The protective effect of fish n-3 fatty acids on cerebral ischemia in rat hippocampus

Neurochemistry International 50 (2007) 548–554 www.elsevier.com/locate/neuint

The protective effect of fish n-3 fatty acids on cerebral ischemia in rat hippocampus Orhan Bas a,*, Ahmet Songur a, Onder Sahin b, Hakan Mollaoglu c, Oguz Aslan Ozen a, Mehmet Yaman d, Olcay Eser f, Huseyin Fidan e, Murat Yagmurca g a

Kocatepe University, School of Medicine, Department of Anatomy, Afyonkarahisar, Turkey Kocatepe University, School of Medicine, Department of Pathology, Afyonkarahisar, Turkey c Kocatepe University, School of Medicine, Department of Physiology, Afyonkarahisar, Turkey d Kocatepe University, School of Medicine, Department of Neurology, Afyonkarahisar, Turkey e Kocatepe University, School of Medicine, Department of Anesthesiology, Afyonkarahisar, Turkey f Kocatepe University, School of Medicine, Department of Neurosurgery, Afyonkarahisar, Turkey g Kocatepe University, School of Medicine, Department of Histology & Embryology, Afyonkarahisar, Turkey b

Received 21 August 2006; received in revised form 12 October 2006; accepted 6 November 2006 Available online 21 December 2006

Abstract Reactive oxygen species (ROS) have been implicated in the pathogenesis of cerebral injury after ischemia–reperfusion (I/R). Fish n-3 essential fatty acids (EFA), contain eicosapentaenoic acids (EPA) and docosahexoenoic acids (DHA), exhibit antioxidant properties. DHA is an important component of brain membrane phospholipids and is necessary for the continuity of neuronal functions. EPA prevents platelet aggregation and inhibits the conversion of arachidonic acid into thromboxane A2 and prostaglandins. They have been suggested to be protective agents against neurological and neuropsychiatric disorders. In this study, the neuroprotective effects of fish n-3 EFA on oxidant–antioxidant systems and number of apoptotic neurons of the hippocampal formation (HF) subjected to cerebral I/R injury was investigated in Sprague–Dawley rats. Six rats were used as control (Group I). Cerebral ischemia was produced by occlusion of both the common carotid arteries combined with hypotension for 45 min, followed by reperfusion for 30 min, in rats either on a standard diet (Group II) or a standard diet plus fish n-3 EFA (Marincap1, 0.4 g/kg/day, by gavage) for 14 days (Group III). At the end of procedures, the rats were sacrificed and their brains were removed immediately. The levels of malonedialdehyde (MDA) and nitric oxide (NO) and activities of superoxide dismutase (SOD) and catalase (CAT) were measured in left HF. In addition, the number of apoptotic neurons was counted by terminal transferase dUTP nick end labelling (TUNEL) assay in histological samples of the right HF. We found that SOD activities and MDA levels increased in Group III rats compared with Group II rats. On the other hand, CAT activities and NO levels were found to be decreased in Group III rats compared with Group II rats. Additionally, the number of apoptotic neurons was lower in Group III in comparison with Group II rats. The present findings suggest that fish n-3 EFA could decrease the oxidative status and apoptotic changes in ischemic rat hippocampal formation. Dietary supplementation of n-3 EFA may be beneficial to preserve or ameliorate ischemic cerebral vascular disease. # 2006 Elsevier Ltd. All rights reserved. Keywords: Fish n-3 fatty acids; Cerebral ischemia; Rat hippocampal formation

1. Introduction Although reactive oxygen species (ROS) are essential to many normal biological processes and are produced physio-

* Corresponding author. Tel.: +90 272 2140152; fax: +90 272 2142067. E-mail address: [email protected] (O. Bas). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.11.005

logically; excessive production and gathering of ROS (or impairment of the antioxidant systems) can become hazardous to cells and tissues (Sarsilmaz et al., 2003a; Tian et al., 2005). ROS have been implicated in brain injury after ischemia. Cerebral blood flow is reduced in the brain regions during cerebral ischemia and results in the increased production of ROS. Following this, spontaneous or thrombolytic reperfusion can increase the oxygen and ROS levels (Chan, 2001; Irmak

