Neurochem Res (2014) 39:853–861 DOI 10.1007/s11064-014-1281-7

ORIGINAL PAPER

Effects of Lithium and Lamotrigine on Oxidative–Nitrosative Stress and Spatial Learning Deficit After Global Cerebral Ischemia Ayca Ozkul • Ahmet Sair • Ali Akyol • Cigdem Yenisey • Turhan Dost • Canten Tataroglu

Received: 28 August 2013 / Revised: 13 February 2014 / Accepted: 13 March 2014 / Published online: 25 March 2014 Ó Springer Science+Business Media New York 2014

Abstract Lithium (Li) and lamotrigine (LTG) have neuroprotective properties. However, the exact therapeutic mechanisms of these drugs have not been well understood. We investigated the antioxidant properties of Li (40 and 80 mg/kg/day) and LTG (20 and 40 mg/kg/day) in a rat model of global cerebral ischemia based on permanent bilateral occlusion of the common carotid arteries (BCAO). Nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), glutathione reductase (GSH-R), catalase (CAT) and superoxide dismutase (SOD) levels were measured as an indicator of oxidative–nitrosative stress in both prefrontal cortex (PFC) and hippocampus after 28 days of treatment. The spatial learning disability was also assessed at the end of the study by Morris water maze (MWM) test. All oxidative–nitrosative parameters were found to be higher in the groups under treatment than in sham. Both drugs caused a decrease in PFC NO and MDA elevation, meanwhile the increase in GSH, GSH-R, CAT and SOD levels was significantly more evident in treated groups. We also found higher PFC GSH-R and hippocampal SOD levels in BCAO ? Li (80 mg/day) treated group when compared with BCAO ? LTG 40 mg/day. MWM test data showed a similar increase in spatial learning ability in all groups under treatment. We found no other statistical difference in comparison of treated groups with different dosages. Our findings suggested that Li and LTG treatments may decrease spatial learning memory deficits accompanied by lower oxidative–nitrosative stress in global cerebral

A. Ozkul (&)  A. Sair  A. Akyol  C. Yenisey  T. Dost  C. Tataroglu Faculty of Medicine, Adnan Menderes University, Aydın, Turkey e-mail: [email protected]

ischemia. Both drugs may have potential benefits for the treatment of vascular dementia in clinical practice. Keywords Oxidative stress  Cerebral ischemia  Neuroprotection  Dementia

Introduction Lithium (Li) and lamotrigine (LTG) are widely used in the treatment of mood disorders [1].They may have neuroprotective properties in other neurological disorders, especially neurodegenerative diseases. Recently Li has been shown to improve behavioral and cognitive deficits in animal models of neurodegenerative diseases, including stroke, amyotrophic lateral sclerosis, fragile X syndrome, and Huntington’s, Alzheimer’s, and Parkinson’s diseases [2]. In rats exposed to focal ischemia, chronic Li treatment has a significant neuroprotective role by reducing apoptotic death, brain infarct volume and hemiplegia [3]. Additionally Li inhibits lipid peroxidation and protein oxidation, suggesting that it has neuroprotective effects against oxidative–nitrosative stress [4, 5]. Likewise, LTG, a phenyltriazine derivative blocks voltage-sensitive sodium channels and inhibits the release of excitatory amino acids [6]. It is commonly used as an antiepileptic drug as well as a mood stabilizer. It is also used in migraine, attention deficiency hyperactivity disorder, neuropathic pain treatments [7]. In animal models of cerebral ischemia LTG administration improves neurological outcome and has therefore been used in numerous studies of acute ischemia [5, 6]. Although the neuroprotective effects of both drugs have been shown in acute ischemia [3, 6, 8], there is still insufficient data in chronic ischemic conditions. Moreover the effects of antioxidant Li and LTG on functional or

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behavioral alterations in global cerebral ischemia are still undetermined. They may have an expanded use in treating other neurological diseases like vascular dementia caused by chronic global cerebral hypoperfusion due to low output cardiac failure, bilateral carotid artery occlusion/stenosis and vasculopathy. However such treatment choices are not commonly used in clinical practice because of limited data. In the present study, a rat model of global cerebral ischemia was used, based on the permanent bilateral occlusion of the common carotid arteries (BCAO) leading to chronic cerebral hypoperfusion. Our aim was to assess the effects of chronic Li and LTG treatments at different two dosages on deteriorated spatial learning functions and nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), glutathione reductase (GSH-R), catalase (CAT) and superoxide dismutase (SOD) levels in ischemic prefrontal cortex (PFC) and hippocampal tissue caused by BCAO.

