Modulation of nitrergic pathway by sesamol prevents cognitive deficits and associated biochemical alterations in intracerebroventricular streptozotocin administered rats

European Journal of Pharmacology 659 (2011) 177–186

Contents lists available at ScienceDirect

European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Behavioural Pharmacology

Modulation of nitrergic pathway by sesamol prevents cognitive deficits and associated biochemical alterations in intracerebroventricular streptozotocin administered rats Shubham Misra, Vinod Tiwari, Anurag Kuhad, Kanwaljit Chopra ⁎ Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Center of Advanced Study, Panjab University, Chandigarh-160014, India

a r t i c l e

i n f o

Article history: Received 22 September 2010 Received in revised form 25 February 2011 Accepted 21 March 2011 Available online 2 April 2011 Keywords: Alzheimer's disease Intracerebroventricular streptozotocin L-arginine L-NAME (N(G)-nitro-L-arginine methyl ester) Sesamol TNF-α (tumor necrosis factor-alpha)

a b s t r a c t Alzheimer's disease is a neurodegenerative disorder characterized by progressive cognitive decline and widespread loss of neurons and their synapses in the cerebral cortex and hippocampus. Increasing evidence indicates that factors such as oxidative–nitrergic stress, glutathione depletion, impaired protein metabolism and cholinergic deficit can interact in a vicious cycle, which is central to Alzheimer's disease pathogenesis. Intracerebroventricular (i.c.v.) streptozotocin induced-cognitive impairment has been widely used as an experimental paradigm to study Alzheimer's disease. In the present study, i.c.v. streptozotocin produced significant cognitive deficits as measured in Morris water maze and elevated plus maze task coupled with increased serum TNF-α levels and marked rise in brain acetylcholinesterase and oxidative–nitrergic stress in female Wistar rats. Sesamol (5-hydroxy-1,3-benzodioxole or 3,4-methylenedioxyphenol), a potent antioxidant and anti-inflammatory molecule markedly improved cognitive impairment, reduced acetylcholinesterase activity, TNF-α levels and attenuated oxidative–nitrergic stress in brain of i.c.v.-streptozotocin treated rats. Administration of L-arginine (125 mg/kg i.p), a nitric oxide donor, alone to i.c.v.-streptozotocin treated rats accentuated behavioral and biochemical deficits and also abolished the protective effect of sesamol (8 mg/kg). L-NAME (10 mg/kg i.p.), a non-specific NOS inhibitor significantly restored all the behavioral and biochemical indices in i.c.v.-streptozotocin rats. Moreover, combination of L-NAME with subeffective dose of sesamol (4 mg/kg) potentiated its protective effect. Our findings demonstrate the effectiveness of sesamol in preventing intracerebroventricular streptozotocin-induced cognitive deficits by modulating nitrergic signaling and oxido-inflammatory cascade. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Alzheimer's disease is the most common type of dementia in Western societies and has profound economic and social impact. It accounts for 50% of dementia cases all around the globe (Areosa and Sherriff, 2006). Alzheimer's disease is characterized by marked atrophy of cerebral neocortex, hippocampus and loss of cortical and sub-cortical neurons (Vance et al., 2005). Neuroscientists all around the world are trying their best to develop a sure-shot remedy for Alzheimer's disease and related dementia. Current strategies are mainly focused on two aspects; one is to develop agents, which improve cognitive deficits of Alzheimer's disease and second to find appropriate experimental models for screening of such agents (Vance et al., 2005). The intracerebroventricular streptozotocin (i.c.v. streptozotocin) injected rat has been described as an appropriate animal model for sporadic dementia characterized by progressive deteriora-

⁎ Corresponding author. Tel.: + 91 172 2534105; fax: + 91 172 2541142. E-mail address: [email protected] (K. Chopra). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.03.026

tion of memory and cerebral glucose and energy metabolism, along with oxidative stress (Lannert and Hoyer, 1998; Sharma and Gupta, 2001a; Sonkusare et al., 2005). Nitric oxide mediated nitrergic signaling, an important bioregulatory signaling in the nervous, immune and cardiovascular system, plays a pivotal role in brain homeostasis. The involvement of nitric oxide in a number of neurological disorders is well established. Researchers have begun to recognize and to explore the putative link between nitric oxide and Alzheimer's disease (Dorheim et al., 1994; Norris et al., 1996). Although existing evidence regarding their association is not abundant, emerging data are showing Alzheimer's disease-related changes in the nitric oxide synthase system, and it appears that nitric oxide could be related to many of the pathological mechanisms of the disease. The ability of nitric oxide to exert cellular damage due to its reactive oxidative properties is perhaps the primary neurotoxic mechanism. The presence of a stimulus that leads to the overproduction of nitric oxide will likely cause neuronal damage (Law et al., 2001). Since oxidative damage is implicated in the etiology of neurological complications, treatment with antioxidants has been used as a

178

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

therapeutic approach in various types of neurodegenerative diseases (Ahmad et al., 2005; Ansari et al., 2004). Sesamol (5-hydroxy-1,3benzodioxole or 3,4-methylenedioxyphenol) is the major constituent of sesame seed oil Sesamum indicum L and is a powerful antioxidant that inhibits ultra violet and Fe3+/ascorbate-induced lipid peroxidation in rat brain (Prasad et al., 2005). Sesamol also possess neuroprotective (Hou et al., 2006), hepatoprotective (Hsu et al., 2006), anti-inflammatory (Hou et al., 2006), chemo-preventive (Prasad et al., 2005) and anti-aging properties (Sharma and Kaur, 2006). With this background, the present study was designed to investigate the possible protective effect of sesamol on streptozotocin-induced neurotoxicity and to explore the possible involvement of nitrergic signaling. The functional interaction of sesamol with nitrergic signaling was investigated using nitric oxide precursor, L-arginine, and non selective nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). 2. Material and methods 2.1. Animals care Adult female Wistar rats (250–300 g) bred in Central Animal House facility of Panjab University were used with 5–8 animals in each group. The female rats were used as they are more prone to the cognitive impairment (Roof and Stein, 1999). The rats were housed in polyacrylic cages [38 × 23 × 10 cm] and maintained under standard laboratory conditions with natural dark and light (12:12 h) cycle and had free access to food (Ashirwad Industries, Chandigarh, India) and water ad libitum. Animals were acclimatized to laboratory conditions before all the behavioral tests. All experiments were carried out between 0900 and 1700 h and performed in accordance with the Guidelines of EC Directive 86/609/EEC for animal experiments. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Panjab University and performed in accordance with the guidelines of the Committee for Control and Supervision of Experimentation on Animals (CPCSEA), Government of India.

