European Journal of Pharmacology 659 (2011) 177–186

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European Journal of Pharmacology

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Behavioural Pharmacology

Modulation of nitrergic pathway by sesamol prevents cognitive deficits andassociated biochemical alterations in intracerebroventricular streptozotocinadministered 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

⁎ Corresponding author. Tel.: +91 172 2534105; fax:E-mail address: dr_chopra_k@yahoo.com (K. Chopra

0014-2999/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.ejphar.2011.03.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 September 2010Received in revised form 25 February 2011Accepted 21 March 2011Available online 2 April 2011

Keywords:Alzheimer's diseaseIntracerebroventricular streptozotocinL-arginineL-NAME (N(G)-nitro-L-arginine methyl ester)SesamolTNF-α (tumor necrosis factor-alpha)

Alzheimer's disease is a neurodegenerative disorder characterized by progressive cognitive decline andwidespread loss of neurons and their synapses in the cerebral cortex and hippocampus. Increasing evidenceindicates that factors such as oxidative–nitrergic stress, glutathione depletion, impaired protein metabolismand 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 anexperimental paradigm to study Alzheimer's disease. In the present study, i.c.v. streptozotocin producedsignificant cognitive deficits as measured in Morris water maze and elevated plus maze task coupled withincreased serum TNF-α levels and marked rise in brain acetylcholinesterase and oxidative–nitrergic stress infemale Wistar rats. Sesamol (5-hydroxy-1,3-benzodioxole or 3,4-methylenedioxyphenol), a potent anti-oxidant and anti-inflammatory molecule markedly improved cognitive impairment, reduced acetylcholin-esterase activity, TNF-α levels and attenuated oxidative–nitrergic stress in brain of i.c.v.-streptozotocintreated rats. Administration of L-arginine (125 mg/kg i.p), a nitric oxide donor, alone to i.c.v.-streptozotocintreated rats accentuated behavioral and biochemical deficits and also abolished the protective effect ofsesamol (8 mg/kg). L-NAME (10 mg/kg i.p.), a non-specific NOS inhibitor significantly restored all thebehavioral and biochemical indices in i.c.v.-streptozotocin rats. Moreover, combination of L-NAME with sub-effective dose of sesamol (4 mg/kg) potentiated its protective effect. Our findings demonstrate theeffectiveness of sesamol in preventing intracerebroventricular streptozotocin-induced cognitive deficits bymodulating nitrergic signaling and oxido-inflammatory cascade.

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1. Introduction

Alzheimer's disease is the most common type of dementia inWestern societies and has profound economic and social impact. Itaccounts for 50% of dementia cases all around the globe (Areosa andSherriff, 2006). Alzheimer's disease is characterized by markedatrophy of cerebral neocortex, hippocampus and loss of cortical andsub-cortical neurons (Vance et al., 2005). Neuroscientists all aroundthe world are trying their best to develop a sure-shot remedy forAlzheimer's disease and related dementia. Current strategies aremainly focused on two aspects; one is to develop agents, whichimprove cognitive deficits of Alzheimer's disease and second to findappropriate experimental models for screening of such agents (Vanceet al., 2005). The intracerebroventricular streptozotocin (i.c.v. strep-tozotocin) injected rat has been described as an appropriate animalmodel for sporadic dementia characterized by progressive deteriora-

tion of memory and cerebral glucose and energy metabolism, alongwith oxidative stress (Lannert and Hoyer, 1998; Sharma and Gupta,2001a; Sonkusare et al., 2005).

Nitric oxide mediated nitrergic signaling, an important bioregula-tory signaling in the nervous, immune and cardiovascular system,plays a pivotal role in brain homeostasis. The involvement of nitricoxide in a number of neurological disorders is well established.Researchers have begun to recognize and to explore the putative linkbetween nitric oxide and Alzheimer's disease (Dorheim et al., 1994;Norris et al., 1996). Although existing evidence regarding theirassociation is not abundant, emerging data are showing Alzheimer'sdisease-related changes in the nitric oxide synthase system, and itappears that nitric oxide could be related to many of the pathologicalmechanisms of the disease. The ability of nitric oxide to exert cellulardamage due to its reactive oxidative properties is perhaps the primaryneurotoxic mechanism. The presence of a stimulus that leads to theoverproduction of nitric oxide will likely cause neuronal damage (Lawet al., 2001).