Material and Method The animals, Wistar female rats aged 6 months [188–260 g (230 ± 24.9 g)], were housed in groups of five per cage at a temperature of 23 ± 1 °C with a 12 h light–dark cycle (light on 7 a.m.–7 p.m.), and had free access to food and water. All experiments were performed in accordance with the guidelines for care and local ethical regulations established by the National Institutes of Health. Experimental Design Bilateral Common Carotid Occlusion (BCAO) Bilateral common carotid arteries were occluded under ketamine hydrochloride (80 mg/kg, i.p.) and xylazine (12 mg/kg, i.p.) anaesthetics. After the rats were anesthetized, the bilateral common carotid arteries were exposed and carefully separated from the carotid sheath and the cervical sympathetic and vagal nerves through a ventral cervical incision. The bilateral common carotid arteries were ligated with 4-0 type surgical silk in ischemia rats, but not ligated in sham-operated rats (group 1). The operation was performed on a heating pad to maintain body temperature at 37.5 ± 0.5 °C and the animal was kept on the pad until recovery from anesthesia. Starting the day after BCAO, the rats were treated daily with either Li [40 mg/kg ip (group 3) and 80 mg/kg ip (group 4) dissolved in saline] or LTG [20 mg/kg ip (group 5) and 40 mg/kg ip (group 6) dissolved in saline] or physiological saline (group-2). The injections were continued for 4 weeks. Each of the six groups [sham, BCAO, BCAO with Li treatment (40 and 80 mg/kg), and BCAO with LTG treatment (20 and 40 mg/ kg)] consisted of 9 rats with identical mean body weights.

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Morris Water Maze Test The Morris water maze (MWM) test was performed as previously described by Morris [9]. It is one of the most widely used tasks in behavioral neuroscience for studying the psychological processes and neural mechanisms of spatial learning and memory. Place navigation in the water maze is often used as a general assay of cognitive function. MWM test was started on the 23rd day of the study and continued for five consequent days. Li and LTG treatments continued till the end of 4 weeks. We used a circular water tank 180 cm in diameter and 70 cm in height, filled to a depth of 40 cm with water at 21 ± 1 °C to cover a black platform (10 cm in diameter). Distinctive, geometric, extramaze cues were affixed at specific locations surrounded the tank at a height of 120–150 cm to facilitate orientation, and were visible to the rats while in the maze. Maze performance was recorded by a video camera suspended above the maze. The tank was divided into four quadrants called north, east, south and west at equal distances on the rim. The platform was located in the center of the northeast quadrant during training. The top of the platform was approximately 1.5 cm below the surface of water. The rat was placed in the water facing the wall at one random start location of four (north, south, east and west, locating at equal distances from each other on the pool rim). The rats were given one trial per day to find the hidden platform on five consecutive days (maximum trial duration 60, 20 s reinforcement on the platform). The experimenter conducting the MWM was blinded to the treatment groups. The rats were gently placed into the water facing the side walls of the maze at one of the four starting positions. For the training trial, the latency to escape onto the hidden platform was recorded. A maximum of 60 s was given to each rat to find the hidden platform. If the rat failed to find the platform within 60 s, the training was terminated and a maximum score of 60 s was assigned. The rat was then guided to the hidden platform by hand, and it was allowed to stay on the platform for 20 s. After the last learning trial on training day 5, a probe test, in which the hidden platform was removed, was conducted in the next day. The rats were allowed to swim freely in the pool for 60 s and a percentage of the time in the target quadrant was recorded as an assessment of spatial memory. Sample Preparation and Biochemical Evaluations After completing the behavioral tests, at the end of 4 weeks of treatment the same rats were sacrificed under anaesthesia with ketamine hydrochloride (80 mg/kg, i.p.) and xylazine (12 mg/kg, i.p). The brain was quickly dissected out, weighed and stored over ice. Brains were removed from the skull and placed in a stainless steel brain