was injected bilaterally in two divided doses on first and third day making the dose of 1.5 mg/kg each day. The concentration of streptozotocin in aCSF was adjusted so as to deliver 10 μl of the solution. Control animals received intracerebroventricular injection of the same volume of aCSF on the first and third day. The skin was sutured after second injection followed by daily application of antiseptic powder (Neosporin). Postoperatively, the rats were fed with oral glucose and normal pellet diet for 4 days, followed by normal pellet diet alone. 2.4. Experimental design Rats were randomly divided into ten different groups containing 5–8 animals in each group viz Group 1: control animals received an equivalent volume of vehicle for streptozotocin i.e. artificial CSF (aCSF) on day 1 and day 3; Groups 2 and 3: animals received intracerebroventricular injections of aCSF on day 1 and day 3 along with L-arginine (125 mg/kg; intraperitoneal) and L-NAME (10 mg/kg; intraperitoneal) respectively for 21 days; Group 4: animals received intracerebroventricular injection of streptozotocin 1.5 mg/kg on each day 1 and day 3; Groups 5, 6 and 7: i.c.v.-streptozotocin treated rats administered sesamol (4 mg/kg), L-NAME (10 mg/kg; intraperitoneal) and L-NAME (10 mg/kg; intraperitoneal) + sesamol (4 mg/kg) respectively for 21 days; Groups 8, 9 and 10: i.c.v.-streptozotocin treated rats received sesamol (8 mg/kg; oral gavage), L-arginine (125 mg/kg; intraperitoneal) and L-arginine (125 mg/kg; intraperitoneal) + sesamol (8 mg/kg; oral gavage) respectively for 21 days. From the preliminary data of the present study, we have selected two doses of sesamol (4 and 8 mg/kg) for further drug interaction studies with nitric oxide modulators. The possible participation of the nitric oxide signaling in the neuroprotective effect of sesamol was investigated. Mice were pretreated with L-arginine, a precursor of nitric oxide (125 mg/kg; intraperitoneal) daily 30 min before sesamol (8 mg/kg; oral gavage) for 21 days. In another set of experiments, we investigated the synergistic effect of sesamol (4 mg/kg; oral gavage) with L-NAME, a non specific nitric oxide synthase inhibitor (10 mg/kg; intraperitoneal). L-NAME was administered daily 30 min before sesamol (4 mg/kg; oral gavage) for 21 days.

2.2. Drugs and treatment 2.5. Behavioral tests Sesamol, L-arginine, L-NAME and streptozotocin were purchased from Sigma Aldrich, St. Louis, MO, USA. Sesamol, L-arginine and L-NAME were dissolved in double distilled water while streptozotocin was dissolved in artificial cerebrospinal fluid (aCSF) (2.9 mM KCl, 147 mMNaCl, 1.7 mMCaCl2, 1.6 mM MgCl2, and 2.2 mM D-glucose). The doses of sesamol and nitric oxide modulators were selected according to the previous studies conducted in our laboratory (Kuhad and Chopra, 2008; Chander et al., 2005). All drug solutions were freshly prepared immediately prior to injection. Sesamol was administered per orally whereas L-arginine and L-NAME were injected intraperitoneally. 2.3. Surgical procedures: intracerebroventricular injection of streptozotocin Intracerebroventricular injection of streptozotocin was performed according to the procedure of Sonkusare et al. (2005). Rats were anesthetized with thiopentone (Neon Laboratories, India, 45 mg/kg, i.p.). The scalp was shaved, cleaned and cut to expose the skull. The head was positioned in a stereotaxic frame and a midline sagittal incision was made in the scalp. Burr holes were drilled in the skull on both sides over the lateral ventricles by using the following coordinates: 0.8 mm posterior to bregma; 1.5 mm lateral to sagittal suture and 3.6 mm beneath the surface of the brain (Sharma and Gupta, 2002). Streptozotocin (3 mg/kg, intracerebroventricular)

2.5.1. Morris water maze test Animals were tested in a spatial version of Morris water maze test (Morris et al., 1982; Tuzcu and Baydas, 2006). The apparatus consisted of a circular water tank (180 cm in diameter and 60 cm high). A platform (12.5 cm in diameter and 38 cm high) invisible to the rats, was set 2 cm below the water level inside the tank with water maintained at 28.5 ± 2 °C at a height of 40 cm. The tank was located in a large room where there were several brightly colored cues external to the maze; these were visible from the pool and could be used by the rats for spatial orientation. The position of the cues remained unchanged throughout the study. The water maze task was carried out for five consecutive days from 15th to 19th day. The rats received four consecutive daily training trials in the following 5 days, with each trial having a ceiling time of 90 s and a trial interval of approximately 30 s. For each trail, each rat was put into the water at one of four starting positions, the sequence of which being selected randomly. During test trials, rats were placed into the tank at the same starting point, with their heads facing the wall. The rat had to swim until it climbed onto the platform submerged underneath the water. After climbing onto the platform, the animal remained there for 20 s before the commencement of the next trial. The escape platform was kept in the same position relative to the distal cues. If the rat failed to reach the escape platform within the maximally allowed time of 90 s, it was guided with the help of a rod and allowed to remain on the platform

