Edaravone Reduces Iron-Mediated Hydrocephalus and Behavioral Disorder in Rat by Activating the Nrf2/HO-1 Pathway

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Abstract

Our previous studies have demonstrated that hemorrhage-derived iron has a key role in causing brain injury after intraventricular hemorrhage (IVH). Based on this finding, we hypothesized that edaravone, a free-radical scavenger, has the potential to alleviate hydrocephalus and neurological deficits post-IVH by suppressing iron-induced oxidative stress. Thus, this study aimed to investigate the efficacy of edaravone for rats with FeCl3 injection, as well as to explore the related molecular mechanism. An experimental model was established in adult male Sprague-Dawley rats via FeCl3 injection into the right lateral ventricle. Edaravone or vehicle was administered immediately, 1 day and 2 days after intraventricular injection. Brain water content, magnetic resonance imaging, neurological score, oxidative stress assays, Western blot analysis, and electron microscopy were employed to evaluate brain injury in these rats. Intraventricular injection of FeCl3 induced brain edema, ventricular dilation, and neurobehavioral disorder in rats. Edaravone treatment significantly attenuated the above symptoms, reduced ependymal cilia and neuron damage, and inhibited oxidative stress (elevated levels of an antioxidant, superoxide dismutase; decreased levels of an oxidant, malondialdehyde). Moreover, edaravone administration effectively activated the Nrf2/HO-1 signaling pathway in rat brain following FeCl3 injection. These results showed that edaravone treatment alleviated brain edema, ventricular expansion, and neurological disorder after FeCl3 injection. The possible mechanism is by protecting ependymal cilia and neurons from oxidative stress injury and activating the Nrf2/HO-1 signaling pathway. These results provide further experimental evidence for edaravone application in the treatment of IVH.

Introduction

Intraventricular hemorrhage (IVH) is one of the most common and severe complications in patients with intracerebral hemorrhage (ICH). As a negative prognostic factor for ICH, IVH is closely related to the high mortality, morbidity, and disability of ICH patients, which seriously endangers public health.1 Half of these patients will experience chronic hydrocephalus, leading to varying degrees of motor function disorder and neurocognitive impairments. Due to the unclear etiology and pathogenesis of this condition, the therapeutic options for IVH are relatively limited.2 An external ventricular drainage operation to export intraventricular hemorrhage as well as cerebrospinal fluid is employed to maintain intracranial pressure stability in the early stage of IVH, but external ventricular drainage fails to promote clot resolution and reduce the incidence of chronic hydrocephalus.3 In past decades, clot lysis has been proposed as a potential method to clear ventricular clots and prevent hydrocephalus after IVH. Recently, a series of intraventricular thrombolysis clinical trials named Evaluating Accelerated Resolution of IVH (CLEAR-IVH) were performed to test intraventricular injection of recombinant tissue plasminogen activator.4, 5 However, the results showed no significant benefit in attenuating hydrocephalus and improving the outcome after IVH.6

To date, there remains no comprehensive strategy for IVH management, and thus, it is imperative to identify new therapeutic targets. Over past decades, increasing evidences have indicated that hemorrhage-derived blood and subsequent metabolic products, plasma components, the coagulation cascade, platelets, and leukocytosis are involved in the pathogenesis of IVH.7, 8 Especially, more and more preclinical studies have suggested that the iron, a toxic degradation product of hemoglobin, has a key role in brain injury and hydrocephalus development following IVH,9, 10, 11 but the underlying molecular mechanism remains unclear. In 2008, Nakamura et al12 reported that iron could promote oxidative stress in the rat brain after ICH, which further led to edema and behavioral dysfunction. Edaravone, a free-radical scavenger, effectively reduced ICH-induced oxidative stress and alleviated secondary brain injury. Recently, we also observed that edaravone administration exerted similar neuroprotective effects in a rat model of IVH,13 but the mechanism is still unknown.

Thus, we hypothesized that edaravone treatment may alleviate brain injury after IVH by suppressing iron-induced oxidative stress. Therefore, we established a rat model to mimic iron-mediated brain injury by intraventricular injection of FeCl3. Then, we further investigated the neuroprotective effect of edaravone on rats that received FeCl3 injection, as well as the related potential mechanism.