Since oxidative damage is implicated in the etiology of neurolog-ical 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-1,3-benzodioxole or 3,4-methylenedioxyphenol) is the major constituentof sesame seed oil Sesamum indicum L and is a powerful antioxidantthat inhibits ultra violet and Fe3+/ascorbate-induced lipid peroxida-tion in rat brain (Prasad et al., 2005). Sesamol also possessneuroprotective (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 studywas designed to investigatethe possible protective effect of sesamol on streptozotocin-inducedneurotoxicity and to explore the possible involvement of nitrergicsignaling. The functional interaction of sesamol with nitrergic signalingwas investigated using nitric oxide precursor, L-arginine, and nonselective nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methylester (L-NAME).

2. Material and methods

2.1. Animals care

Adult female Wistar rats (250–300 g) bred in Central AnimalHouse facility of Panjab University were used with 5–8 animals ineach group. The female rats were used as they are more prone to thecognitive impairment (Roof and Stein, 1999). The rats were housed inpolyacrylic cages [38×23×10 cm] and maintained under standardlaboratory conditions with natural dark and light (12:12 h) cycle andhad free access to food (Ashirwad Industries, Chandigarh, India) andwater ad libitum. Animals were acclimatized to laboratory conditionsbefore all the behavioral tests. All experiments were carried outbetween 0900 and 1700 h and performed in accordance with theGuidelines of EC Directive 86/609/EEC for animal experiments. Theexperimental protocols were approved by the Institutional AnimalEthics Committee (IAEC) of Panjab University and performed inaccordance with the guidelines of the Committee for Control andSupervision of Experimentation on Animals (CPCSEA), Government ofIndia.

2.2. Drugs and treatment

Sesamol, L-arginine, L-NAME and streptozotocin were purchasedfrom Sigma Aldrich, St. Louis, MO, USA. Sesamol, L-arginine andL-NAME were dissolved in double distilled water while streptozoto-cin was dissolved in artificial cerebrospinal fluid (aCSF) (2.9 mMKCl,147 mMNaCl, 1.7 mMCaCl2, 1.6 mM MgCl2, and 2.2 mM D-glucose).The doses of sesamol and nitric oxide modulators were selectedaccording to the previous studies conducted in our laboratory(Kuhad and Chopra, 2008; Chander et al., 2005). All drug solutionswere freshly prepared immediately prior to injection. Sesamolwas administered per orally whereas L-arginine and L-NAME wereinjected intraperitoneally.

2.3. Surgical procedures: intracerebroventricular injectionof streptozotocin

Intracerebroventricular injection of streptozotocin was performedaccording to the procedure of Sonkusare et al. (2005). Rats wereanesthetized with thiopentone (Neon Laboratories, India, 45 mg/kg,i.p.). The scalp was shaved, cleaned and cut to expose the skull. Thehead was positioned in a stereotaxic frame and a midline sagittalincision was made in the scalp. Burr holes were drilled in the skullon both sides over the lateral ventricles by using the followingcoordinates: 0.8 mm posterior to bregma; 1.5 mm lateral to sagittalsuture and 3.6 mm beneath the surface of the brain (Sharma andGupta, 2002). Streptozotocin (3 mg/kg, intracerebroventricular)

was injected bilaterally in two divided doses on first and third daymaking the dose of 1.5 mg/kg each day. The concentration ofstreptozotocin in aCSF was adjusted so as to deliver 10 μl of thesolution. Control animals received intracerebroventricular injectionof the same volume of aCSF on the first and third day. The skin wassutured after second injection followed by daily application ofantiseptic powder (Neosporin). Postoperatively, the rats were fedwith oral glucose and normal pellet diet for 4 days, followed bynormal pellet diet alone.