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microtome. Then they were sectioned coronally with razor blades for the dissection of PFC at 2.7 mm from bregma. The hippocampal tissue manually dissected out with guidance from the rat brain atlas of Paxinos and Watson [10]. The brains were further processed within half an hour of dissection and the oxidative–nitrosative stress markers were studied in the same working day. Tissue Preparation and Determination of Oxidative– Nitrosative Stress Parameters Tissues were homogenized in 50 mM phosphate buffer (pN 7.4) containing protease inhibitor, 0.2 lM PMSF and 1 mM EDTA, at 4 °C using a Polytron homogenizer (B. BRAUN, Germany). The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C. The resultant supernatant was used for measurement for free radicals determination. NO Measurement NO (nitrite ? nitrate) was assayed by a modification of cadmium-reduction method as mentioned by Navarro-Gonzalves et al. [11]. The nitrite produced was determined by diazotization of sulphanilamide and coupling to naphthlethylene diamine. For the measurement of NO (nitrite ? nitrate), 400 ll sample was denatured by adding 80 ll 30 % ZnSO4 solution, stirring and then centrifuging at 10,0009g for 20 min at 4 °C. First, we activated Cd granules using CuSO4 solution in glycine-NaOH buffer. Then 100 ll of deproteinized samples and standards were added. This reaction using pre-treatment of samples to reduce nitrate to nitrite, which can be accomplished by catalytic reactions using enzyme or Cd. The samples were analyzed spectrophotometrically using a microplate reader and quantified automatically against KNO3 standard curve and the results were expressed as lmol/g.

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of NADPH at 340 nm at 37 °C. The reaction was initiated by the addition of 50 ll sample to 1 ml of assay mixture containing 50 mmol/l Tris, pH 7.6, 100 lmol/l ETDA, 4 mmol/l GSSG, 120 lmol/l NADPH. A blank cuvette was prepared in which the sample was replaced by buffer. The reaction was record 2–3 min. The result were given as U/g. Catalase (CAT) Measurement CAT activity measurement in erytrocyte lysate was measured by the method of Aebi [15]. The reaction mixture was 50 mM phosphate buffer(pH 7.0), 10 mM H202 and erytrocyte lysate. The reduction rate of H202 was follewed at 240 nm for 30 sn at room temperature. Catalase activity was expressed in U/g wet tissue. Cu,Zn-SOD Determination We add 2.45 ml of SOD assay reagent to each 40 tubes, then 0.5 ml of pure Cu,ZnSOD (0–270 ng), or samples were added to tubes. The final volume of the reaction system is 3.0 ml and it contains, per liter, 0.1 mmol of EDTA,i 50 mg of BSA, 25 lmol of NBT, 9.9 nmol of XOD, and 40 mmol of Na2CO3 (pH 10.2). Placed the rack of 40 tubes into a water bath adjusted to 25 °C. Then we added 50 ll of XOD solution to each tube at 30-s intervals. Then each tube was incubated 20 min. The reaction was terminated bu adding CuCl2. In this way, 40 tube assay can be done within 40 min. The production of formazan was determined at 560 nm using Shimadzu UV160 spectrophotometer. Under these conditions, the absorbance at 560 nm of the blank tube is about 0.25. The percent inhibition is calculated as below. % inhibition = (Ablank - Asample)/Ablank 9 100. We calculated Cu,Zn-SOD activity via standart curve [16]. Statistical Analyses