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

for 20 s. The time to reach the platform (escape latency in seconds) was measured. 2.5.2. Memory consolidation test A probe trial was performed (Tuzcu and Baydas, 2006) wherein the extent of memory consolidation was assessed. The time spent in the target quadrant indicates the degree of memory consolidation that has taken place after learning. In the probe trial, the rat was placed into the pool as in the training trial, except that the hidden platform was removed from the pool. The total time spent in target quadrant in a time period of 90 s was recorded. 2.5.3. Elevated plus maze test Memory acquisition and retention were tested using elevated plus maze test on days 19 and 20. The apparatus consisted of two crossed arms, one closed and the other, open. Each rat was placed on the open arm, facing outwards. The time taken by the rat to enter the closed arm in the first trial (acquisition trial) on 19th day was noted and was called as initial transfer latency. Cut-off time was fixed at 90 s and in case a rat could not find the closed arm within this period, it was gently pushed in to one of the closed arms and allowed to explore the maze for 30 s. Second trial (retention trial) was performed 24 h after the acquisition trial and retention transfer latency was noted (Sharma and Gupta, 2002; Mh and Gupta, 2002). The retention trial latency was expressed as percentage of initial trial latency. 2.5.4. Closed field activity Closed field activity was measured to rule out the interference of change in locomotor activity in the parameters of learning and memory. Spontaneous locomotor activity was measured on day 20 using digital photoactometer and values expressed as counts per 5 min. The apparatus was placed in a darkened, light and sound attenuated and ventilated testing room (Sharma and Gupta, 2001a,b). 2.6. Biochemical estimation 2.6.1. Brain homogenate preparation Brain samples were rinsed with ice cold saline (0.9% sodium chloride) and homogenized in chilled phosphate buffer (pH 7.4). The homogenates were centrifuged at 800 × g for 5 min at 4 °C to separate the nuclear debris. The supernatant thus obtained was centrifuged at 10,500 ×g for 20 min at 4 °C to get the post mitochondrial supernatant, which was used to assay acetylcholinesterase activity, lipid peroxidation, reduced glutathione, nitrite, catalase and superoxide dismutase activity. 2.6.2. Acetylcholinesterase activity Cholinergic dysfunction was assessed by acetylcholinesterase activity. The quantitative measurement of acetylcholinesterase levels in the whole brain homogenate was estimated according to the method of Ellman and Courtney (1961). The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide and 0.10 ml 5,5′-dithiobis (2nitro benzoic acid) (Ellman reagent). The change in absorbance was measured at 412 nm for 5 min. Results were calculated using molar extinction coefficient of chromophore (1.36 × 104 M− 1 cm− 1) and expressed as percentage of control.

179

boiling water for 10 min. After cooling, 1 ml double distilled water was added and absorbance was measured at 532 nm. Thiobarbituric acid-reactive substances were quantified using an extinction coefficient of 1.56 × 105 M− 1 cm− 1 and expressed as nmol of malondialdehyde per mg protein. Tissue protein was estimated using the Biuret method and the brain malondialdehyde content expressed as percentage of control. 2.6.4. Estimation of reduced glutathione Reduced glutathione was assayed by the method of Jollow et al. (1974). Briefly, 1.0 ml of post-mitochondrial supernatant (10%) was precipitated with 1.0 ml of sulphosalicylic acid (4%). The samples were kept at 4 °C for at least 1 h and then subjected to centrifugation at 1200 ×g for 15 min at 4 °C. The assay mixture contained 0.1 ml supernatant, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml 5,5′dithiobis (2-nitro benzoic acid) (Ellman's reagent, 0.1 mM, pH 8.0) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm. 2.6.5. Estimation of superoxide dismutase Cytosolic superoxide dismutase activity was assayed by the method of Kono (1978). The assay system consisted of 0.1 mM EDTA, 50 mM sodium carbonate and 96 mM of nitro blue tetrazolium (NBT). In the cuvette, 2 ml of above mixture was taken and to it 0.05 ml of post mitochondrial supernatant and 0.05 ml of hydroxylamine hydrochloride (adjusted to pH 6.0 with NaOH) were added. The auto-oxidation of hydroxylamine was observed by measuring the change in optical density at 560 nm for 2 min at 30/60 s intervals. 2.6.6. Estimation of catalase Catalase activity was assayed by the method of Claiborne (1985). Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml post mitochondrial supernatant (10%) in a final volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of K min− 1 and expressed as percentage of control. 2.6.7. Nitrite estimation Nitrite was estimated in the whole brain using the Greiss reagent and served as an indicator of nitric oxide production. A measure of 500 μl of Greiss reagent (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% napthaylamine diamine dihydrochloric acid in water) was added to 100 μl of post mitochondrial supernatant and absorbance was measured at 546 nm (Green et al., 1982). Nitrite concentration was calculated using a standard curve for sodium nitrite. Nitrite levels were expressed as percentage of control. 2.7. Data analysis Results were expressed as mean ± S.E.M. The intergroup variation was measured by one way analysis of variance (ANOVA) followed by Tukey's test. Escape latency was analyzed by using two way analysis of variance (ANOVA) followed by Tukey's test. The statistical analysis was done using the SPSS Statistical Software version 16. Statistical significance was considered at P b 0.05. 3. Results

2.6.3. Estimation of lipid peroxidation The malondialdehyde content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances by the method of Wills (1966). Briefly, 0.5 ml of post-mitochondrial supernatant and 0.5 ml of Tris–HCl were incubated at 37 °C for 2 h. After incubation 1 ml of 10% trichloro acetic acid was added and centrifuged at 1000 × g for 10 min. To 1 ml of supernatant, 1 ml of 0.67% thiobarbituric acid was added and the tubes were kept in

3.1. Behavioral observations 3.1.1. Effect of sesamol and its combination with nitric oxide modulators on performance in Morris water maze task The change in escape latency was observed onto a hidden platform produced by training trials. Although latency to reach the submerged platform decreased gradually in all the groups during 5 days of

180

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

training in Morris water maze test however the mean latency (from 2nd to 5th day) was significantly (P b 0.05) prolonged in i.c.v.streptozotocin group, as compared to control group, showing a poorer learning performance due to i.c.v.-streptozotocin injection. This disrupted performance of i.c.v.-streptozotocin group was significantly [F(5,24) = 57.061 (P b 0.001)] improved by the chronic treatment with sesamol (4 and 8 mg/kg). L-arginine (125 mg/kg) pretreatment with effective dose of sesamol (8 mg/kg) significantly reversed the protective effect of sesamol. Combination of L-NAME (10 mg/kg) with sub-effective dose of sesamol (4 mg/kg) significantly potentiated the protective effect of sesamol, which was significantly different as compared to the effect of sesamol (4 mg/kg) and L-NAME alone treated group (Fig. 1A). In the probe trial, with the hidden platform removed, i.c.v.streptozotocin group failed to memorize the precise location of the platform, spending significantly (P b 0.05) less time (20.40 ± 1.03 s) in the target quadrant than control group (68.33 ± 0.88 s). The total time spent in the target quadrant was significantly [F(5,24) = 120.183 (P b 0.001)] increased by the chronic sesamol (4 and 8 mg/kg) treatment (38.00 ± 2.68 s and 54.00 ± 2.65 s respectively) as compared to i.c.v.-streptozotocin treated rats. While in case of animals receiving L-arginine pretreatment to sesamol (8 mg/kg) significantly decreased the total time spent in target quadrant (25.84 ± 0.88 s) than the animals receiving sesamol (8 mg/kg) alone. However, combination of L-NAME (10 mg/kg) with sub-effective dose of sesamol (4 mg/kg) significantly increased the time spent in target quadrant (63.90 ± 2.91 s) as compared to the effect of sesamol (4 mg/kg) and L-NAME alone treated group (Fig. 1B).