2.4. Experimental design

Rats were randomly divided into ten different groups containing5–8 animals in each group viz Group 1: control animals received anequivalent volume of vehicle for streptozotocin i.e. artificial CSF(aCSF) on day 1 and day 3; Groups 2 and 3: animals receivedintracerebroventricular injections of aCSF on day 1 and day 3 alongwith L-arginine (125 mg/kg; intraperitoneal) and L-NAME (10 mg/kg;intraperitoneal) respectively for 21 days; Group 4: animals receivedintracerebroventricular injection of streptozotocin 1.5 mg/kg on eachday 1 and day 3; Groups 5, 6 and 7: i.c.v.-streptozotocin treated ratsadministered sesamol (4 mg/kg), L-NAME (10 mg/kg; intraperitone-al) and L-NAME (10 mg/kg; intraperitoneal)+sesamol (4 mg/kg)respectively for 21 days; Groups 8, 9 and 10: i.c.v.-streptozotocintreated rats received sesamol (8 mg/kg; oral gavage), L-arginine(125 mg/kg; intraperitoneal) and L-arginine (125 mg/kg; intraperito-neal)+sesamol (8 mg/kg; oral gavage) respectively for 21 days.

From the preliminary data of the present study, we have selectedtwo doses of sesamol (4 and 8 mg/kg) for further drug interactionstudies with nitric oxide modulators. The possible participation of thenitric oxide signaling in the neuroprotective effect of sesamol wasinvestigated. Mice were pretreated with L-arginine, a precursor ofnitric oxide (125 mg/kg; intraperitoneal) daily 30 min before sesamol(8 mg/kg; oral gavage) for 21 days. In another set of experiments, weinvestigated 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 beforesesamol (4 mg/kg; oral gavage) for 21 days.

2.5. Behavioral tests

2.5.1. Morris water maze testAnimals were tested in a spatial version of Morris water maze test

(Morris et al., 1982; Tuzcu and Baydas, 2006). The apparatus consistedof a circular water tank (180 cm in diameter and 60 cm high). Aplatform (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 watermaintained at 28.5±2 °C at a height of 40 cm. The tank was located ina large room where there were several brightly colored cues externalto themaze; these were visible from the pool and could be used by therats for spatial orientation. The position of the cues remainedunchanged throughout the study. The water maze task was carriedout for five consecutive days from 15th to 19th day. The rats receivedfour consecutive daily training trials in the following 5 days, with eachtrial having a ceiling time of 90 s and a trial interval of approximately30 s. For each trail, each rat was put into the water at one of fourstarting positions, the sequence of which being selected randomly.During test trials, rats were placed into the tank at the same startingpoint, with their heads facing the wall. The rat had to swim until itclimbed onto the platform submerged underneath the water. Afterclimbing onto the platform, the animal remained there for 20 s beforethe commencement of the next trial. The escape platform was kept inthe same position relative to the distal cues. If the rat failed to reachthe escape platform within the maximally allowed time of 90 s, it wasguided with the help of a rod and allowed to remain on the platform

179S. 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 testA probe trial was performed (Tuzcu and Baydas, 2006) wherein

the extent of memory consolidation was assessed. The time spent inthe target quadrant indicates the degree ofmemory consolidation thathas taken place after learning. In the probe trial, the rat was placedinto the pool as in the training trial, except that the hidden platformwas removed from the pool. The total time spent in target quadrant ina time period of 90 s was recorded.

2.5.3. Elevated plus maze testMemory acquisition and retention were tested using elevated plus

maze test on days 19 and 20. The apparatus consisted of two crossedarms, one closed and the other, open. Each rat was placed on the openarm, facing outwards. The time taken by the rat to enter the closedarm in the first trial (acquisition trial) on 19th day was noted and wascalled as initial transfer latency. Cut-off time was fixed at 90 s and incase a rat could not find the closed arm within this period, it wasgently pushed in to one of the closed arms and allowed to explore themaze for 30 s. Second trial (retention trial) was performed 24 h afterthe acquisition trial and retention transfer latency was noted (Sharmaand Gupta, 2002; Mh and Gupta, 2002). The retention trial latencywas expressed as percentage of initial trial latency.

2.5.4. Closed field activityClosed field activity was measured to rule out the interference of

change in locomotor activity in the parameters of learning andmemory. Spontaneous locomotor activity was measured on day 20using digital photoactometer and values expressed as counts per5 min. The apparatus was placed in a darkened, light and soundattenuated and ventilated testing room (Sharma and Gupta, 2001a,b).