MDA Measurement The MDA production and hence lipid peroxidation were assessed in the tissues by the method of Ohkawa et al. [12]. MDA forms a colored complex in the presence of TBA, which is detectable by measurement of absorbance at 532 nm. Absorbance was measured with Shimadzu UV-160 spectrophotometer. 1,10 ,3,30 -Tetraethoxypropane was used as a standard and the results were expressed as nM/g wet tissue. Glutathione (GSH) Measurement Total glutathione content in the samples were measured according to the method of Beutler et al. [13], using metaphosphoric acid to precipitate the protein and 5,50 dithiobis (2-nitrobenzoic acid) for colour development. The standart curve was used to calculate GSH content. Glutathione Reductase (GSH-R) Measurement We used the method described by Racker E for GSH-R measurement [14]. GSH-R was assayed by following the oxidation

All statistical analysis was performed by using SPSS for Windows statistical software (SPSS Inc, version 14.0, Chicago, Illinois). The results were evaluated using Kruskal–Wallis test for comparison of multiple groups. Then for the parameters having statistical difference we compare each group with each other by using Mann– Whitney U test. Differences were considered significant when p \ 0.05. Nonparametric tests were used since normal distrubution couldn’t be detected in studied parameters. All values were expressed as mean ± standard deviation.

Results Body weights at baseline and 4 weeks after BCAO revealed no significant differences between the six study groups. Biochemically studied parameters (NO, CAT,

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Table 1 The concentrations of oxidative stress parameters in the hippocampal and PFC tissues of the groups Sham (group-1)

BCAO (group-2)

BCAO ? Li 40 mg/kg (group-3)

BCAO ? Li 80 mg/kg (group-4)

BCAO ? LTG 20 mg/kg (group-5)

BCAO ? LTG 40 mg/kg (group-6)

p

NO (lmol/g) PFC Hippocampus MDA (nM/g)

0.059 ± 0.047

0.51 ± 0.44

0.27 ± 0.25

0.22 ± 0.13

0.203 ± 0.062

0.22 ± 0.086

0.03

0.212 ± 0.18

0.197 ± 0.23

0.15 ± 0.13

0.15 ± 0.08

0.207 ± 0.027

0.26 ± 0.03

NS

PFC

19.51 ± 3.90

40.78 ± 11.16

27.70 ± 4.59

30.53 ± 7.15

24.53 ± 3.68

28.03 ± 4.11

0.003

Hippocampus

20.92 ± 2.09

39.75 ± 4.28

33.15 ± 3.44

37.45 ± 4.75

29.28 ± 4.88

27.40 ± 2.12

0.000

GSH (mg/g) PFC

0.91 ± 0.343

0.60 ± 0.064

1.25 ± 0.30

1.45 ± 0.74

1.37 ± 0.37

1.41 ± 0.29

0.002

1.835 ± 0.650

1.55 ± 0.51

1.84 ± 0.64

2.13 ± 1.05

1.83 ± 0.5

1.49 ± 0.30

NS

PFC

124.85 ± 51.13

185.93 ± 99.73

276.76 ± 43.25

310.25 ± 32.08

220.32 ± 46.12

225.93 ± 46.12

0.002

Hippocampus

211.65 ± 43.90

215.4 ± 29.51

242.73 ± 35.98

249.96 ± 24.07

251.042 ± 27.55

243.29 ± 33.35

NS

PFC

6.73 ± 1.68

8.39 ± 3.86

15.08 ± 7.37

18.08 ± 9.1

9.86 ± 1.82

11.29 ± 1.9

0.008

Hippocampus

4.19 ± 1.86

5.02 ± 1.63

10.60 ± 7.36

10.25 ± 4.41

10.69 ± 1.68

13.45 ± 5.69

0.001

Hippocampus GSH-R (mU/g)

CAT (ng/g)

SOD (U/g) PFC

22.75 ± 4.08

27.06 ± 5.91

31.97 ± 4.25

33.14 ± 9.27

30.01 ± 3.95

31.71 ± 3.26

0.026

Hippocampus

26.70 ± 6.35

31.56 ± 4.51

36.03 ± 2.14

38.1 ± 1.84

36.11 ± 3.47

34.23 ± 3.04

0.003

Values are given as mean ± SD BCAO bilateral carotid artery occlusion, NS nonsignificant