A

CTRL S S+SML(4)+LN S+LA+SML(8)

Escape Latency (s)

90

3.1.2. Effect of sesamol and its combination with nitric oxide modulators on initial transfer latency in elevated plus maze test Initial transfer latency (ITL) did not differ significantly in any of the groups. Retention transfer latency (RTL) of control group was significantly less than that of i.c.v.-streptozotocin injected group. Treatment with sesamol (4 and 8 mg/kg) significantly [F(5,24) = 31.370 (Pb 0.001)] lowered the RTL that lowered the %ITL which is calculated by [(RTL/ITL) 100] in i.c.v.-streptozotocin injected rats. L-arginine (125 mg/kg) pretreatment with effective dose of sesamol (8 mg/kg) significantly reversed the protective effect of sesamol. However, L-NAME (10 mg/kg) pretreatment with sub-effective dose of sesamol (4 mg/kg) significantly potentiated the protective effect of sesamol (Fig. 2). 3.1.3. Effect of sesamol and its combination with nitric oxide modulators on the locomotor activity The spontaneous locomotor activity did not differ significantly between the control, i.c.v.-streptozotocin group, sesamol (4 and 8 mg/ kg), L-arginine and L-NAME treated i.c.v.-streptozotocin groups on the 20th day [F(5,24) = 1.071 (P N 0.001)]. The mean values in the control vehicle treated group, i.c.v.-streptozotocin group, sesamol (4 and 8 mg/kg) treated i.c.v.-streptozotocin group were 281.00 ± 10.11 s, 271.80 ± 33.00 s, 245.5 ± 13.33 s, and 267.75 ± 13.6 s respectively. 3.2. Biochemical observations 3.2.1. Effect of sesamol and its combination with nitric oxide modulators on acetylcholinesterase activity Acetylcholinesterase activity was increased in i.c.v.-streptozotocin treated rat brain as compared to control group. Sesamol (4 and 8 mg/

CTRL+LA S+SML(4) S+SML(8)

CTRL+LN S+LN S+LA

75 *

60

* *

**,#

45

**,#

30

**,#

15 0

1

2

3

4

5

Days

B

80

TSTQ (s)

60

**,#,$

**,#

40

**,#

**

* 20

0 CTRL

CTRL+LA

CTRL+LN

S

S+SML(4)

S+LN S+SML(4)+LN S+SML(8)

S+LA

S+LA+SML(8)

Fig. 1. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide modulators on the performance of spatial memory acquisition phase using Morris water maze (A) and time spent in target quadrant (TSTQ) (B) in intracerebroventricular streptozotocin (i.c.v.-streptozotocin) treated rats. Data is expressed as mean ± S.E.M. ⁎P b 0.05 compared to CTRL from the second day of the training sessions; ⁎⁎P b 0.05 compared to S; #P b 0.05 compared to one another; $P b 0.05 compared to S + SML(4) and S + LN. CTRL: control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

181

100

80

* **,#

% ITL

60

**,$

**,#

40

20

0

Fig. 2. Effect of sesamol (4 and 8 mg/kg) and its combination with nitric oxide modulators on percentage initial transfer latency (%ITL) in intracerebroventricular streptozotocin treated rats. Data is expressed as mean ± S.E.M. ⁎P b 0.05 compared to CTRL from the second day of the training sessions; ⁎⁎P b 0.05 compared to S; #P b 0.05 compared to one another; $ P b 0.05 compared to S + SML(4) and S + LN. CTRL: control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

kg) treatment significantly decreased acetylcholinesterase activity in the brain of i.c.v.-streptozotocin injected rats [F(5,24) = 107.192 (P b 0.001)]. L-arginine (125 mg/kg) pretreatment with effective dose of sesamol (8 mg/kg) reversed the protective effect of sesamol. However, L-NAME (10 mg/kg) pretreatment with sub-effective dose of sesamol (4 mg/kg) significantly potentiated the protective effect of sesamol (Fig. 3). 3.2.2. Effect of sesamol and its combination with nitric oxide modulators on antioxidant profile The enzymatic activities of catalase (Fig. 4A) and superoxide dismutase (Fig. 4B) and reduced glutathione levels (Fig. 4C) were significantly decreased in the brain of i.c.v.-streptozotocin treated rats as compared control group. These alterations were significantly (P b 0.05) restored by sesamol (4 and 8 mg/kg) treatment. L-arginine (125 mg/kg) pretreatment with effective dose of sesamol (8 mg/kg) significantly reversed the protective effect of sesamol. However,

L-NAME (10 mg/kg) pretreatment with sub-effective dose of sesamol (4 mg/kg) significantly potentiated the protective effect of sesamol.

3.2.3. Effect of sesamol and its combination with nitric oxide modulators on lipid peroxidation Malondialdehyde (MDA) levels were significantly increased in the brain of i.c.v.-streptozotocin treated rats as compared to control group. Chronic treatment with sesamol (4 and 8 mg/kg) produced significant (P b 0.05) reduction in MDA levels in streptozotocintreated rat brain [F(5,24) = 150.301 (P b 0.001)]. L-arginine (125 mg/ kg) pretreatment with effective dose of sesamol (8 mg/kg) significantly reversed the protective effect of sesamol. However, L-NAME (10 mg/kg) pretreatment with sub-effective dose of sesamol (4 mg/ kg) significantly potentiated the protective effect of sesamol, which was significant as compared to the effect of sesamol (4 mg/kg) and L-NAME alone treated group (Fig. 5).