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et al., 2003). Since the rates of oxidative metabolic activities are high and the antioxidant enzymes activities are low in the brain, neurons are more vulnerable to toxic or ischemic occurrences (Irmak et al., 2003; Tian et al., 2005). There is a lot of evidence that ROS are involved in membrane pathology in the brain and may play a role in neurological and neuropsychiatric disorders (Halliwell, 1992; Irmak et al., 2003; Sarsilmaz et al., 2003a; Lohr, 1991). It is known that cerebral ischemia and reperfusion (I/R) enhance the formation of ROS in brain tissue. In many studies, it has been asserted that accumulation of ROS through I/R cause neuronal damage (Choi-Kwon et al., 2004; Chan, 1996; Evans, 1993). ROS can also increase the occurrence of apoptotic cell death following cerebral I/R (Choi-Kwon et al., 2004; Chan, 2001; Fujimura et al., 2000; Sugawara et al., 2002). In the course of time, this vicious circle can increase the apoptosis, necrosis and ROS in neurons. ROS are scavenged by antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT). Superoxide anions are converted to a less harmful product, H2O2, by catalyst SOD (Irmak et al., 2003). Fish oil, found especially in herring and salmon fishes, contains n-3 essential fatty acids (EFA) including eicosapentaenoic acids (EPA) and docosahexoenoic acids (DHA). These fatty acids are important for the histological, anatomical and biochemical integrity of the brain. In addition, the dietary intake of DHA and EPA is essential because mammals are incapable of synthesizing fatty acids with a double bond past the -9 position (Sarsilmaz et al., 2003a; Songur et al., 2004; Zararsiz et al., 2006). DHA (C22:6n-3) is a major component of brain membrane phospholipids and is necessary for continuity of neuronal functions (Ahmad et al., 2002a). DHA protects neurons from apoptotic cell death, controls gene expression in the brain and maintains the structural integrity of neural membranes (Black et al., 1984; Cao et al., 2004; Choi-Kwon et al., 2004). Lose of brain DHA results in the extensive loss of sensory, behavioral, and cognitive function both in animals and humans (Ahmad et al., 2002a; Carlson et al., 1993; Gamoh et al., 1999; Greiner et al., 2001; Pawlosky et al., 1997; Wainwright et al., 1998). EPA (C20:5n-3) prevents platelet aggregation and inhibits the conversion of arachidonic acid to thromboxane A2 and prostaglandins (Black et al., 1984). DHA and EPA are nutritional antioxidants and decrease cerebral lipid peroxides (Choi-Kwon et al., 2004; Hossain et al., 1999). They have been suggested to be protective agents against neurological and neuropsychiatric disorders (Black et al., 1979, 1984). Our literature search revealed a dearth of studies on the effects of fish n-3 EFA on the ischemic brain (Black et al., 1979, 1984; Cao et al., 2004; Choi-Kwon et al., 2004). Cao et al. (2004) found that chronic DHA administration improves brain oxidative damage, reduces the degree of locomotor hyperactivity and reduces neuronal cell damage in hippocampal CA1 areas induced by transient cerebral ischemia and reperfusion in gerbils. However, to our knowledge there is no report on the number of apoptotic cell as a marker of neuron damage and antioxidative functions of n-3 EFA treatment in ischemic hippocampal formation. Hippocampal formation, located in the temporal lobe,

