Elsevier

Brain Research

Volume 1519, 26 June 2013, Pages 95-104
Brain Research

Research Report
Therapeutic impact of eicosapentaenoic acid on ischemic brain damage following transient focal cerebral ischemia in rats

https://doi.org/10.1016/j.brainres.2013.04.046Get rights and content

Highlights

  • Protective effects of EPA on ischemic brain damage were examined in rats.

  • EPA pretreatment achieved infarct volume reduction and neurological improvement.

  • EPA pretreatment reduced oxidative stress following brain ischemia.

  • EPA pretreatment inhibited endothelial Rho-kinase activation after brain ischemia.

Abstract

Long-chain n-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have been shown to reduce ischemic neuronal injury. We investigated the effects of ethyl-EPA (EPA-E) on ischemic brain damage using a rat transient focal cerebral ischemia model. Male Sprague–Dawley rats (n=105) were subjected to 90 min of focal cerebral ischemia. EPA-E (100 mg/kg/day) or vehicle was administered once a day for 3, 5 or 7 days prior to ischemia. Different withdrawal intervals of 3, 5, and 7 days prior to ischemia following 7-day pretreatment with EPA-E or vehicle were also examined. In addition, post-ischemic administration of EPA-E was investigated. Pretreatment with EPA-E for 7 and 5 days, but not 3 days, showed significant infarct volume reduction and neurological improvements when compared with vehicle pretreatment. In addition, withdrawal of EPA-E administration for 3 days, but not 5 and 7 days, also demonstrated significant infarct volume reduction and neurological improvements when compared with vehicle treatment. Post-ischemic treatment of EPA-E did not show any neuroprotection. Immunohistochemistry revealed that 7-day pretreatment with EPA-E significantly reduced cortical expression of 8-hydroxydeoxyguanosine (maker for oxidative DNA damage), 4-hydroxy-2-nonenal (maker for lipid peroxidation), phosphorylated adducin (marker for Rho-kinase activation) and von Willebrand factor (endothelial marker) when compared with vehicle pretreatment. In addition, phosphorylated adducin expression co-localized with von Willebrand factor immunoreactivity. The present study established the neuroprotective effect of EPA-E on ischemic brain damage following transient focal cerebral ischemia in rats, which may be involved in the suppression of oxidative stress and endothelial Rho-kinase activation.

Introduction

Long-chain n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are derived from marine products, have attracted considerable attention. Early epidemiological studies in Greenland discovered that the Inuit people showed a lower incidence of myocardial infarction than Danish people (Bang et al., 1971), and EPA from a fish-rich diet was considered to be responsible for preventing thrombosis and atherosclerosis in the Inuit (Dyerberg et al., 1978). Furthermore, a recent meta-analysis of cohort studies disclosed an inverse relationship between fish consumption and stroke risk (He et al., 2004). A large amount of highly purified EPA can be prepared in an ethyl-ester form (EPA-E) with an approximately 90% purity (Mizuguchi et al., 1993), and EPA-E has been clinically used as a lipid-lowering medication in Japan. In addition, a recent clinical controlled trial of the Japan EPA lipid intervention study (JELIS) has shown that EPA-E reduces stroke recurrence in Japanese hypercholesterolemic patients (Tanaka et al., 2008).

We have previously shown that long-term administration of EPA-E (100 mg/kg/day) ameliorated the age-related decline of cerebral blood flow (CBF) in stroke-prone spontaneously hypertensive rats (Katayama et al., 1997). Furthermore, we have also reported that post-ischemic delayed administration of EPA-E (100 mg/kg/day) for 4 weeks increased local CBF within the peri-infarct areas in a rat chronic cerebral infarction model (Katsumata et al., 1999). In addition, several investigations have described the neuroprotective effects of EPA on forebrain ischemia (Okabe et al., 2011, Bas et al., 2007, Ozen et al., 2008, Ajami et al., 2011). However, the protective effects of EPA on acute focal cerebral ischemia remain unclear.