0.10

0.08

* **,#

0.06

**,$ **,#

0.04

0.02

0.00

Fig. 3. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide modulators on acetylcholinesterase (AChE) activity in intracerebroventricular streptozotocin treated rats. Data is expressed as mean ± S.E.M. ⁎P b 0.05 compared to CTRL from second day of the training sessions; ⁎⁎P b 0.05 compared to S; #P b 0.05 compared to one another; $ P b 0.05 compared to S + SML(4) and S + LN. CTRL: control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

182

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

A Catalase µmol of H2O2decomposed/min/mg protein

20

B

**,$ **,#

16

**,#

12

* 8

4

0

CTRL

CTRL+LA

CTRL+LN

S

S+SML(4)

S+LN S+SML(4)+LN S+SML(8)

S+LA

S+LA+SML(8)

S+LA

S+LA+SML(8)

S+LA

S+LA+SML(8)

3.5

SOD (units/mg protein)

3.0

**,$

2.5

**,# 2.0 1.5

**,# *

1.0 0.5 0.0

CTRL

CTRL+LA

CTRL+LN

S

S+SML(4)

S+LN S+SML(4)+LN S+SML(8)

C

GSH (µmol/mgprotein)

0.025

0.020

**,$

0.015

**,#

**,#

0.010

*

0.005

0.000 CTRL

CTRL+LA

CTRL+LN

S

S+SML(4)

S+LN

S+SML(4)+LN S+SML(8)

Fig. 4. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide modulators on (A) catalase activity (B) superoxide dismutase and (C) reduced glutathione levels in intracerebroventricular streptozotocin treated rats. Data is expressed as mean ± S.E.M. ⁎P b 0.05 compared to CTRL from the second day of the training sessions; ⁎⁎P b 0.05 compared to S; #P b 0.05 compared to one another; $P b 0.05 compared to S + SML(4) and S + LN. CTRL: control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

3.2.4. Effect of sesamol and its combination with nitric oxide modulators on nitrergic stress Nitrite levels were significantly elevated in the brain of i.c.v.streptozotocin treated animals as compared to control group. Sesamol (4 and 8 mg/kg) treatment significantly inhibited this rise in brain nitrite levels [F(5,24) = 153.884 (P b 0.001)]. L-arginine (125 mg/kg) pretreatment with effective dose of sesamol (8 mg/kg) significantly reversed the protective effect of sesamol. However, L-NAME (10 mg/ kg) pretreatment with sub-effective dose of sesamol (4 mg/kg) significantly potentiated the protective effect of sesamol, which

was significant as compared to the effect of sesamol (4 mg/kg) and alone treated group (Fig. 6).

L-NAME

3.2.5. Effect of sesamol and its combination with nitric oxide modulators on tumor necrosis factor-alpha (TNF-α) TNF-α levels were significantly elevated in the serum of i.c.v.streptozotocin treated animals as compared to control group. Sesamol (4 and 8 mg/kg) treatment significantly (P b 0.05) inhibited this increase in TNF-α levels in the serum of i.c.v.-streptozotocin treated rats. L-arginine (125 mg/kg) pretreatment with effective dose of

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

183

n moles of MDA/mg protein

4.0

3.0

* **,#

**,#

2.0

**,$ 1.0

0.0 CTRL

CTRL+LA

CTRL+LN

S

S+SML(4)

S+LN

S+SML(4)+LN S+SML(8)

S+LA

S+LA+SML(8)

Fig. 5. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide modulators on malonaldehyde level in brain of intracerebroventricular streptozotocin treated rats. Data is expressed as mean ± S.E.M. ⁎P b 0.05 compared to CTRL from the second day of the training sessions; ⁎⁎P b 0.05 compared to S; #P b 0.05 compared to one another; $ P b 0.05 compared to S + SML(4) and S + LN. CTRL: control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

sesamol (8 mg/kg) significantly reversed the protective effect of sesamol. However, L-NAME (10 mg/kg) pretreatment with subeffective dose of sesamol (4 mg/kg) significantly potentiated the protective effect of sesamol, which was significant when compared to the effect of each compound alone (Fig. 7). 4. Discussion The present study for the first time demonstrated the protective effect of sesamol in the intracerebroventricular streptozotocininduced cognitive impairment and indicated possible involvement of nitrergic pathway in ameliorating the cognitive deficit associated with i.c.v. streptozotocin. Morris water maze and elevated plus maze tests were used as behavioral paradigm for assessment of learning and memory. Decreased escape latency in Morris water maze task in repeated trials demonstrates intact learning and memory function. The escape latency of i.c.v.-streptozotocin treated rats could not decrease significantly in repeated trials ranging from day 1 to day 5 suggesting significant impairment in memory of i.c.v.-streptozotocin treated rats. However, sesamol administration to i.c.v.-streptozotocin injected rats significantly decreased the time to reach the hidden platform in water maze task. In probe trial also, the time spent in target quadrant was significantly decreased in streptozotocin treated rats as compared to control group, which was significantly reversed on treatment with sesamol. The results from elevated plus maze further substantiated the findings of Morris water maze test as

sesamol treatment significantly reduced the increased percent initial transfer latencies after 24 h in i.c.v. streptozotocin administered rats. The locomotor activity of any of the group did not differ significantly suggesting that decrease in latency in both the behavioral paradigms was not affected by locomotion and the results are in accordance with findings from Sharma and Gupta (2002). The intracerebroventricular streptozotocin model produces cognitive deficits similar to those seen in sporadic dementia of Alzheimer's type (Ganguli et al., 2000; Salkovic-Petrisic and Hoyer, 2007). Streptozotocin is thought to be responsible for the pathogenesis of the neurodegenerative changes observed in this in vivo model of Alzheimer's disease, even in the absence of amyloid β (Aβ) aggregation (Grünblatt et al., 2007). The neurodegeneration in Alzheimer's disease is associated with oxidative stress, mitochondrial dysfunction, impaired energy metabolism, progressive Aβ agglutination and formation of neurofibrillary tangles (Li and Hölscher, 2007). Intracerebroventricular injection of streptozotocin in sub-diabetogenic dose reduced energy metabolism, leading to cognitive dysfunction by inhibiting the synthesis of adenosine triphosphate and acetyl-CoA. This ultimately results into cholinergic deficiency supported by reduced cholineacetyltransferase activity in hippocampus and an increased acetylcholinesterase activity in the brain of i.c.v. streptozotocin administered rats (Sharma and Gupta, 2001b; Sonkusare et al., 2005). Nitric oxide is a very important signaling molecule which is involved in various physiological functions within our body. But when

250

* Nitrite (µmol/mg protein)

200

**,# 150

**,$

**,#

100

50

0 CTRL

CTRL+LA

CTRL+LN

S

S+SML(4)

S+LN

S+SML(4)+LN S+SML(8)

S+LA

S+LA+SML(8)

Fig. 6. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide modulators on brain nitrite levels. Data is expressed as mean ± S.E.M. ⁎P b 0.05 compared to CTRL from the second day of the training sessions; ⁎⁎P b 0.05 compared to S; #P b 0.05 compared to one another; $P b 0.05 compared to S + SML(4) and S + LN. CTRL: control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

