640 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
Chongqing, China) weighing 250–350 g were used in this study. The animals were anesthetized with pentobarbital (40 mg/kg intraperitoneally). Body temperature was maintained at 37.51C using a feedback-controlled heating pad. A polyethylene catheter (PE-50) was inserted into the right femoral artery as a source for collecting the arterial blood. Rats were then positioned in a stereotaxic frame and a cranial burr hole (1 mm) was drilled (coordinates: 0.6 mm posterior and 1.6 mm lateral to the bregma). Nonheparinized arterial blood (from the femoral artery catheterization) or saline was collected in a 1 ml syringe with a 26 G needle, which was positioned in the infusion pump and stereotaxically inserted 4.5 ventrally into the right lateral ventricle through the burr hole. Autologous arterial blood (200 ml) or saline was infused at a rate of 14 ml/min using a microinfusion pump. The needle was removed after injection and the skin incision was closed with sutures.
Intracerebral hemorrhage (ICH) is the most severe of the major stroke subtypes. Extension of the hemorrhage into the ventricles after ICH results in intraventricular hemorrhage (IVH), which may occur in up to 50% of patients with ICH [1,2]. Recent studies have confirmed that IVH is an independent predictor of poor outcome after ICH [2–4]. However, the mechanisms of brain injury after IVH are not well understood, and no valid medical treatment is available at present. It is known that brain iron overload is associated with free-radical formation, and contributes toward brain edema, oxidative injury, and brain atrophy after ICH [5,6]. Our recent study has shown that IVH resulted in brain iron accumulation and enlargement of the lateral ventricles in a rat model [7]. However, there is limited information on the role of oxidative stress and neurobehavioral deficits after IVH. The free-radical scavenger edaravone is known to exert antioxidant effects by inhibiting hydroxyl radical-dependent and radical-independent lipid peroxidation. Therefore, the present study investigated whether systemic edaravone treatment could attenuate the IVH-induced acute brain injury and improve behavioral deficits after IVH in a rat model.
Materials and methods Animal preparation and intraventricular infusion
Animal use protocols were approved by the Animal Committee of the Third Military Medical University. A total of 48 male Sprague–Dawley rats (the animal center of the Third Military Medical University, c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
NeuroReport 2014, 25:635–640 Keywords: behavior, cilia, edaravone, intraventricular hemorrhage, oxidative stress Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing, China Correspondence to Hua Feng, MD, PhD, Department of Neurosurgery, Southwest Hospital, Third Military Medical University, No. 30, Gaotanyan Street, Chongqing 400038, China Tel: + 86 23 68765759; fax: + 86 23 68765265; e-mail: [email protected] Received 18 August 2013 accepted 18 September 2013
Experimental groups
These experiments were conducted in two parts. In the first part (short-term study), rats were divided into three groups including intraventricular saline injection (saline control), intraventricular blood injection with vehicle treatment (IVH + Veh), and intraventricular blood injection with edaravone treatment (IVH + Edv). Rats received an injection of 200 ml saline or autologous whole blood into the right lateral ventricle and were treated with either edaravone (6 mg/kg subcutaneously; SigmaAldrich, St Louis, Missouri, USA) or vehicle injection immediately (15 min) after blood injection. This dose of edaravone can reduce ICH-induced brain edema [8]. Rats were euthanized at day 1 for brain water content DOI: 10.1097/WNR.0000000000000050
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
636
NeuroReport 2014, Vol 25 No 9
determination (n = 6 for each group) as well as malondialdehyde (MDA) level and superoxide dismutase (SOD) activity (n = 5 for each group). In the second part (long-term study), rats were divided into three groups as in the first part (n = 5 for each group). The rats with blood injection received either edaravone (6 mg/kg/day subcutaneously) or vehicle treatment every 24 h up to 3 days (immediately, 1 and 2 days after intraventricular injection). Behavioral tests were performed from day 23 after IVH, and these rats were then euthanized for morphological observation at day 28 after injection. Brain water content