2.6. Biochemical estimation

2.6.1. Brain homogenate preparationBrain samples were rinsed with ice cold saline (0.9% sodium

chloride) and homogenized in chilled phosphate buffer (pH 7.4). Thehomogenates were centrifuged at 800×g for 5 min at 4 °C to separatethe nuclear debris. The supernatant thus obtained was centrifuged at10,500×g for 20 min at 4 °C to get the post mitochondrial superna-tant, which was used to assay acetylcholinesterase activity, lipidperoxidation, reduced glutathione, nitrite, catalase and superoxidedismutase activity.

2.6.2. Acetylcholinesterase activityCholinergic dysfunction was assessed by acetylcholinesterase

activity. The quantitative measurement of acetylcholinesterase levelsin the whole brain homogenate was estimated according to themethod of Ellman and Courtney (1961). The assay mixture contained0.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH8), 0.10 ml of acetylthiocholine iodide and 0.10 ml 5,5′-dithiobis (2-nitro benzoic acid) (Ellman reagent). The change in absorbance wasmeasured at 412 nm for 5 min. Results were calculated using molarextinction coefficient of chromophore (1.36×104 M−1 cm−1) andexpressed as percentage of control.

2.6.3. Estimation of lipid peroxidationThe malondialdehyde content, a measure of lipid peroxidation,

was assayed in the form of thiobarbituric acid-reactive substances bythe method of Wills (1966). Briefly, 0.5 ml of post-mitochondrialsupernatant 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 andcentrifuged at 1000×g for 10 min. To 1 ml of supernatant, 1 ml of0.67% thiobarbituric acid was added and the tubes were kept in

boiling water for 10 min. After cooling, 1 ml double distilled waterwas added and absorbance was measured at 532 nm. Thiobarbituricacid-reactive substances were quantified using an extinction coeffi-cient of 1.56×105 M−1 cm−1 and expressed as nmol of malondial-dehyde per mg protein. Tissue protein was estimated using the Biuretmethod and the brain malondialdehyde content expressed aspercentage of control.

2.6.4. Estimation of reduced glutathioneReduced glutathione was assayed by the method of Jollow et al.

(1974). Briefly, 1.0 ml of post-mitochondrial supernatant (10%) wasprecipitated with 1.0 ml of sulphosalicylic acid (4%). The sampleswere kept at 4 °C for at least 1 h and then subjected to centrifugationat 1200×g for 15 min at 4 °C. The assay mixture contained 0.1 mlsupernatant, 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) ina total volume of 3.0 ml. The yellow color developed was readimmediately at 412 nm.

2.6.5. Estimation of superoxide dismutaseCytosolic superoxide dismutase activity was assayed by the

method of Kono (1978). The assay system consisted of 0.1 mMEDTA, 50 mM sodium carbonate and 96 mM of nitro blue tetrazolium(NBT). In the cuvette, 2 ml of above mixture was taken and to it0.05 ml of post mitochondrial supernatant and 0.05 ml of hydroxyl-amine hydrochloride (adjusted to pH 6.0with NaOH)were added. Theauto-oxidation of hydroxylamine was observed by measuring thechange in optical density at 560 nm for 2 min at 30/60 s intervals.

2.6.6. Estimation of catalaseCatalase 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 mlpost mitochondrial supernatant (10%) in a final volume of 3.0 ml.Changes in absorbance were recorded at 240 nm. Catalase activitywas calculated in terms of K min−1 and expressed as percentage ofcontrol.

2.6.7. Nitrite estimationNitrite was estimated in the whole brain using the Greiss reagent

and served as an indicator of nitric oxide production. A measure of500 μl of Greiss reagent (1:1 solution of 1% sulphanilamide in 5%phosphoric acid and 0.1% napthaylamine diamine dihydrochloricacid in water) was added to 100 μl of post mitochondrial supernatantand absorbance was measured at 546 nm (Green et al., 1982). Nitriteconcentration was calculated using a standard curve for sodiumnitrite. Nitrite levels were expressed as percentage of control.

2.7. Data analysis

Results were expressed as mean±S.E.M. The intergroup variationwas measured by one way analysis of variance (ANOVA) followed byTukey's test. Escape latency was analyzed by using two way analysisof variance (ANOVA) followed by Tukey's test. The statistical analysiswas done using the SPSS Statistical Software version 16. Statisticalsignificance was considered at Pb0.05.