184

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186 75

*

Concentration (pg/ml)

60

**,# 45

**,$

**,#

30

15

0

CTRL

CTRL+LA

CTRL+LN

S

S+SML(4)

S+LN

S+SML(4)+LN S+SML(8)

S+LA

S+LA+SML(8)

Fig. 7. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide modulators in serum TNF-α levels. Data is expressed as mean ± S.E.M. ⁎P b 0.05 compared to CTRL from the second day of the training sessions; ⁎⁎P b 0.05 compared to S; #P b 0.05 compared to one another; $P b 0.05 compared to S + SML(4) and S + LN. CTRL: control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

it generates in excess in the presence of free radicals, can produce very toxic effects. Nitric oxide exerts its neurotoxic effects via several mechanisms. Nitric oxide is a free radical and can combine with superoxide anions to form peroxynitrite, a highly destructive radical moiety (Eliasson et al., 1999). The resultant reactive oxygen/nitrogen species can induce significant oxidative/nitrergic stress that causes lipid peroxidation and produces functional alterations in proteins and DNA, eventually leading to neuronal death (Beckman et al., 1994). Nitric oxide can also cause nitrosylation in a variety of proteins, including PKC and glyceraldehyde-3 phosphate dehydrogenase, thereby inhibiting phosphorylation and the glycolytic pathway, respectively (Zhang and Snyder, 1995). Furthermore, nitric oxide or peroxynitrite can impair glycolysis and subsequent cellular energy production by potentiating ADP-ribosylation of glyceraldehyde-3 phosphate dehydrogenase (Zhang and Snyder, 1993). Nitric oxide, by damaging DNA, could also lead to poly ADP-ribose polymerase over-activation, causing neuronal ATP depletion and death (Ha and Snyder, 1999). The activation of microglia causes inducible nitric oxide synthase mediated nitric oxide release (Hu et al., 1998; Kröncke et al., 1995). In addition, the activated microglia produces TNF-α, which also potentiates nitric oxide production (Kröncke et al., 1995). Moreover, the aggregation of reactive astrocytes in the proximity of Aβ has been demonstrated and the presence of cytokines can directly induce astrocytic inducible nitric oxide synthase mediated nitric oxide production or act in synergy with Aβ to induce astrocytic inducible nitric oxide synthase expression (Hu et al., 1998; Rossi and Bianchini, 1996). The resultant increase in nitric oxide production by these different means would most likely lead to increased free radical production and possibly cell death. In the Alzheimer's disease brain, both nitric oxide and peroxynitrite mediated neuronal damage have been observed (Chabrier et al., 1999; Koppal et al., 1999). In our study, we also observed a significant increase in malondialdehyde and nitrite levels along with marked reduction in reduced glutathione and enzymatic activity of superoxide dismutase and catalase in the brain of i.c.v.-streptozotocin treated rats. The cognitive restoration by sesamol was coupled with marked inhibition of reactive oxygen and nitrogen species. Our results are supported by findings from other authors who reported that sesamol inhibited the enzymatic activity of nitric oxide synthase (Chu et al., 2009; Hsu et al., 2006). Our previous laboratory findings have also shown that sesamol inhibited the oxidative–nitrergic stress in diabetic neuropathy and diabetes associated cognitive decline (Chopra et al., 2010; Kuhad and Chopra, 2008). Acetylcholine, a neurotransmitter associated with learning and memory, is degraded by the enzyme acetylcholinesterase, terminat-

ing the physiological action of the neurotransmitter. In addition to their role in cholinergic transmission, cholinesterases may also play a role during morphogenesis and neurodegenerative diseases (Hellweg et al., 1992; Reyes et al., 1997; Layer et al., 2006). Sesamol has been shown to inhibit acetylcholinesterase activity in brain of diabetic rats with cognitive deficits (Kuhad and Chopra, 2008). In the present study, the acetylcholinesterase activity in brain of i.c.v.-streptozotocin treated rats was significantly increased as compared to aCSF treated rats, which is in accordance with the findings of Sonkusare et al. (2005). This increase in acetylcholinesterase activity may lead to diminished cholinergic transmission due to a decrease in acetylcholine levels. In the present study, sesamol treatment significantly inhibited acetylcholinesterase activity in the brains of intracerebroventricular streptozotocin treated rats. In our previous study also, we found significant inhibition of acetylcholinesterase activity in the brain of diabetic rats with cognitive deficits on treatment with sesamol (Kuhad and Chopra, 2008). Pro-inflammatory cytokines are known to be elevated in several neuropathological states that are associated with learning and memory. Experimental studies reported that the inhibition of long term potentiation in the dentate gyrus region of the rat hippocampus, by tumor necrosis factor (TNF)-alpha, represents a biphasic response, an early phase dependent on p38 mitogen activated protein kinase activation and a later phase, possibly dependent on protein synthesis (Cumiskey et al., 2007). In our very recent study, we also found a significant increase in TNF-α and IL-1β levels in cerebral cortex and hippocampus of rats with cognitive deficits due to chronic alcohol consumption (Tiwari et al., 2009). In the present study, we observed a significant increase in TNF-α level in i.c.v. streptozotocin treated rats which were significantly reduced on treatment with sesamol. Experimental studies have shown that administration of L-arginine generated neurotoxicity that leads to neuronal death in animal models of depression (Kulkarni and Dhir, 2007) and epilepsy (Akula et al., 2008). Khavandgar et al. (2003) showed that treatment with L-NAME (3 mg/kg and 10 mg/kg) restored the memory impairment induced by pre-training morphine, and this effect was blocked by concomitant L-arginine (60 mg/kg) treatment. In the present study also, L-arginine produced marked deterioration in behavioral and biochemical parameters in intracerebroventricular streptozotocin treated rats. Combination of L-arginine with effective dose of sesamol significantly attenuated the protective effect of sesamol on behavioral and biochemical alterations in intracerebroventricular streptozotocin treated rats. Various laboratories have reported the protective effect of L-NAME on experimental paradigms of neurological diseases (Boultadakis et al., 2010; Choopani et al., 2008). In the present study,

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186 L-NAME has been shown to improve behavioral and biochemical deficits in intracerebroventricular streptozotocin treated rats. When we combined sub-effective dose of sesamol with L-NAME, the protective effect of sesamol was potentiated. Loss of protection by nitric oxide precursor suggests that increasing levels of nitric oxide enhanced nitrergic signaling and this enhanced nitrergic signaling precipitated behavioral and biochemical deficits. However, inhibition of nitrergic signaling by L-NAME could be the possible reason for restoration of learning and memory dysfunction. This suggests that antioxidant property of sesamol may be responsible for protecting against the oxidative stress, possibly by increasing the endogenous defensive capacity of the brain to combat oxidative stress induced by i.c.v. streptozotocin. In addition to potent antioxidant activity the inhibition of acetylcholinesterase activity and TNF-α level along with modulation of nitrergic signaling contributes significantly in preventing the cognitive impairment in i.c.v. streptozotocin model in rats.