Animals were decapitated under deep anesthesia 1 day after IVH for brain water content measurement. The brains were removed immediately and the frontal poles (4 mm) were cut off. The remaining brains were divided into three tissue samples: bilateral cerebral hemispheres and the cerebellum. Brain samples were weighted immediately to obtain the wet weight, and were then dried at 1001C for 24 h to obtain the dry weight. Tissue water content was calculated as (wet weight – dry weight)/wet weight. Malondialdehyde assay and superoxide dismutase activities
The rats were reanesthetized and perfused with 0.1 mol/l PBS, and then the brains were removed immediately and the frontal poles (4 mm) were cut off. Then, the whole right cortex and hippocampus were dissected and stored at – 701C to determine the level of MDA and SOD enzyme activities. The level of lipid peroxidation products (MDA) in the right cerebral cortex and hippocampus was measured by the thiobarbituric acid method using the assay kit following the manufacturer’s instruction (Nanjing Jiancheng Biochemistry Co., Nanjing, China) [9]. The protein concentration was determined using Commassie blue assay. The level of MDA was calculated as nmol/mg protein in the sample according to the standard curve derived. Meanwhile, SOD activity was measured using the kit following the manufacturer’s instruction (Nanjing Jiancheng Biochemistry Co.) [9]. It uses a xanthine–xanthine oxidase system to determine the inhibition of nitroblue tetrazolium reduction because of superoxide anion generation. Total nitrite produced by the oxidation of oxyamine was measured by detecting the absorbance at 550 nm. One unit of SOD was defined as the enzyme amount causing 50% inhibition of the nitroblue tetrazolium reduction rate. SOD activity was expressed in U/mg protein. Learning and memory test
The Morris water maze was used to assess learning and memory of the animals [10]. The test was performed in a metal pool (200 cm in diameter, 50 cm in depth) filled with water. The spatial acquisition training was performed at day 23 after IVH. All the rats received
5 days of acquisition training with the platform remaining in a single location. Each training day consisted of four trials from one of four start positions with a 10 min interval, and the latencies and the distances to the goal were averaged across four trials per day. The probe trial to assess reference memory was conducted 1 day after the last acquisition with the platform removed. The time and distance traveled in each of the four quadrants was recorded. The percent time and the percent distance in the target quadrant were calculated. Dissection of lateral ventricles and scanning electron microscopy
Animals were decapitated under deep anesthesia, and the brains were removed immediately. The right lateral ventricles were exposed widely using the whole-mount dissection technique as described previously [11]. The distribution of the yellow-stained area was measured using Image J software (National Institutes of Health, Bethesda, Maryland, USA), and presented as a percentage of the total area of the right lateral ventricle. Brain samples containing the dorsal–lateral wall of the right lateral ventricle were then prepared for scanning electron microscopy (FEI Quanta 450; FEI Company, Hillsboro, Oregon, USA) as described previously [12]. Briefly, the tissues were fixed in Sorensen’s phosphate-buffered glutaraldehyde (4%, pH 7.4) for 48 h. After postfixation in 1% osmium tetroxide, samples were dehydrated through ethanol gradients and dried. After sputter coating with platinum, the mounted samples were ready for examination. Statistical analysis
The data in this study are given as means±SD. Data were analyzed by one-way analysis of variance, followed by Scheffe’s post-hoc test. Differences were considered statistically significant at a P-value of less than 0.05.
Results Edaravone treatment attenuates intraventricular hemorrhage-induced brain edema and free-radical stress
Brain water content was significantly increased in bilateral cerebral hemispheres of the vehicle-treated rats. Treatment with a single dose of edaravone at 6 mg/kg reduced the brain edema in the right hemisphere (78.1±0.3 vs. 78.5±0.2% in the IVH + Veh group, P < 0.01, Fig. 1a). MDA activity and SOD level were measured to evaluate lipid peroxidation and cellular antioxidant potential 1 day after IVH (Fig. 1b and c). IVH led to an increased level of MDA and decreased activity of SOD in the right cortex and hippocampus (P < 0.01, Fig. 1b and c). Edaravone treatment significantly reduced the MDA level in the right cortex (5.2±0.4 vs. 5.8±0.6 nmol/mg protein in the IVH + Veh group, P < 0.05, Fig. 1b) and the hippocampus (5.2±0.6 vs. 5.9±0.5 nmol/mg protein in the IVH + Veh group, P < 0.05, Fig. 1b), and significantly increased SOD
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Protective effects of edaravone after IVH Chen et al. 637
activity only in the right cortex (72.7±2.6 vs. 67.8±3.3 U/mg protein in the IVH + Veh group, P < 0.05, Fig. 1c).