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is a part of the limbic system. It is composed of the entorhinal cortex, the hippocampus itself, the subicular complex and the dentate gyrus. Hippocampal formation has a role in emotional life, learning and especially short term memory and is crucial for normal brain functions. Most of neurological, neuropsychiatric disorders, such as transient ischemic attack, epilepsy, behavioral disorders, amnesia, dementia, schizophrenia, etc., are associated with structural and functional abnormalities of specific hippocampal neurons or pathway (Amaral and Witter, 1989; Eyre et al., 2003; Malberg, 2004; Matsunaga et al., 2003; Songur et al., 2003; Sarsilmaz et al., 2003a). The aim of this study was to investigate the antioxidant and neuroprotective effects of fish n-3 EFA on cerebral I/R injury in Sprague–Dawley rats’ hippocampal formation. So, we examined the effects of fish n-3 EFA on activities of SOD and CAT, levels of MDA and NO in rat hippocampal formation following I/R. In addition, we counted the apoptotic neurons by TUNEL assay in histological samples. 2. Materials and methods 2.1. Animals and keeping Our study was approved by Kocatepe University Animal Ethical Committee and was performed in accordance with the ‘‘Animal Welfare Act and the Guide for the Care and Use of Laboratory animals prepared by the Kocatepe University, Animal Ethical Committee’’. Male Sprague–Dawley rats (aged 8–12 weeks) weighing 300  20 g (mean  standard deviation) obtained from Laboratory Animal Production Unit of Suleyman Demirel University were used in the experiment. The rats were placed in a temperature (21  2 8C) and humidity (50  5%) controlled room in which a 12 h light:12 h dark cycle were maintained for 1 week before the start of experiment. A standard diet (Afyon Yem Sanayi Ltd., Afyonkarahisar, Turkey) and tap water were provided ad libitum. Rats were randomly divided into three experimental groups as follows: group I was control and non-ischemic rats (n = 6); group II was ischemic rats fed on a standard diet (n = 6); group III was ischemic rats fed on the standard diet plus fish oil including fish n-3 EFA (Marincap1, 0.4 g/kg/day, by gavage) for 14 days (n = 6). The fatty acid composition of Marincap1 capsule (500 mg) is formed by EPA (18%) and DHA (12%). In the control and ischemic groups, saline was given in the same way for 14 days.

2.2. Cerebral ischemia model For Groups II and III rats, bilateral femoral vein and artery were canalized with cannula (no: 24) (Datex/Ohmeda S/5, Helsinki, Finland) by inguinal incision. The arterial pressure and arterial pulse of the rats were monitored and measured with cannula. Glycerol trinitrate (PerlinganitTM, Adeka, Turkey) (IV) infusion was also initiated via femoral veins. Cerebral ischemia was produced by occlusion both of the common carotid artery combined with hypotension for 45 min, followed by reperfusion for 30 min according to a modified method described by Ali et al. (2004). In brief, Groups II and III rats were anesthetized with ketamine–xylazine combination (50 and 6 mg/kg, respectively). After a median incision of the neck skin, the right and left common carotid arteries (CCA) were exposed whilst leaving the vagus nerve intact. Bilateral CCA were clamped with aneurysm clips (Sugita, temporary aneurysm clip, Mizuho, Japan) for 45 min. Following surgery, the clips were removed and anesthesia was discontinued for 30 min. The control rats underwent a surgical procedure similar to the other groups but the arteries were not occluded. Biopac MP150 device was used for monitoring. During ischemia, tension arterial (TA) was held at about 50 mmHg (mean) through perfusion of glycerol trinitrate solution (100 mg/kg/min) through the femoral vein. During ischemia period, the mean arterial pulse was 385 min 1 and body temperature was maintained with a heating pad at 36.5–37.5 8C as monitored via a rectal probe.

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At the end of the procedure, the rats were administered with anesthetics containing ketamine hydrochloride (Ketalar, Eczacibasi, Istanbul, Turkey, 50 mg/kg/im) and sacrificed. Their brains were removed immediately. The left hippocampus and dentate gyrus were dissected and stored at 80 8C until biochemical analysis. The right hemispheres were blocked for histopathological analysis.

2.3. Biochemical analysis For biochemical analysis, hippocampuses were weighed and were homogenized (Ultra Turrax T25, Germany) in a five volumes of ice cold Tris–HCl buffer (50 mM/L, pH 7.4) for 2 min at 13,000 rpm. NO and MDA measurements were carried out at that stage of homogenization. The homogenate was then centrifuged at 5000  g for 30 min to remove debris. For a further extraction procedure, the supernatant was extracted with ethanol/chloroform mixture (5/3, v/v). After a second centrifugation at 5000  g for 20 min, the clear upper layer (the ethanol phase) was taken and used in SOD activity determination. All procedures were performed at +4 8C. Protein measurements of the hippocampal formation were made at all stages according to Lowry’s method (Lowry et al., 1951).