5. Conclusion The findings suggest the neuroprotective potential of sesamol against intracerebroventricular streptozotocin-induced cognitive impairment in rats possibly through nitrergic signaling and thus may find a place in therapeutic armamentarium for the treatment of patients with Alzheimer's disease. Conflict of interest There is no actual or potential conflict of interest between any of the authors. Acknowledgment The junior research fellowship granted to Mr. Shubham Misra by the University Grants Commission is gratefully acknowledged. References Ahmad, M., Saleem, S., Ahmad, A.S., Yousuf, S., Ansari, M.A., Khan, M.B., Ishrat, T., Chaturvedi, R.K., Agrawal, A.K., Islam, F., 2005. Ginkgo biloba affords dosedependent protection against 6-hydroxydopamine-induced parkinsonism in rats: neurobehavioural, neurochemical and immunohistochemical evidences. J. Neurochem. 93, 94–104. Akula, K.K., Dhir, A., Kulkarni, S.K., 2008. Nitric oxide signaling pathway in the anticonvulsant effect of adenosine against pentylenetetrazol-induced seizure threshold in mice. Eur. J. Pharmacol. 587, 129–134. Ansari, M.A., Ahmad, A.S., Ahmad, M., Salim, S., Yousuf, S., Ishrat, T., Islam, F., 2004. Selenium protects cerebral ischemia in rat brain mitochondria. Biol. Trace Elem. Res. 101, 73–86. Areosa, S.A., Sherriff, F., 2006. Memantine for dementia. Cochrane Database Syst. Rev. 3, CD003154. Beckman, J.S., Chen, J., Crow, J.P., Ye, Y.Z., 1994. Reactions of nitric oxide, superoxide and peroxynitrite with superoxide dismutase in neurodegeneration. Prog. Brain Res. 103, 371–376. Boultadakis, A., Georgiadou, G., Pitsikas, N., 2010. Effects of the nitric oxide synthase inhibitor L-NAME on different memory components as assessed in the object recognition task in the rat. Behav. Brain Res. 207, 208–214. Chabrier, P.E., Demerle-Pallardy, C., Auguet, M., 1999. Nitric oxide synthases: targets for therapeutic strategies in neurological diseases. Cell. Mol. Life Sci. 55, 1029–1035. Chander, V., Tirkey, N., Chopra, K., 2005. Resveratrol, a polyphenolic phytoalexin protects against cyclosporine-induced nephrotoxicity through nitric oxide dependent mechanism. Toxicology 210, 55–64. Choopani, S., Moosavi, M., Naghdi, N., 2008. Involvement of nitric oxide in insulin induced memory improvement. Peptides 29, 898–903. Chopra, K., Tiwari, V., Arora, V., Kuhad, A., 2010. Sesamol suppresses neuro-inflammatory cascade in experimental model of diabetic neuropathy. J. Pain 11, 950–957. Chu, P.Y., Chien, S.P., Hsu, D.Z., Liu, M.Y., 2009. Protective effect of sesamol on the pulmonary inflammatory response and lung injury in endotoxemic rats. Food Chem. Toxicol. 48, 1821–1826. Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), CRC Handbook of Methods for Oxygen Radical Research, pp. 283–284. Cumiskey, D., Butler, M.P., Moynagh, P.N., O'Connor, J.J., 2007. Evidence for a role for the group I metabotropic glutamate receptor in the inhibitory effect of tumor necrosis factor-[alpha] on long-term potentiation. Brain Res. 1136, 13–19.