Fig. 1 Comparison of NO (lmol/g) (*p \ 0.005, **p \ 0.01, ***p \ 0.05 vs sham) (a) and MDA (nM/g) (*p \ 0.005, **p B 0.01, ***p \ 0.05 vs sham; # p \ 0.005, ##p B 0.01, ### p \ 0.05 vs BCAO) (b) levels in PFC and hippocampal tissues of the studied groups

SOD, MDA, GSH and GSH-R) were evaluated in hippocampal and PFC tissues. NO and MDA Levels in Rat Brain NO as a nitrosative marker showed significant differences between groups in PFC, but not in hippocampal tissue. In PFC tissue NO levels (lmol/g) of BCAO, Li and LTG treated groups were higher than the sham operated group

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(Table 1; Fig. 1a). Although this elevation was more evident in BCAO, there was no statistical difference when compared with groups under treatment. MDA (nM/g) levels were also increased along with the cortical diffuse ischemia in both PFC and hippocampal tissue. In the groups receiving Li and LTG therapy this increase was less prominent but still higher than sham operated group (Table 1; Fig. 1b). Administration of Li and LTG was found to decrease the elevation of MDA but

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Fig. 2 The figure showing the differences of GSH (mg/g) (*p \ 0.005, **p B 0.02 vs sham; #p \ 0.005 vs BCAO) (a); GSH-R (mU/g) (*p \ 0.005, **p \ 0.02 vs sham; #p \ 0.02 vs BCAO; d p \ 0.01 vs BCAO ? Li 80 mg/kg) (b) in hippocampal and PFC tissues between groups

not NO. However, there was no significant difference between groups treated with two different drugs at different dosages. Endogen Protective Antioxidants; CAT, SOD, GSH and GSH-R GSH (mg/g), which is an important substrate in cell protection against oxidative stress, was found to be lower in the PFC tissues of the BCAO group when compared with the sham operated group. We found significantly increased levels of PFC GSH in treated groups without any effect of dosage when compared with the BCAO and sham operated groups (Table 1; Fig. 2a). However similar significant differences couldn’t be detected in hippocampal tissue. Likewise, there was no significant difference in hippocampal GSH-R levels between groups. In PFC, GSH-R (mU/g) was found elevated in groups under treatment when compared with sham operated rats. However there was no effect of BCAO on PFC GSH-R except group 4. In comparison with the BCAO group, the only marked elevation of GSH-R could be found in PFC tissue of group 4 (BCAO ? Li 80 mg/kg). (Table 1; Fig. 2b). CAT and SOD have antioxidant properties and work against reactive oxygen species. Both hippocampal and PFC CAT (ng/g) and SOD (U/g) levels were also found increased in the groups receiving Li and LTG therapy when compared with the sham operated rats (Table 1; Fig. 3a, b). When we compared the treated groups with the BCAO hippocampal CAT levels of all treated groups except group 3 (BCAO ? Li 40 mg/kg) were found statistically elevated. In analyses of SOD levels we found elevation in all treated groups in comparison with the sham operated group. PFC SOD level detected in group 5 (BCAO ? LTG 20 mg/kg) was higher than sham but this didn’t show any statistical difference.