2.3.1. Determination of malonedialdehyde Measurement of MDA levels was based on the coupling of MDA with thiobarbituric acid at +95 8C (Wasowicz et al., 1993). Results were expressed as nanomole per gram wet tissue (nmol/g wet tissue). 2.3.2. Determination of nitric oxide NO measurement is very difficult in biological specimens, because it is easily oxidized to nitrite (NO2 ) and subsequently to nitrate (NO3 ) which serve as index parameters of NO production (Sahin et al., 2002). The method for determination of hippocampal nitrite and nitrate levels was based on the Griess reaction (Cortas and Wakid, 1990). Samples were initially deproteinized with Somogyi reagent. Total nitrite (NO2 + NO3 ) was measured by spectrophotometry (Shimadzu, UV-Pharmaspec 1700, Japan) at 545 nm after the conversion of NO2 to NO3 by copperized cadmium (Cd) granules. Results were expressed as micromole per gram tissue protein (mmol/g protein). 2.3.3. Determination of SOD activity Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al. (1988), with a slight modification by Durak et al. (1993). The principle of the method is based on the inhibition of NBT reduction

Fig. 1. Terminal transferase dUTP nick end labeling (TUNEL) staining of hippocampus in control (I), ischemia (II) and ischemia plus fish n-3 fatty acids (III) groups. CA, cells of the hippocampus proper; DG, dentate gyrus. Scale bar: 200 mm.

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by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant after 1.0 ml of ethanol– chloroform mixture (5:3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. The SOD activity was expressed as units per mg tissue protein (U/mg protein).

color segmentation for quantitative color analysis (Kumrala et al., 2003). The number of apoptotic cells were calculated as an average per rat. An example of a cell counting area and typical images of damaged and undamaged neuronal cells are shown in Fig. 1.

2.3.4. Determination of CAT activity Tissue CAT (EC 1.11.1.6) activity was determined according to Aebi’s method (Aebi, 1974). The principle of the assay is based on the determination of the rate constant k (dimension: s-1) of the hydrogen peroxide decomposition. By measuring the absorbance changes per minute, the rate constant of the enzyme was determined. Results were expressed as k (rate constant) per gram of protein (k/g protein).

All biochemical and histopathological data were presented as means  standard deviations (SD). A computer program (SPSS 9.0, SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Distribution of the groups was analyzed with one sample Kolmogorov–Smirnov test. Since the number of rats in each group was 6 and showed abnormal distribution, the data were considered to be non-parametric. They were therefore analyzed using Kruskal–Wallis H-test. Differences between two groups were determined with Mann–Whitney U-test. p < 0.05 was considered statistically significant.

2.4. Immunohistochemical analysis 2.4.1. TUNEL staining The right hemispheres of the brain were fixed in 4% paraformaldehyde solution with phosphate-buffered saline (PBS). After dehydration procedures, the samples were embedded and blocked in paraffin. Five micrometer thick sagittal sections of hemispheres including the hippocampal formation were cut by a microtome and mounted on poly-L-lysine-coated glass slides. The FragELTM DNA Fragmentation Detection Kit, Calorimetric–Terminal deoxynucleotidyl transferase (TdT) Enzyme (Calbiochem, Darmstadt, Germany) was used to apoptosis in the mounted sections according to the manufacturer’s instructions. Briefly, residues of digoxigenin nucleotide were catalytically added by Proteinase K to the 3X–OH ends of double- or singlestranded DNA. The labeling product was visualized using diaminobenzidine (DAB), which yielded brown granules mainly localized to apoptotic cells. Following this, the sections were counterstained with 1% methyl green. Omission of TdT or digoxigenin-nucleotide gave completely negative results not shown. 2.4.2. Histological examination Comparisons of levels of neuronal cell death were determined from the number of morphologically intact cells and the number of deep brown-stained (TUNEL+) apoptotic cells in the hippocampal CA1, CA2, CA3, and dentate girus (DG) regions. The numbers of cells in each group were counted separately in four hippocampal areas (two areas each per portion) of the hippocampal regions per section. An area was defined as 800 mm (W)  600 mm (H), with the neuronal cell layer of the hippocampal CA1, CA2, CA3, and DG region set up parallel to the major axis. Mounted slides were examined under a light microscope (Nikon Microscope ECLIPSE E600W, Tokyo, Japan) and photographed using a digital camera (Microscope Digitale Camera DP70, Tokyo, Japan). The photographs were analyzed by image analysis system. The system used is composed of a PC, hardware and software. The images were processed by an IBM-compatible personal computer, high-resolution video monitor and image analysis software (BS200Docu Version 2.0, BAB Imaging Systems, Ankara, Turkey) camera and optical microscope. The method requires preliminary software procedures of spatial calibration (micron scale) and setting of