Fig. 1
(a) 79
∗∗
Brain water content (%)
∗∗
Edaravone treatment improved learning and memory ability after intraventricular hemorrhage
#
78
77
76
Left brain
Right hemisphere
Cerebellum
(b) 8
MDA level (nmol/mg protein)
7
∗∗
6
∗∗ #
#
5 4 3
Morphological findings after intraventricular hemorrhage
2
Hemosiderin deposit in the ventricular wall was an obvious finding in both of the vehicle-treated rats and the edaravone-treated rats at 28 days after IVH (Fig. 3a–c). There was a significant reduction in the yellow-stained area in edaravone-treated rats (17.9±2.7 vs. 12.5±3.4% in vehicle-treated rats, P < 0.01). Scanning electron microscopy of the surface of the dorsal–lateral wall of the lateral ventricle indicated injury of ependymal cilia after IVH (Fig. 3d–f). The ependyma of the salineinjected rats (Fig. 3d) was covered with normal cilia evenly, whereas the ependyma of vehicle-treated rats (Fig. 3e) was largely devoid of cilia. The loss of cilia on ependyma was attenuated in all edaravone-treated rats (Fig. 3f) compared with vehicle-treated rats.
1 0
Cortex
Hippocampus
(c) 100
SOD activity (U/mg protein)
In acquisition training, there was a trend toward a decrease in the latencies and distances to the goal after IVH, suggesting IVH-induced learning deficits (Fig. 2a and b). For latencies, the differences between the saline group and the IVH + Veh group reached significance at 1, 3, 4, and 5 training days (P < 0.05 or 0.01, Fig. 2a), but only reached significance at 2, 3, and 5 training days for distances (P < 0.01, Fig. 2b). Repeated edaravone treatment (6 mg/kg/day 3 days) significantly improved the latencies at 2, 3, and 5 training days (P < 0.05 or 0.01, Fig. 2a), and the distance at 2, 3, 4, and 5 training days (P < 0.05 or 0.01, Fig. 2b). In the probe trial, the vehicle-treated animals spent less percent time and remained less percent distance in the target quadrant than the saline group (P < 0.01, Fig. 2c and d), indicating IVH-induced memory deficits. Repeated edaravone treatment improved the percent time and percent distance in the target quadrant significantly (P < 0.01 and 0.05, Fig. 2c and d). Thus, repeated edaravone treatment improved memory deficits after IVH.
80
∗∗
# ∗∗
60
40
Discussion
20
0
Cortex Saline
Hippocampus IVH+Veh
IVH+Edv
Bar graph showing the effect of edaravone (Edv) treatment on the brain water content (a), MDA assay (b), and SOD activity (c) at 1 day after intraventricular hemorrhage. Values are expressed as the means±SD, n = 5–6; **P < 0.01 versus the saline group; #P < 0.05 versus the IVH + Veh group. IVH, intraventricular hemorrhage; MDA, malondialdehyde; SOD, superoxide dismutase; Veh, vehicle.
The major findings of the current study are as follows: (a) an intraventricular injection of autologous whole blood resulted in brain edema, oxidative stress, and cognitive deficits; (b) systemic edaravone treatment can reduce IVH-induced brain edema and cognitive deficits. It is known that IVH may result in direct brain injury as well as posthemorrhagic hydrocephalus [4]. Most researches on the adult experimental IVH have focused on hydrocephalus. In contrast, IVH-induced acute brain injuries and neurobehavioral deficits have seldom been described [7,13,14]. Iron overload and iron-induced oxidative injury after ICH and other neurological
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
638
NeuroReport 2014, Vol 25 No 9
Fig. 2
(a)
(b) 3000
∗
120
IVH+Veh IVH+Edv
Latency (s)
100 80 #
60
∗∗ ##
40
∗
∗∗ #
4
5
20 0
∗∗
Saline
1
2
3 Training day
(c)
Saline IVH+Veh
2500 Distance (cm)
140
IVH+Edv
2000 ##
1500
∗∗ ## ∗∗
1000 500 0
#
# 1
2
3 Training day
4
5
(d) ##
70 60 50
∗∗
40 30 20 10 0
Saline
IVH+Veh
60 Percent distance in quadrant
Percent time in quadrant
80
∗∗
40 30 20 10 0
IVH+Edv
#
50
Saline
IVH+Veh
IVH+Edv
(e)
Saline
IVH+Veh
IVH+Edv
Effect of edaravone (Edv) treatment on the learning and memory function (water maze) after intraventricular hemorrhage. (a) Escape latency in training trials. (b) Escape distance in training trials. (c) Percent time in the target quadrant. (d) Percent distance in the target quadrant. (e) Representative swim traces in probe trials. Values are expressed as the means±SD, n = 5; *P < 0.01, **P < 0.01 versus the saline group; #P < 0.05, ##P < 0.01 versus the IVH + Veh group. IVH, intraventricular hemorrhage; Veh, vehicle.