When we compare treated groups with the BCAO group, the only significant difference was found in elevated hippocampal SOD levels of group 4 (BCAO ? Li 80 mg/kg). Although hippocampal SOD levels of other treated groups seemed to be higher than BCAO, this elevation was not statistically significant. The only significant difference between two drugs could be detected in PFC GSH-R and hippocampal SOD levels. In PFC GSH-R level of group 4 (BCAO ? Li 80 mg/day) was higher than group 6 (BCAO ? LTG 40 mg/day) (Fig. 2b). Similar difference was also found in hippocampal SOD levels (Fig. 3b). In hippocampal tissue SOD level of group 4 (BCAO ? Li 80 mg/day) was higher when compared with group 6 (BCAO ? LTG 40 mg/day). Treatment with high dosage of Li caused elevated endogen antioxidant response when compared with high dosage of LTG in terms of PFC GSH-R and hippocampal SOD. However the other studied parameters did not differ between groups under treatment. MWM Test Results Apart from the biochemical aspect, MWM test data showed an increase in learning ability in groups receiving treatment (Table 2). The time spent by the rats to find the hidden platform on the last day of training differed between groups respectively as follows: 19.22 ± 8.24, 46.77 ± 18.11, 31.55 ± 6.61, 29.44 ± 6.5, 26.11 ± 3.21, 28.22 ± 2.77 s (Fig. 4). When the hidden platform was removed, we assessed the time spent searching for the platform in the same quadrant of the pool. The group results in seconds were as follows: 30.33 ± 4.87, 12.22 ± 3.73, 17.11 ± 3.21, 16.77 ± 3.5, 19.66 ± 3.5, 20 ± 2.8 (Fig. 5). All of the treated groups showed better performance when compared with group 2 (BCAO). However the groups under Li and LTG treatment showed similar performance in MWM test.

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Fig. 3 The figure showing the differences of CAT (U/g) (*p \ 0.005, **p B 0.01, ***p \ 0.05 vs sham; # p \ 0.005, ##p B 0.01 vs BCAO) (a) and SOD levels (ng/ g) (*p \ 0.005,**p \ 0.01, ***p \ 0.05 vs sham; #p \ 0.01 vs BCAO; dp = 0.01 vs BCAO ? Li 80 mg/kg) (b) in hippocampal and PFC tissues between groups

Table 2 Time parameters from MWM test including time to locate the hidden platform on the fifth day of training and MWM post training probe test Sham (group-1)

BCAO (group-2)

BCAO ? Li 40 mg/kg (group-3)

BCAO ? Li 80 mg/kg (group-4)

BCAO ? LTG 20 mg/kg (group-5)

BCAO ? LTG 40 mg/kg (group-6)

p

MWM test results of the fifth day (s)

19.22 ± 8.24

46.77 ± 18.11

31.55 ± 6.61

29.44 ± 6.5

26.11 ± 3.21

28.22 ± 2.77

0.004

MWM post training probe test (s)

30.33 ± 4.87

12.22 ± 3.73

17.11 ± 3.21

16.77 ± 3.5;

19.66 ± 3.53

20 ± 2.8

0.00

The values are given as mean ± SD BCAO bilateral carotid artery occlusion

Fig. 4 The figure showing MWM test results. At the end of five training days time to locate hidden platform was found reduced in treated groups when compared with BCAO group (*p \ 0.05 vs sham, #p \ 0.05, ##p \ 0.00 vs BCAO)

Discussion In this study, the rat model of global cerebral ischemia due to BCAO was used to assess oxidative–nitrosative stress in brain tissue. We also used lower and higher dosages of both Li and LTG treatments and evaluated their effects on oxidative–nitrosative stress in PFC and hippocampus. PFC NO and both PFC and hippocampal MDA were increased in rats with BCAO. This increase was seen to be less

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Fig. 5 In MWM post training probe test groups having Li or LTG treatments spent longer time in target quadrant when compared with BCAO group (*p \ 0.00 vs sham, #p \ 0.05, ##p \ 0.00 vs BCAO)

prominent in treated groups. Additionally the imbalance between the rate of free radical production and the effect of protective antioxidants can lead to oxidative damage. Therefore increased endogenous antioxidant capacity via GSH, GSH-R, SOD and CAT, and a smaller increase in