2.5. Statistical analysis

3. Results 3.1. Biochemical analysis The values of the biochemical analysis in three groups are summarized in Table 1. According to this table, the levels of MDA and NO, and the activities of SOD and CAT in the hippocampal formation were significantly higher in Group II rats than in Group I rats ( p values 0.002 in all groups). With the exception of CAT, similar increased of MDA and NO levels as well as SOD activity were obtained for Groups I and III rats ( p values 0.002, 0.009, 0.002 and 0.002, respectively). CAT activity was decreased in Group III compared to Group I. On the other hand, the MDA levels significantly increased and NO levels decreased in the Group III compared to Group II rats. In addition, SOD activity increased and CAT activity decreased in Group III compared to Group II rats. 3.2. Immunohistochemical analysis The results of apoptotic neurons count in the hippocampal formation are summarized in Table 2 and Fig. 1. According to the results, the number of apoptotic neurons (TUNEL+) was significantly increased in Group II rats compared to Group I in the CA1, CA2, CA3 and dentate gyrus areas of rats’ hippocampal formation. On the other hand, the number of apoptotic neurons was decreased significantly in Group III rats compared to Group II rats’ hippocampal areas.

Table 1 The levels of malonedialdehyde (MDA) and nitric oxide (NO), and activities of superoxide dismutase (SOD) and catalase (CAT) in control (I), ischemia (II) and ischemia plus fish n-3 fatty acids (III) groups in rats’ hippocampal formation Groups I (n: 6) II (n: 6) III (n: 6)

MDA (nmol/g wet tissue)

NO (mmol/g protein)

SOD (U/mg protein)

CAT (k/g protein)

47.71  4.96 72.92  6.71 103.26  7.60

6.42  0.90 9.08  0.85 7.92  0.50

0.637  0.040 0.861  0.072 2.442  0.227

0.335  0.036 0.438  0.031 0.264  0.024

0.002 0.009 0.015

0.002 0.002 0.002

0.002 0.002 0.002

p values I–II I–III II–III Data represent means  S.D.

0.002 0.002 0.002

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Table 2 The number of apoptotic cells in control (I), ischemia (II) and ischemia plus fish n-3 fatty acids (III) groups in rats’ hippocampal formation Groups I (n: 6) II (n: 6) III (n: 6) p values I–II I–III II–III

CA1

CA2

CA3

DG

0.58  0.67 13.08  4.37 5.25  2.01

0.41  0.51 13.91  3.20 6.58  1.51

0.33  0.49 8.50  3.06 4.58  1.44

0.67  0.65 24.33  4.42 12.66  10.33

0.000 0.000 0.000

0.000 0.000 0.000

0.000 0.000 0.001

0.000 0.000 0.000

Data represent means  S.D.