3. Results

3.1. Behavioral observations

3.1.1. Effect of sesamol and its combination with nitric oxide modulatorson performance in Morris water maze task

The change in escape latency was observed onto a hidden platformproduced by training trials. Although latency to reach the submergedplatform 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 (from2nd to 5th day) was significantly (Pb0.05) prolonged in i.c.v.-streptozotocin group, as compared to control group, showing apoorer learning performance due to i.c.v.-streptozotocin injection.This disrupted performance of i.c.v.-streptozotocin group wassignificantly [F(5,24)=57.061 (Pb0.001)] improved by the chronictreatment with sesamol (4 and 8 mg/kg). L-arginine (125 mg/kg)pretreatment with effective dose of sesamol (8 mg/kg) significantlyreversed the protective effect of sesamol. Combination of L-NAME(10 mg/kg) with sub-effective dose of sesamol (4 mg/kg) significantlypotentiated the protective effect of sesamol, which was significantlydifferent as compared to the effect of sesamol (4 mg/kg) and L-NAMEalone 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 theplatform, spending significantly (Pb0.05) less time (20.40±1.03 s) inthe target quadrant than control group (68.33±0.88 s). The total timespent in the target quadrant was significantly [F(5,24)=120.183(Pb0.001)] increased by the chronic sesamol (4 and 8 mg/kg)treatment (38.00±2.68 s and 54.00±2.65 s respectively) as com-pared to i.c.v.-streptozotocin treated rats.

While in case of animals receiving L-arginine pretreatment tosesamol (8 mg/kg) significantly decreased the total time spent intarget 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 increasedthe time spent in target quadrant (63.90±2.91 s) as compared tothe effect of sesamol (4 mg/kg) and L-NAME alone treated group(Fig. 1B).

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Fig. 1. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide mmaze (A) and time spent in target quadrant (TSTQ) (B) in intracerebroventricular streptozcompared to CTRL from the second day of the training sessions; ⁎⁎Pb0.05 compared to S; #

control group; S: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg);

3.1.2. Effect of sesamol and its combination with nitric oxide modulatorson initial transfer latency in elevated plus maze test

Initial transfer latency (ITL) did not differ significantly in any of thegroups. Retention transfer latency (RTL) of control group was signifi-cantly less than that of i.c.v.-streptozotocin injected group. Treatmentwith sesamol (4 and 8 mg/kg) significantly [F(5,24)=31.370(Pb0.001)] lowered the RTL that lowered the %ITL which is calculatedby [(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 theprotective 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 modulatorson the locomotor activity

The spontaneous locomotor activity did not differ significantlybetween 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 the20th day [F(5,24)=1.071 (PN0.001)]. The mean values in the controlvehicle treated group, i.c.v.-streptozotocin group, sesamol (4 and8 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 modulatorson acetylcholinesterase activity

Acetylcholinesterase activity was increased in i.c.v.-streptozotocintreated rat brain as compared to control group. Sesamol (4 and 8 mg/

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odulators on the performance of spatial memory acquisition phase using Morris waterotocin (i.c.v.-streptozotocin) treated rats. Data is expressed as mean±S.E.M. ⁎Pb0.05Pb0.05 compared to one another; $Pb0.05 compared to S+SML(4) and S+LN. CTRL:SML(8): sesamol (8 mg/kg).

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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 streptozotocintreated rats. Data is expressed asmean±S.E.M. ⁎Pb0.05 compared to CTRL from the second day of the training sessions; ⁎⁎Pb0.05 compared to S; #Pb0.05 compared to one another;$Pb0.05 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).

181S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

kg) treatment significantly decreased acetylcholinesterase activity inthe brain of i.c.v.-streptozotocin injected rats [F(5,24)=107.192(Pb0.001)]. L-arginine (125 mg/kg) pretreatment with effectivedose of sesamol (8 mg/kg) reversed the protective effect of sesamol.However, L-NAME (10 mg/kg) pretreatment with sub-effective doseof sesamol (4 mg/kg) significantly potentiated the protective effect ofsesamol (Fig. 3).