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MDA and PFC NO levels in groups under treatment suggest that these two drugs may also reduce oxidative– nitrosative stress by enhancing reactive antioxidative defence system in global cerebral ischemia. However, there was no significant difference between two drugs at two different dosages. NO and highly toxic free radical formations as a consequence of glutamate receptor overstimulation are related to neuronal membrane damage by lipid peroxidation [17, 18]. Although NO seems to be a nontoxic mediator of cerebral vasodilatation, it has neurotoxic effects at high concentrations [17]. In a previous study it has been also shown that shear stress induced by BCAO, upregulates endothelial NO synthase (eNOS) but not neuronal nitric oxide synthase (nNOS). Hyperactivated vascular NO appears in the first 2 weeks as a reaction to maintain homeostasis of local cerebral blood flow. However vascular NO level decreases in long term of ischemia and this may have triggered metabolic changes within hippocampal cells resulting in hippocampal dysfunction as reflected by spatial memory impairment [17]. Free radical damage is assumed to be initiated by increased production of superoxides and their by-products, and has been shown to be involved in membrane damage in cerebral hypoxia and ischemia. Although both drugs have antioxidant properties in ischemic conditions, there are limited data in literature to explain the mechanism. In a recent study, LTG, which blocks ischemia-induced glutamate release, significantly decreased the lipid peroxidation products in cerebral ischemia [18]. The neuroprotective role of LTG has been shown in terms of MDA as a lipid peroxidation product in rats with permanent cerebral ischemia in this study. Additionally LTG improves the neurological outcome in animal models of global and focal cerebral ischemia, and it has been used in numerous studies of acute phase of ischemic stroke models [8, 18]. Recent data suggest that the possible neuroprotective action of LTG is realized through its interactions with processes of NO production and inhibition of lipid peroxidation [19]. In literature it has been shown that LTG has neuroprotective effects in acute cerebral ischemia [5]. The mechanism by which LTG prevents brain damage may be largely attributed to its ability to inhibit the release of glutamate by blocking presynaptic sodium channels [6]. It has been also shown that LTG treatment before and simultaneously with spinal cord injury increased the level of protective antioxidants SOD and GSH-peroxidase [6]. Likewise in intestinal ischemia rat model glutathione peroxidase and SOD levels were found to be higher in LTG treated rats [19]. Chronic treatment with Li also inhibits lipid peroxidation and protein oxidation in primary cultured rat cerebral

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cortical cells [4, 5]. Increased expression of GRP78 and bcl-2, which have been found to stabilize mitochondrial function and suppress oxyradicals, implies that Li may inhibit ROS overproduction by stabilizing mitochondrial function [20]. There are several mechanisms to explain the neuroprotective effects of Li through oxidative stress. In the literature it has been found that treatment with Li significantly increases both GSH and the protein levels of the glutamate-cysteine ligase (GCL) catalytic subunit [5]. Jornada et al. [21] have also shown that Li treatment increases CAT activity in prefrontal and hippocampus of rats in animal model of mania. In addition to this Li treatment causes an increase in both GSH levels and glutathione S-transferase (GST) activity, indicating that Li may accelerate the process of GST-catalyzed GSH conjugation in order to limit oxidative damage [22]. These findings together suggest that Li and LTG may produce neuroprotective effects against oxidative stress. However the exact mechanism is not clear yet. In our study PFC NO and both hippocampal and PFC MDA increase in rats with BCAO was seen to be less prominent under Li and LTG treatment. In addition to this we found increased levels of protective endogen antioxidants in groups under both treatments. This may be due to antioxidant effects of both drugs which have been shown in several studies before [4, 5, 18– 20]. All these studies are about acute ischemic conditions. However our data suggest that their antioxidant effects can be also beneficial in chronic global cerebral ischemia which hasn’t been studied before. Although parameters having antioxidant properties seemed to be higher in groups under Li treatment, statistical difference between two drugs could be detected only in PFC GSH-R and hippocampal SOD but not in other studied parameters. PFC GSH-R and hippocampal SOD levels were found elevated in group 4 which was under Li (80 mg/day) treatment when compared with group 6 (BCAO ? LTG 40 mg/day). In spite of the fact that parameters belong to antioxidative defence system seemed to be higher in Li treated groups when compared with groups under LTG treatment, it is difficult to make a comment on which therapy and dosage may be more effective in reducing oxidative stress since statistical difference could be detected only in PFC GSH-R and hippocampal SOD. Therefore our data only suggest that elevation in SOD and GSH-R may be more evident with high dosage of Li treatment when compared with high dosage of LTG treatment in global cerebral ischemia. BCAO may cause oxidative–nitrosative stress not only in PFC but also hippocampus. In our study hippocampal NO, GSH and GSH-R levels showed no difference between groups, this may be due to BCAO causing more evident