4. Discussion In this study, we examined the effects of fish n-3 EFA including DHA and EPA on cerebral I/R in the rats’ hippocampal formation. The results of the present study indicated that bilateral CCA occlusion for 45 min followed by reperfusion for 30 min caused a significant increase in the number of apoptotic neurons, the levels of MDA and NO and the activities of SOD and CAT in rat’s hippocampal formation. In addition, our study showed that administration of fish n-3 EFA for 14 days prior to cerebral I/R had a protective effect on the biochemical and histopathological parameters. It is suggested that the oxidative damage in membrane lipids and proteins is increased during I/R. ROS can cause cellular damage by oxidizing membrane lipids, essential cellular proteins and DNA (Cao et al., 2004; Mason et al., 2000). Our findings, mentioned above, suggest that fish n-3 EFA play a role as an antioxidant and neuroprotective agent. In our previous studies, we showed that dietary fish n-3 EFA may act on the oxidant/antioxidant parameters in hippocampus (Sarsilmaz et al., 2003a), corpus striatum (Sarsilmaz et al., 2003b), hypothalamus (Songur et al., 2004) and liver (Yilmaz et al., 2004) of rats. In our study, we observed that MDA levels were higher in Group II rats when compared to Group I rats. One possible reason for this could be the oxidation of membrane lipid by ROS, causing cell damage (Irmak et al., 2003). Previous studies have indicated that cerebral I/R induce lipid oxidation (Cao et al., 2004; Choi-Kwon et al., 2004; Irmak et al., 2003). The MDA levels significantly increased in the Group III compared to Group II rats. It is a known fact that fish oil, especially DHA, is taken up by the brain and is incorporated into the neuron membranes (Choi-Kwon et al., 2004; Sarsilmaz et al., 2003a; Songur et al., 2004). Therefore, it may be surmised that an extra polyunsaturated fatty acid (PUFA) load by fish n-3 EFA supplementation may increase lipid metabolism species especially thiobarbituric acid reactive substances (TBARS). We have observed this effect in our previous studies (Sarsilmaz et al., 2003a,b; Songur et al., 2004; Yilmaz et al., 2004), and it agrees with other previous studies (Choi-Kwon et al., 2004; Miyasaka et al., 2001; Song and Miyazawa, 2001). It is questionable whether MDA is really a reliable marker for oxidative stress (Irmak et al., 2003). On the other hand, it is possible that fish n-3 EFA may have made the brains more

susceptible to lipid peroxidation, thus inducing the antioxidant enzymes and leading to a beneficial effect (Choi-Kwon et al., 2004). The brain is generally relatively poor in antioxidant enzymes (Reiter, 1995), so an increase in antioxidant enzyme capacity of the brain following I/R is important for the primary endogenous defense against free radical-induced injury (Weisbrot-Lefkowitz et al., 1998). The levels of NO in the hippocampal formation were significantly lower in Group III than in Group II rats. NO is involved in the cascade of metabolic events that causes or contributes to the occurrence of ischemic brain damage (Irmak et al., 2003; Szabo, 1996). NO appears to be intimately involved in superoxide mediated tissue injury. The presence of free radicals potentiates systems in the cell that may lead to increased production and release of NO. The concurrent production of mitochondrial superoxide and cytoplasmic NO leads to the rapid formation of peroxynitrite. While decreasing the steady-state concentration of superoxide, peroxynitrite itself is the major toxic product leading to extensive damage (Irmak et al., 2003). The results of the present study indicate that cerebral I/R induce significant increase in peroxynitrite levels in the hippocampus. In contrast, fish n-3 EFA might reduce NO levels in ischemic events. Our previous study showed that the NO levels reduces in hippocampus of rats fed with fish n-3 EFA diet (0.4 g/kg/day) for 30 days compared to control rats (Sarsilmaz et al., 2003a). The SOD activity was significantly higher in Group III rats than in Group II rats ( p = 0.002). SOD specifically detoxifies O2 to H2O2, which is then scavenged by peroxisomal catalase. Briefly, during the I/R, the H2O2 cannot be readily scavenged because of low activities of SOD, CAT, and glutathione peroxidase (Cafe´ et al., 1993). We have surmised that the elevation of SOD activity contributed to the neuroprotective effect in Group III rats. Our findings are in accordance with previous studies where fish n-3 EFA increased the SOD activity in brains (Choi-Kwon et al., 2004), hippocampus (Sarsilmaz et al., 2003a), and corpus striatum (Sarsilmaz et al., 2003b) of rats. Previous studies have reported that SOD may directly affect mitochondrial function by initiating a free radicalmediated chain reaction and plays a role against superoxide anions (Patel et al., 1996; Choi-Kwon et al., 2004). Our study showed that the CAT activities in Group III rats were significantly decreased compared to Group II rats ( p = 0.002). CAT metabolizes peroxides including H2O2 and protects the cellular membranes from lipid peroxidation (ChoiKwon et al., 2004). In Group III rats, the high content of MDA indicates enhanced lipid peroxidation. This is may be because CAT is consumed to detoxify these peroxides. Based on the fact that the TUNEL staining is a sensitive indicator of apoptotic neurons; in our study, the number of TUNEL+ neurons (apoptotic cells) was decreased significantly in Group III rats compared to Group II rats’ hippocampal areas. Brain damage due to I/R is an evolving process. It begins during the insult and extends into the recovery period after the reperfusion interval (Irmak et al., 2003). Cerebral injury takes the form of selective neuronal necrosis or infarction, the latter with destruction of all cellular elements including neurons, glia,