3.2.2. Effect of sesamol and its combination with nitric oxide modulatorson antioxidant profile

The enzymatic activities of catalase (Fig. 4A) and superoxidedismutase (Fig. 4B) and reduced glutathione levels (Fig. 4C) weresignificantly decreased in the brain of i.c.v.-streptozotocin treated ratsas compared control group. These alterations were significantly(Pb0.05) 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,

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Fig. 3. Effect of sesamol (4 and 8 mg/kg) treatment and its combinationwith nitric oxidemodtreated rats. Data is expressed as mean±S.E.M. ⁎Pb0.05 compared to CTRL from second day$Pb0.05 compared to S+SML(4) and S+LN. CTRL: control group; S: streptozotocin; LA: L-a

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 modulatorson lipid peroxidation

Malondialdehyde (MDA) levels were significantly increased in thebrain of i.c.v.-streptozotocin treated rats as compared to controlgroup. Chronic treatment with sesamol (4 and 8 mg/kg) producedsignificant (Pb0.05) reduction in MDA levels in streptozotocin-treated rat brain [F(5,24)=150.301 (Pb0.001)]. L-arginine (125 mg/kg) pretreatment with effective dose of sesamol (8 mg/kg) signifi-cantly 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, whichwas significant as compared to the effect of sesamol (4 mg/kg) andL-NAME alone treated group (Fig. 5).

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ulators on acetylcholinesterase (AChE) activity in intracerebroventricular streptozotocinof the training sessions; ⁎⁎Pb0.05 compared to S; #Pb0.05 compared to one another;

rginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesamol (8 mg/kg).

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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 glutathionelevels in intracerebroventricular streptozotocin treated rats. Data is expressed as mean±S.E.M. ⁎Pb0.05 compared to CTRL from the second day of the training sessions; ⁎⁎Pb0.05compared to S; #Pb0.05 compared to one another; $Pb0.05 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

3.2.4. Effect of sesamol and its combination with nitric oxide modulatorson 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 brainnitrite levels [F(5,24)=153.884 (Pb0.001)]. L-arginine (125 mg/kg)pretreatment with effective dose of sesamol (8 mg/kg) significantlyreversed 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) andL-NAME alone treated group (Fig. 6).

3.2.5. Effect of sesamol and its combination with nitric oxide modulatorson 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 (Pb0.05) inhibited thisincrease in TNF-α levels in the serum of i.c.v.-streptozotocin treatedrats. L-arginine (125 mg/kg) pretreatment with effective dose of

*

**,#

**,$**,#

0.0

1.0

2.0

3.0

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)

n m

ole

s o

f M

DA

/mg

pro

tein

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 streptozotocintreated rats. Data is expressed asmean±S.E.M. ⁎Pb0.05 compared to CTRL from the second day of the training sessions; ⁎⁎Pb0.05 compared to S; #Pb0.05 compared to one another;$Pb0.05 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).

183S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

sesamol (8 mg/kg) significantly reversed the protective effect ofsesamol. However, L-NAME (10 mg/kg) pretreatment with sub-effective dose of sesamol (4 mg/kg) significantly potentiated theprotective effect of sesamol, which was significant when compared tothe effect of each compound alone (Fig. 7).

4. Discussion

The present study for the first time demonstrated the protectiveeffect of sesamol in the intracerebroventricular streptozotocin-induced cognitive impairment and indicated possible involvementof nitrergic pathway in ameliorating the cognitive deficit associatedwith i.c.v. streptozotocin. Morris water maze and elevated plus mazetests were used as behavioral paradigm for assessment of learning andmemory. Decreased escape latency in Morris water maze task inrepeated trials demonstrates intact learning and memory function.The escape latency of i.c.v.-streptozotocin treated rats could notdecrease significantly in repeated trials ranging from day 1 to day 5suggesting significant impairment in memory of i.c.v.-streptozotocintreated rats. However, sesamol administration to i.c.v.-streptozotocininjected rats significantly decreased the time to reach the hiddenplatform in water maze task. In probe trial also, the time spent intarget quadrant was significantly decreased in streptozotocin treatedrats as compared to control group, which was significantly reversedon treatment with sesamol. The results from elevated plus mazefurther substantiated the findings of Morris water maze test as

*

**,#

0

50

100

150

200

250

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

Nit

rite

(µm

ol/m

g p

rote

in)

Fig. 6. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxide modCTRL from the second day of the training sessions; ⁎⁎Pb0.05 compared to S; #Pb0.05 compaS: streptozotocin; LA: L-arginine; LN: L-NAME; SML(4): sesamol (4 mg/kg); SML(8): sesam

sesamol treatment significantly reduced the increased percent initialtransfer latencies after 24 h in i.c.v. streptozotocin administered rats.The locomotor activity of any of the group did not differ significantlysuggesting that decrease in latency in both the behavioral paradigmswas not affected by locomotion and the results are in accordance withfindings from Sharma and Gupta (2002).