disorders have been well established [15–17]. Recently, obvious iron accumulation was also observed by T2* MRI sequences and decreased slowly up to 28 days in an adult rat model of IVH [7]. Therefore, we consider that the observed oxidative stress and brain injury may be at least partially related to the iron accumulation after IVH. In the present study, MDA as a product of lipid peroxide decomposition was measured as an indicator of oxidative stress, and SOD as one of the intrinsic antioxidant systems was measured to evaluate the cellular antioxidant
potential after IVH. Our findings showed that IVH led to an increase in the MDA level, as well as decreased activity of SOD in both of the right cortex and the hippocampus at 1 day after injection. Brain edema also developed at 1 day after blood injection. These shortterm findings showed the acute brain injury and the potential role of oxidative stress. To our knowledge, cognitive functions have not been evaluated in an adult IVH model before. This study showed that IVH induced spatial learning and memory deficits in 3–4 weeks after injury. Activities of normal ependymal cilia are considered
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Protective effects of edaravone after IVH Chen et al.
639
Fig. 3
(a–c) Representative gross photograph of the right brain hemisphere from saline-injected rats (a), vehicle-treated rats (b), and edaravone-treated rats (c) at day 28 after injection, showing the obvious hemosiderin-staining in the dorsal–lateral wall of the right lateral ventricle. (d–f) Representative images by scanning electron microscopy from saline-injected rats (d), vehicle-treated rats (e), and edaravone-treated rats (f) at day 28 after injection, showing the presence and density of the ependyma cilia. Scale bar = 10 mm.
to direct cerebrospinal fluid flow, and their absence or abnormal function may cause hydrocephalus. Ependymal surface damage has been described after IVH previously and our study further confirmed the extensive loss of cilia on the ventricular wall at 28 days after IVH. Edaravone has been used for ischemic stroke in some countries and has also gradually been investigated for use in hemorrhagic stroke. In a rat ICH model, a single dose of edaravone (6 mg/kg) administered immediately or 2 h after ICH reduced brain edema, neurologic deficits, and oxidative injury after ICH [8]. In contrast to the recommended clinical regimen of edaravone (30 mg/12 h for 14 days) for the treatment of adult acute cerebral infarction, the periods of edaravone treatment in animal models were mostly shorter. A 3-day period of edaravone treatment has been shown to be neuroprotective in adult rat models of a chronic hypoperfusion model or a traumatic brain injury model [18,19], which was our
treatment period as well. In the present study, edaravone treatment immediately after IVH reduced brain edema and oxidative stress. More importantly, repeated edaravone treatment for 3 days could improve the IVH-induced learning and memory damage as improvement of neurologic deficits is always a key point for stroke therapy. In addition, less yellow staining and loss of cilia on the ventricular surface was observed in the edaravonetreated animals compared with vehicle-treated animals. It is known that ciliated epitheliums are vulnerable to oxidative damage. It has been shown that very shortduration exposure to H2O2 (3%), which was routinely used intraoperatively in neurosurgery as a hemostatic agent, could also cause cessation of ciliary beat frequency and damage of the ependyma [12]. Therefore, we presumed that edaravone might protect the ependymal cilia against oxidative injury after ICH and facilitate the clearance of hemosiderin deposits on the ventricular surface.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
640 NeuroReport 2014, Vol 25 No 9
Conclusion Our results indicate that oxidative injury occurs after IVH, and edaravone treatment reduced IVH-induced brain injury and neurobehavioral deficits. These effects suggest that edaravone may be a potential therapeutic agent for IVH, especially those ICH patients with ventricular extension.