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and blood vessels. When infarction occurs, the immediate area surrounding the infarct (penumbra) consists of neurons undergoing either necrosis or apoptosis. It is the penumbral area that appears most amenable to reversal of cellular injury through therapeutic intervention (Linnik et al., 1993; Irmak et al., 2003). The studies related with the beneficial effect of fish n-3 EFA on ischemic brain have significantly increased in the last 30 years. Black et al. (1979) found that the volume of brain infarction and neurological deficit in the group of rats given fish oil supplementation was less than that of the focal ischemic cats. In addition, they observed that cerebral blood flow did not fall during reperfusion and edema did not appear in the ischemic gerbils fed on menhaden fish oil for 2 months. They suggest that EPA prevents post-ischemic cerebral edema and hypoperfusion, without affecting the levels of brain diene prostaglandin and thromboxane (Black et al., 1984) and moderate dietary supplements of fish oil may be beneficial in the prophylactic treatment of ischemic cerebral vascular disease (Black et al., 1979). In addition, Ahmad et al. found that DHA deficiency caused a decrease in cell body size of neurons in hippocampus pyramidal neurons. They suggested that in younger rats neurons in the DHA deficient brain grew at a slower rate than rats on a fish n-3 EFA supplemented diet (Ahmad et al., 2002b). Lately, Cao et al. (2004) reported that ethyl DHA (200 mg/kg/day) pretreatment for 10 weeks produced beneficial effects on post-ischemic histological, behavioral, and biochemical changes induced by transient cerebral ischemia and reperfusion in gerbils. They demonstrated that chronic DHA supplementation caused inhibition of lipid peroxidation, attenuation of the depletion of GSH level, prevention of decline in the activities of GSH-PX and CAT, reduction of locomotor hyperactivity, and decrease in the hippocampal CA1 neuronal loss depending on the ischemic injury. In conclusion, the present findings suggest that I/R injury cause accumulation of oxidation products, such as MDA and NO, alterations in endogenous free radical scavengers and induction of apoptosis in rat hippocampal formation. Also fish n-3 EFA ameliorates the oxidative status and apoptotic changes in rat hippocampus following I/R. We suggest that changes in the activities of SOD and CAT are attributable, in part, to the decrease in the number of the apoptotic neurons in the Group III rats. Additional studies into the neuroprotective effects of dietary fish oil in humans are warranted. We conclude that dietary supplementation of fish n-3 EFA may be beneficial to preserve or ameliorate on ischemic cerebral vascular disease. References Aebi, H., 1974. Catalase. In: Bergmeyer, H.-U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 673–677. Ahmad, A., Moriguchi, T., Salem, N., 2002a. Decrease in neuron size in docosahexaenoic acid-deficient brain. Pediatr. Neurol. 26, 210–218. Ahmad, A., Murthy, M., Greiner, R.-S., Moriguchi, T., Salem FN. Jr., 2002b. A decrease in cell size accompanies a loss of docosahexaenoate in the rat hippocampus. Nutr. Neurosci. 5, 103–113. Ali, H., Nakano, T., Saino-Saito, S., Hozumi, Y., Katagiri, Y., Kamii, H., Sato, S., Kayama, T., Kondo, H., Goto, K., 2004. Selective translocation of

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