The intracerebroventricular streptozotocin model produces cog-nitive deficits similar to those seen in sporadic dementia ofAlzheimer's type (Ganguli et al., 2000; Salkovic-Petrisic and Hoyer,2007). Streptozotocin is thought to be responsible for the pathogen-esis of the neurodegenerative changes observed in this in vivo modelof Alzheimer's disease, even in the absence of amyloid β (Aβ) aggre-gation (Grünblatt et al., 2007). The neurodegeneration in Alzheimer'sdisease is associated with oxidative stress, mitochondrial dysfunction,impaired energy metabolism, progressive Aβ agglutination andformation of neurofibrillary tangles (Li andHölscher, 2007). Intracere-broventricular injection of streptozotocin in sub-diabetogenic dosereduced energy metabolism, leading to cognitive dysfunction byinhibiting the synthesis of adenosine triphosphate and acetyl-CoA.This ultimately results into cholinergic deficiency supported byreduced cholineacetyltransferase activity in hippocampus and anincreased acetylcholinesterase activity in the brain of i.c.v. streptozo-tocin administered rats (Sharma and Gupta, 2001b; Sonkusare et al.,2005).

Nitric oxide is a very important signaling molecule which isinvolved in various physiological functions within our body. But when

**,$ **,#

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

ulators on brain nitrite levels. Data is expressed as mean±S.E.M. ⁎Pb0.05 compared tored to one another; $Pb0.05 compared to S+SML(4) and S+LN. CTRL: control group;ol (8 mg/kg).

*

**,#

**,$ **,#

0

15

30

45

60

75

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

Co

nce

ntr

atio

n (

pg

/ml)

Fig. 7. Effect of sesamol (4 and 8 mg/kg) treatment and its combination with nitric oxidemodulators in serum TNF-α levels. Data is expressed as mean±S.E.M. ⁎Pb0.05 compared toCTRL from the second day of the training sessions; ⁎⁎Pb0.05 compared to S; #Pb0.05 compared to one another; $Pb0.05 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

it generates in excess in the presence of free radicals, can produce verytoxic effects. Nitric oxide exerts its neurotoxic effects via severalmechanisms. Nitric oxide is a free radical and can combine withsuperoxide anions to form peroxynitrite, a highly destructive radicalmoiety (Eliasson et al., 1999). The resultant reactive oxygen/nitrogenspecies can induce significant oxidative/nitrergic stress that causeslipid peroxidation and produces functional alterations in proteins andDNA, 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 orperoxynitrite can impair glycolysis and subsequent cellular energyproduction by potentiating ADP-ribosylation of glyceraldehyde-3phosphate dehydrogenase (Zhang and Snyder, 1993). Nitric oxide,by damaging DNA, could also lead to poly ADP-ribose polymeraseover-activation, causing neuronal ATP depletion and death (Ha andSnyder, 1999). The activation of microglia causes inducible nitricoxide synthase mediated nitric oxide release (Hu et al., 1998; Krönckeet 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 ofAβ has been demonstrated and the presence of cytokines can directlyinduce astrocytic inducible nitric oxide synthasemediated nitric oxideproduction or act in synergy with Aβ to induce astrocytic induciblenitric oxide synthase expression (Hu et al., 1998; Rossi and Bianchini,1996). The resultant increase in nitric oxide production by thesedifferent means would most likely lead to increased free radicalproduction and possibly cell death. In the Alzheimer's disease brain,both nitric oxide and peroxynitrite mediated neuronal damage havebeen observed (Chabrier et al., 1999; Koppal et al., 1999).

In our study, we also observed a significant increase in mal-ondialdehyde and nitrite levels along with marked reduction inreduced glutathione and enzymatic activity of superoxide dismutaseand catalase in the brain of i.c.v.-streptozotocin treated rats. Thecognitive restoration by sesamol was coupled with marked inhibitionof reactive oxygen and nitrogen species. Our results are supported byfindings from other authors who reported that sesamol inhibited theenzymatic activity of nitric oxide synthase (Chu et al., 2009; Hsu et al.,2006). Our previous laboratory findings have also shown that sesamolinhibited the oxidative–nitrergic stress in diabetic neuropathy anddiabetes associated cognitive decline (Chopra et al., 2010; Kuhad andChopra, 2008).