Sixty-nine adult male Sprague-Dawley rats (250-350 g; the Third Military Medical University) were used. Animal use procedures followed the Guide for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (SCXK-PLA-20120011). This experiment was conducted in 2 parts. In the first part (short-term study), rats were divided into 3 groups, including intraventricular saline injection (saline control), intraventricular FeCl3 injection with vehicle treatment (FeCl3+Vehi), and intraventricular FeCl3 injection with edaravone treatment (FeCl3+Ed). Rats received an injection of FeCl3 (2 mmol/L, 50 µL) or saline (50 µL) into the right lateral ventricle and were treated with either edaravone (6 mg/kg subcutaneously; Sigma-Aldrich) or vehicle injection immediately (15 minutes) after FeCl3 injection. Intraventricular injections were carried out as described previously.10 This dosage of edaravone was based on our previous study in a rat model of IVH.13 Rats were euthanized on day 1 for brain water content measurement (n = 6 for each group), malondialdehyde (MDA) level and superoxide dismutase (SOD) activity (n = 6 for each group), as well as protein expression levels of Keap1, Nrf2, and HO-1 in periventricular brain tissues (n = 4 for each group). In the second part (long-term study), rats were divided into 3 groups as in the first part (n = 7 for each group). The rats with FeCl3 injection received either edaravone (6 mg/kg/day subcutaneously) or vehicle treatment every 24 hours up to 3 days (immediately, 1 day and 2 days after FeCl3 injection). Behavioral assessments were performed from days 0-7 to days 23-28 following injury. These animals were assessed with MRI scanning for lateral ventricles and hippocampus observation and then euthanized for electron microscopy examination on day 28 after injection.

Animals were decapitated under deep anesthesia 24 hours after FeCl3 injection for brain water content measurement. Brains were removed quickly, and the frontal poles (4 mm) were cut off. The remaining brains were divided into 3 parts: the cerebellum and the ipsilateral and contralateral cerebral hemispheres. Brain samples were weighed immediately to record the wet weight and were then dried at 100°C for 24 hours to record the dry weight. Tissue water content was calculated as (wet weight–dry weight)/wet weight.

Section snippets

MRI and Volume Measurement

Animals were anesthetized with a 2% isoflurane/air mixture during MRI examination. The MRI scans were performed in a 7.0-T Varian MR scanner (Bruker) with a T2* gradient-echo sequence and a T2 fast spin-echo sequence using a view field of 35 mm × 35 mm and 17 coronal slices (1.0 mm thickness). Volumes were calculated as previously described.14 Bilateral ventricles and the hippocampus were outlined and measured; volumes were measured by calculating the areas of all slices and multiplying by the

Assessment of Neurological Abnormalities

Neurological dysfunction of rats was evaluated using a modified Neurological Severity Score (mNSS) method as described previously.16 Briefly, the assessment was performed before and on days 1, 3, and 7 after FeCl3 injection. The mNSS is a composite test of motor, sensory, and balance functions. Neurological function was graded on a scale of 0-18 (normal score, 0; maximal deficit score, 18). The mean neurological score was calculated by 2 blinded observers.

Neurocognitive Function Assessment

Twenty-three days after FeCl3 infusion, the Morris water maze test was performed to assess learning and memory of the animals, as previously described.17 Animals were placed in a metal pool (50 cm in depth, 200 cm in diameter) filled with water and were allowed for find the submerged platform within 120 seconds. Then, they were given 5 days of acquisition training using a random set of start locations in the 4 quadrants. The latency time was monitored and averaged across 4 trials per day. On

Western Blot Analysis

Western blot analysis was performed as previously described.18 The brains were perfused with saline before decapitation. The hippocampus and PVZ brain tissue (1-mm-thick brain tissue around the ventricle) were sampled. The primary antibodies were polyclonal rabbit anti-rat HO-1 IgG (1:2000 dilution; StressGene), mouse monoclonal antibody to Nrf2 (1:500 dilution; Abcam) and polyclonal rabbit anti-Keap1 (1:500 dilution; bovine). The relative densities of the bands were analyzed with NIH ImageJ.

Edaravone Administration Reduced FeCl3-Induced Brain Edema

We previously showed that edaravone significantly attenuated brain edema in a rat model of IVH.13 To explore whether iron has a role in this process, in this study, we tested the efficacy of edaravone in a rat model that received intraventricular FeCl3 injection. Twenty-four hours later, FeCl3 injection increased the brain water content in the bilateral hemispheres (P < .05; Fig 1), and significantly decreased brain water content was observed in the edaravone treatment group compared to the

Discussion

In this report, we showed that intraventricular injection of FeCl3-induced brain edema, ventricular dilation and neurobehavioral disorder in rats. Edaravone treatment effectively improved the above symptoms by suppressing oxidative stress in brain tissues. Moreover, Nrf2/HO-1 signaling pathway activation is involved in the edaravone-mediated neuroprotective effects following FeCl3 injection.

ICH patients always develop brain edema in the early stage, which is closely associated with poor

Conclusions

In summary, edaravone treatment significantly alleviated brain edema, ventricular expansion, and neurological disorder in rats with FeCl3 injection and may act in part by protecting ependymal cilia and neurons from oxidative stress injury by activating the antioxidation Nrf2/HO-1 signaling pathway. Our data provide further experimental evidence for edaravone application in the treatment of IVH.

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    Funding: This work was supported by Grant No. 81701147 (QW.C.) from the National Natural Science Foundation of China and Grant No. 2014CB541606 (H.F.) from the National Key Basic Research Development Program (973 Program) of China.

    Conflicts of Interest: The authors indicate no potential conflicts of interest.

    1

    Drs. J.B. Zhang and X. Shi contributed equally to this work.

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