Cerebral ischemia consists of complex pathological processes, and various factors can exacerbate ischemic brain damage. Oxidative stress is known to be involved in ischemia-reperfusion injury owing to an increase in reactive oxygen species (ROS), which result in DNA damage and lipid peroxidation (Floyd and Carney, 1992, Halliwell, 1992). In addition, Rho-kinase activity is associated with various cerebral vascular diseases, including ischemic brain injury (Chrissobolis and Sobey, 2006). A recent investigation revealed that endothelial Rho-kinase activation following cerebral ischemia played an important role for infarct expansion (Yagita et al., 2007). Furthermore, ROS-induced brain endothelial dysfunction involves the Rho-kinase signaling pathway (Kahles et al., 2007). Therefore oxidative stress and subsequent endothelial Rho-kinase activation may be implicated in ischemic brain injury.

The present study aimed to examine the effects of pre-ischemic and post-ischemic treatments of EPA-E on ischemic brain damage using a rat transient focal cerebral ischemia model. Furthermore we sought to determine whether EPA-E could suppress oxidative DNA damage, lipid peroxidation and endothelial Rho-kinase activation following focal cerebral ischemia.

Section snippets

Plasma fatty acids

Plasma EPA levels were significantly elevated after 7-day pretreatment with EPA-E compared with vehicle treatment (p=0.0019), although plasma arachidonic acid (AA) levels were not different between the groups (p=0.9701) (Table 1). In experiment 2, plasma EPA concentrations were significantly higher even with withdrawal of EPA-E administration for 3 days compared with vehicle administration (p=0.0022); however, EPA-E withdrawal for more than 5 days showed no differences in plasma EPA levels

Discussion

Several investigators have studied the effects of n-3 PUFAs on cerebral ischemia (Okabe et al., 2011, Bas et al., 2007, Ozen et al., 2008, Ajami et al., 2011, Cao et al., 2004, Pan et al., 2009). The examined n-3 PUFAs in each study were EPA, DHA or a mixture of EPA/DHA. Okabe and colleagues reported that EPA inhibited inflammatory responses and oxidative damage following transient forebrain ischemia in gerbils (Okabe et al., 2011), and Bas and colleagues studied the effects of fish oil

Experimental procedure

All experimental protocols were carried out in accordance with the institutional guidelines of Nippon Medical School for animal care and use.

Contributors

MU and YK conceived of the experiments. MU, TI, CN and NK performed the experiments, and analyzed the data with YK. MU wrote the paper. All authors discussed the results and commented on the manuscript.

Acknowledgment

EPA-E was a generous gift from Mochida Pharmaceutical Co., Ltd. (Tokyo, Japan).

References (37)

  • N. Okabe et al.

    Eicosapentaenoic acid prevents memory impairment after ischemia by inhibiting inflammatory response and oxidative damage

    J. Stroke Cerebrovasc. Dis.

    (2011)
  • S. Okubo et al.

    FK-506 extended the therapeutic time window for thrombolysis without increasing the risk of hemorrhagic transformation in an embolic rat stroke model

    Brain Res.

    (2007)
  • Y. Okuda et al.

    Eicosapentaenoic acid enhances nitric oxide production by cultured human endothelial cells

    Biochem. Biophys. Res. Commun.

    (1997)
  • H.C. Pan et al.

    Protective effect of docosahexaenoic acid against brain injury in ischemic rats

    J. Nutr. Biochem.

    (2009)
  • D.J. Philbrick et al.

    Ingestion of fish oil or a derived n-3 fatty acid concentrate containing eicosapentaenoic acid (EPA) affects fatty acid compositions of individual phospholipids of rat brain, sciatic nerve and retina

    J. Nutr.

    (1987)
  • M. Ajami et al.

    Expression of Bcl-2 and Bax after hippocampal ischemia in DHA+EPA treated rats

    Neurol. Sci.

    (2011)
  • H.O. Bang et al.

    Plasma lipid and lipoprotein pattern in Greenlandic West-coast Eskimos

    Lancet

    (1971)
  • S. Chrissobolis et al.

    Recent evidence for an involvement of rho-kinase in cerebral vascular disease

    Stroke

    (2006)
  • Cited by (0)

    View full text