185

Dorheim, M.A., Tracey, W.R., Pollock, J.S., Grammas, P., 1994. Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer's disease. Biochem. Biophys. Res. Commun. 205, 659–665. Eliasson, M.J.L., Huang, Z., Ferrante, R.J., Sasamata, M., Molliver, M.E., Snyder, S.H., Moskowitz, M.A., 1999. Neuronal nitric oxide synthase activation and peroxynitrite formation in ischemic stroke linked to neural damage. J. Neurosci. 19, 5910–5919. Ellman, G.L., Courtney, K.D., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–90. Ganguli, M., Chandra, V., Kamboh, M.I., Johnston, J.M., Dodge, H.H., Thelma, B.K., Juyal, R.C., Pandav, R., Belle, S.H., DeKosky, S.T., 2000. Apolipoprotein E polymorphism and Alzheimer disease: the Indo-US cross-national dementia study. Arch. Neurol. 57, 824–831. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131–138. Grünblatt, E., Salkovic-Petrisic, M., Osmanovic, J., Riederer, P., Hoyer, S., 2007. Brain insulin system dysfunction in streptozotocin intracerebroventricularly treated rats generates hyperphosphorylated tau protein. J. Neurochem. 101, 757–770. Ha, H.C., Snyder, S.H., 1999. Poly (ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc. Natl. Acad. Sci. 96, 13978–13984. Hellweg, R., Nitsch, R., Hock, C., Jaksch, M., Hoyer, S., 1992. Nerve growth factor and choline acetyltransferase activity levels in the rat brain following experimental impairment of cerebral glucose and energy metabolism. J. Neurosci. Res. 31, 479–486. Hou, R.C.W., Chen, Y.S., Chen, C.H., Chen, Y.H., Jeng, K.C.G., 2006. Protective effect of 1, 2, 4-benzenetriol on LPS-induced NO production by BV2 microglial cells. J. Biomed. Sci. 13, 89–99. Hsu, D.Z., Chen, K.T., Li, Y.H., Chuang, Y.C., Liu, M.Y., 2006. Sesamol delays mortality and attenuates hepatic injury after cecal ligation and puncture in rats: role of oxidative stress. Shock 25, 528–537. Hu, J., Akama, K.T., Krafft, G.A., Chromy, B.A., Van Eldik, L.J., 1998. Amyloid-[beta] peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release. Brain Res. 785, 195–206. Jollow, D.J., Mitchell, J.R., Zampaglione, N., Gillette, J.R., 1974. Bromobenze induced liver necrosis: protective role of glutathione and evidence for 3,4-bromobenzenoxide as the hepatotoxic intermediate. Pharmacology 11, 151–169. Khavandgar, S., Homayoun, H., Zarrindast, M.R., 2003. The effect of L -NAME and L-arginine on impairment of memory formation and state-dependent learning induced by morphine in mice. Psychopharmacology 167, 291–296. Kono, Y., 1978. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch. Biochem. Biophys. 186, 189–195. Koppal, T., Drake, J., Yatin, S., Jordan, B., Varadarajan, S., Bettenhausen, L., Butterfield, D.A., 1999. Peroxynitrite-induced alterations in synaptosomal membrane proteins: insight into oxidative stress in Alzheimer's disease. J. Neurochem. 72, 310–317. Kröncke, K.D., Fehsel, K., Kolb-Bachofen, V., 1995. Inducible nitric oxide synthase and its product nitric oxide, a small molecule with complex biological activities. Biol. Chem. 376, 327–343. Kuhad, A., Chopra, K., 2008. Effect of sesamol on diabetes-associated cognitive decline in rats. Exp. Brain Res. 185, 411–420. Kulkarni, S.K., Dhir, A., 2007. Possible involvement of L-arginine-nitric oxide (NO)cyclic guanosine monophosphate (cGMP) signaling pathway in the antidepressant activity of berberine chloride. Eur. J. Pharmacol. 569, 77–83. Lannert, H., Hoyer, S., 1998. Intracerebroventricular administration of streptozotocin causes long-term diminutions in learning and memory abilities and in cerebral energy metabolism in adult rats. Behav. Neurosci. 112, 1199–1208. Law, A., Gauthier, S., Quirion, R., 2001. Say NO to Alzheimer's disease: the putative links between nitric oxide and dementia of the Alzheimer's type. Brain Res. Rev. 35, 73–96. Layer, P.G., Alber, R., Sporns, O., 2006. Quantitative development and molecular forms of acetyl-and butyrylcholinesterase during morphogenesis and synaptogenesis of chick brain and retina. J. Neurochem. 49, 175–182. Li, L., Hölscher, C., 2007. Common pathological processes in Alzheimer disease and type 2 diabetes: a review. Brain Res. Rev. 56, 384–402. Mh, V.K., Gupta, Y.K., 2002. Intracerebroventricular administration of colchicine produces cognitive impairment associated with oxidative stress in rats. Pharmacol. Biochem. Behav. 73, 565–571. Morris, R.G.M., Garrud, P., Rawlins, J.N.P., O'Keefe, J., 1982. Place navigation impaired in rats with hippocampal lesions. Nature 297, 681–683. Norris, P.J., Faull, R.L.M., Emson, P.C., 1996. Neuronal nitric oxide synthase (nNOS) mRNA expression and NADPH-diaphorase staining in the frontal cortex, visual cortex and hippocampus of control and Alzheimer's disease brains. Mol. Brain Res. 41, 36–49. Prasad, N.R., Mahesh, T., Menon, V.P., Jeevanram, R.K., Pugalendi, K.V., 2005. Photoprotective effect of sesamol on UVB-radiation induced oxidative stress in human blood lymphocytes in vitro. Environ. Toxicol. Pharmacol. 20, 1–5. Reyes, A.E., Perez, D.R., Alvarez, A., Garrido, J., Gentry, M.K., Doctor, B.P., Inestrosa, N.C., 1997. A monoclonal antibody against acetylcholinesterase inhibits the formation of amyloid fibrils induced by the enzyme. Biochem. Biophys. Res. Commun. 232, 652–655. Roof, R.L., Stein, D.G., 1999. Gender differences in Morris water maze depend on task parameters. Physiol. Behav. 68 (1–2), 81–86. Rossi, F., Bianchini, E., 1996. Synergistic induction of nitric oxide by [beta]-amyloid and cytokines in astrocytes. Biochem. Biophys. Res. Commun. 225, 474–478. Salkovic-Petrisic, M., Hoyer, S., 2007. Central insulin resistance as a trigger for sporadic Alzheimer-like pathology: an experimental approach. J. Neural. Transm. Suppl. 72, 217–233.

186

S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

Sharma, M., Gupta, Y.K., 2001a. Effect of chronic treatment of melatonin on learning, memory and oxidative deficiencies induced by intracerebroventricular streptozotocin in rats. Pharmacol. Biochem. Behav. 70, 325–331. Sharma, M., Gupta, Y.K., 2001b. Intracerebroventricular injection of streptozotocin in rats produces both oxidative stress in the brain and cognitive impairment. Life Sci. 68, 1021–1029. Sharma, M., Gupta, Y.K., 2002. Chronic treatment with trans resveratrol prevents intracerebroventricular streptozotocin induced cognitive impairment and oxidative stress in rats. Life Sci. 71, 2489–2498. Sharma, S., Kaur, I.P., 2006. Development and evaluation of sesamol as an antiaging agent. Int. J. Dermatol. 45, 200–208. Sonkusare, S., Srinivasan, K., Kaul, C., Ramarao, P., 2005. Effect of donepezil and lercanidipine on memory impairment induced by intracerebroventricular streptozotocin in rats. Life Sci. 77, 1–14.

Tiwari, V., Kuhad, A., Chopra, K., 2009. Suppression of neuro-inflammatory signaling cascade by tocotrienol can prevent chronic alcohol-induced cognitive dysfunction in rats. Behav. Brain Res. 203, 296–303. Tuzcu, M., Baydas, G., 2006. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur. J. Pharmacol. 537, 106–110. Vance, J.E., Hayashi, H., Karten, B., 2005. Cholesterol homeostasis in neurons and glial cells. Semin. Cell Dev. Biol. 16, 193–212. Wills, E.D., 1966. Mechanisms of lipid peroxide formation in animal tissues. Biochem. J. 99, 667–676. Zhang, J., Snyder, S.H., 1993. Purification of a nitric oxide-stimulated ADP-ribosylated protein using biotinylated. beta.-nicotinamide adenine dinucleotide. Biochemistry 32, 2228–2233. Zhang, J., Snyder, S.H., 1995. Nitric oxide in the nervous system. Annu. Rev. Pharmacol. Toxicol. 35, 213–233.