7 8
9
10
Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (81070929, 81271281).
11
12
Conflict of interest
13
There are no conflicts of interest.
14
References 1 2 3
4
5 6
Keep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets. Lancet Neurol 2012; 11:720–731. Hanley DF. Intraventricular hemorrhage: severity factor and treatment target in spontaneous intracerebral hemorrhage. Stroke 2009; 40:1533–1538. Morgan T, Awad I, Keyl P, Lane K, Hanley D. Preliminary report of the clot lysis evaluating accelerated resolution of intraventricular hemorrhage (CLEAR-IVH) clinical trial. Acta Neurochir Suppl 2008; 105:217–220. Strahle J, Garton HJL, Maher CO, Muraszko KM, Keep RF, Xi GH. Mechanisms of hydrocephalus after neonatal and adult intraventricular hemorrhage. Transl Stroke Res 2012; 3:S25–S38. Xi G, Keep RF, Hoff JT. Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 2006; 5:53–63. Hua Y, Nakamura T, Keep RF, Wu J, Schallert T, Hoff JT, Xi G. Long-term effects of experimental intracerebral hemorrhage: the role of iron. J Neurosurg 2006; 104:305–312.
15
16
17
18
19
Chen Z, Gao C, Hua Y, Keep RF, Muraszko K, Xi G. Role of iron in brain injury after intraventricular hemorrhage. Stroke 2011; 42:465–470. Nakamura T, Kuroda Y, Yamashita S, Zhang X, Miyamoto O, Tamiya T, et al. Edaravone attenuates brain edema and neurologic deficits in a rat model of acute intracerebral hemorrhage. Stroke 2008; 39:463–469. Hu J, Luo CX, Chu WH, Shan YA, Qian ZM, Zhu G, et al. 20Hydroxyecdysone protects against oxidative stress-induced neuronal injury by scavenging free radicals and modulating NF-kappaB and JNK pathways. PLoS One 2012; 7:e50764. Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006; 1:848–858. Mirzadeh Z, Doetsch F, Sawamoto K, Wichterle H, Alvarez-Buylla A. The subventricular zone en-face: wholemount staining and ependymal flow. J Vis Exp 2010; 39:e1938. Hirst RA, Rutman A, O’Callaghan C. Hydrogen peroxide at a concentration used during neurosurgery disrupts ciliary function and causes extensive damage to the ciliated ependyma of the brain. Childs Nerv Syst 2009; 25:559–561. Lodhia KR, Shakui P, Keep RF. Hydrocephalus in a rat model of intraventricular hemorrhage. Acta Neurochir Suppl 2006; 96:207–211. Mayfrank L, Kissler J, Raoofi R, Delsing P, Weis J, Kuker W, Gilsbach JM. Ventricular dilatation in experimental intraventricular hemorrhage in pigs. Characterization of cerebrospinal fluid dynamics and the effects of fibrinolytic treatment. Stroke 1997; 28:141–148. Nakamura T, Keep RF, Hua Y, Nagao S, Hoff JT, Xi G. Iron-induced oxidative brain injury after experimental intracerebral hemorrhage. Acta Neurochir Suppl 2006; 96:194–198. Wu J, Hua Y, Keep RF, Schallert T, Hoff JT, Xi G. Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. Brain Res 2002; 953:45–52. Kruck TP, Percy ME, Lukiw WJ. Metal sulfate-mediated induction of pathogenic genes and repression by phenyl butyl nitrone and Feralex-G. Neuroreport 2008; 19:245–249. Ueno Y, Zhang N, Miyamoto N, Tanaka R, Hattori N, Urabe T. Edaravone attenuates white matter lesions through endothelial protection in a rat chronic hypoperfusion model. Neuroscience 2009; 162:317–327. Wang GH, Jiang ZL, Li YC, Li X, Shi H, Gao YQ, et al. Free-radical scavenger edaravone treatment confers neuroprotection against traumatic brain injury in rats. J Neurotrauma 2011; 28:2123–2134.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