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ischemia in cortical regions than in the hippocampal area in rats. Although we found more severe changes in oxidative– nitrosative stress markers in PFC tissue when compared with hippocampus, both regions were affected. This seems enough to cause spatial learning deficits since experimental and clinical evidence indicates that a fronto-hippocampal system may provide an integrated neurological basis for spatial representational ability [23]. We found that Li and LTG treatments enhance spatial learning ability which is impaired by global cerebral ischemia. Both drugs have clearly beneficial effects by causing less oxidative–nitrosative stress and cognitive deterioration. Agarwal et al. studied the effects of LTG, oxcarbazepine and topiramate treatments on cognitive functions and oxidative status in PTZ-kindled mice and found significant alterations in oxidative stress parameters in the topiramate treated group but not in group under LTG treatment. Additionally, long term administration of topiramate caused cognitive impairment during experimental epilepsy, while LTG had no negative effects on it [24]. It has been also shown that LTG treatment significantly protects against the cognitive deficits associated with 15 min of cerebral ischemia [25]. It also attenuates the impairment of neurobehavioral outcome associated with 6.5 min of cerebral ischemia as measured by acquisition of the conditioned eyeblink response [26]. However, all these studies were associated with short term cerebral ischemia. In the present study we found that LTG treatment at two different dosages attenuated spatial learning disability in global cerebral ischemia of 28 days duration. In another study it has been shown that Li pretreatment protected gerbils from global ischemia-induced open field hyperactivity and learning/memory impairment [27]. Moreover Li treatment decreased behavioral and functional deficits by reducing cell death in the hippocampus in transient global cerebral ischemia model. Data suggested that Li may improve learning and memory ability by reducing apoptotic death in the hippocampal CA1 region [26]. In the present study, chronic Li and LTG treatments attenuated the spatial learning disability associated with chronic global cerebral ischemia. We also used two different dosages but failed to show any differences in terms of MWM test results. Additionally pretreatment of both Li and LTG may also have effects on oxidative–nitrosative stress and spatial learning in global cerebral ischemia which may cause interest for further studies. Our data suggested that the possibility that reducing oxidative–nitrosative damage may be one of the mechanisms of Li and LTG in the rat model of vascular dementia. Furthermore, it is the first time that the antioxidative effects of chronic Li and LTG treatment on spatial learning disability in global cerebral ischemia as a chronic ischemic condition have been investigated.

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Conclusion In conclusion, we found that Li and LTG treatments decreased spatial learning memory deficits in global cerebral ischemia without any significant effect of dosage. Our data also suggest that both drugs may reduce oxidative– nitrosative stress and enhance protective antioxidants. Chronic Li and LTG treatments may cause an increase in levels of the antioxidants GSH, GSH-R, SOD and CAT. NO and MDA levels were also increased in global cerebral ischemia, but this response was less prominent in all treated groups. To our knowledge this is the first study investigating the effects of chronic Li and LTG treatment on oxidative–nitrosative stress and cognitive deterioration in global cerebral ischemia. Our data suggested that both drugs may reduce spatial learning memory deficits regardless of dosage. We believe that both Li and LTG may have potential benefits for the treatment of vascular dementia in clinical practice in the future.

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Effects of lithium and lamotrigine on oxidative-nitrosative stress and spatial learning deficit after global cerebral ischemia.

Lithium (Li) and lamotrigine (LTG) have neuroprotective properties. However, the exact therapeutic mechanisms of these drugs have not been well unders...
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