Acetylcholine, a neurotransmitter associated with learning andmemory, is degraded by the enzyme acetylcholinesterase, terminat-

ing the physiological action of the neurotransmitter. In addition totheir role in cholinergic transmission, cholinesterases may also play arole during morphogenesis and neurodegenerative diseases (Hellweget al., 1992; Reyes et al., 1997; Layer et al., 2006). Sesamol has beenshown to inhibit acetylcholinesterase activity in brain of diabetic ratswith cognitive deficits (Kuhad and Chopra, 2008). In the presentstudy, the acetylcholinesterase activity in brain of i.c.v.-streptozotocintreated rats was significantly increased as compared to aCSF treatedrats, which is in accordance with the findings of Sonkusare et al.(2005). This increase in acetylcholinesterase activity may lead todiminished cholinergic transmission due to a decrease in acetylcho-line levels. In the present study, sesamol treatment significantlyinhibited acetylcholinesterase activity in the brains of intracerebro-ventricular streptozotocin treated rats. In our previous study also, wefound significant inhibition of acetylcholinesterase activity in thebrain of diabetic rats with cognitive deficits on treatment withsesamol (Kuhad and Chopra, 2008).

Pro-inflammatory cytokines are known to be elevated in severalneuropathological states that are associated with learning andmemory. Experimental studies reported that the inhibition of longterm 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 kinaseactivation and a later phase, possibly dependent on protein synthesis(Cumiskey et al., 2007). In our very recent study, we also found asignificant increase in TNF-α and IL-1β levels in cerebral cortex andhippocampus of rats with cognitive deficits due to chronic alcoholconsumption (Tiwari et al., 2009). In the present study, we observed asignificant increase in TNF-α level in i.c.v. streptozotocin treated ratswhich were significantly reduced on treatment with sesamol.

Experimental studies have shown that administration of L-argininegenerated neurotoxicity that leads to neuronal death in animalmodelsof 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 thememory impairment induced bypre-training morphine, and this effect was blocked by concomitantL-arginine (60 mg/kg) treatment. In the present study also, L-arginineproduced marked deterioration in behavioral and biochemicalparameters in intracerebroventricular streptozotocin treated rats.Combination of L-arginine with effective dose of sesamol significantlyattenuated the protective effect of sesamol on behavioral andbiochemical alterations in intracerebroventricular streptozotocintreated rats. Various laboratories have reported the protective effectof L-NAME on experimental paradigms of neurological diseases(Boultadakis et al., 2010; Choopani et al., 2008). In the present study,

185S. Misra et al. / European Journal of Pharmacology 659 (2011) 177–186

L-NAME has been shown to improve behavioral and biochemicaldeficits in intracerebroventricular streptozotocin treated rats. Whenwe combined sub-effective dose of sesamol with L-NAME, theprotective effect of sesamol was potentiated. Loss of protection bynitric oxide precursor suggests that increasing levels of nitric oxideenhanced nitrergic signaling and this enhanced nitrergic signalingprecipitated behavioral and biochemical deficits. However, inhibitionof nitrergic signaling by L-NAME could be the possible reason forrestoration of learning and memory dysfunction.

This suggests that antioxidant property of sesamol may beresponsible for protecting against the oxidative stress, possibly byincreasing the endogenous defensive capacity of the brain to combatoxidative stress induced by i.c.v. streptozotocin. In addition to potentantioxidant activity the inhibition of acetylcholinesterase activity andTNF-α level along with modulation of nitrergic signaling contributessignificantly in preventing the cognitive impairment in i.c.v. strepto-zotocin model in rats.

5. Conclusion

The findings suggest the neuroprotective potential of sesamolagainst intracerebroventricular streptozotocin-induced cognitive im-pairment in rats possibly through nitrergic signaling and thus mayfind a place in therapeutic armamentarium for the treatment ofpatients with Alzheimer's disease.

Conflict of interest

There is no actual or potential conflict of interest between any ofthe authors.

Acknowledgment

The junior research fellowship granted to Mr. Shubham Misra bythe University Grants Commission is gratefully acknowledged.

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