Protective effect of curcumin against intracerebral streptozotocin induced impairment in memory and cerebral blood flow

Life Sciences 86 (2010) 87–94

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Life Sciences 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 / l i f e s c i e

Protective effect of curcumin against intracerebral streptozotocin induced impairment in memory and cerebral blood flow Himani Awasthi a, Santoshkumar Tota a, Kashif Hanif a, Chandiswar Nath b, Rakesh Shukla a,⁎ a b

Division of Pharmacology, Central drug Research Institute, (CSIR) Lucknow, India Division of Toxicology, Central drug Research Institute, (CSIR) Lucknow, India

a r t i c l e

i n f o

Article history: Received 22 July 2009 Accepted 7 November 2009 Keywords: Curcumin Cerebral blood flow Memory Oxidative stress Streptozotocin

a b s t r a c t Aims: The aim of the present study is to investigate the effect of curcumin on cerebral blood flow (CBF), memory impairment, oxidative stress and cholinergic dysfunction in intracerebral (IC) streptozotocin (STZ) induced memory impairment in mice. Main methods: Memory impairment was induced by STZ (0.5 mg/kg, IC) administered twice with an interval of 48 h in mice. Memory function was assessed by Morris water maze and passive avoidance test. CBF was measured by Laser Doppler Flowmetry (LDF). To study the preventive effect, curcumin (10, 20 and 50 mg/kg, PO) was administered for 21 days starting from the first dose of STZ. In another set of experiment, curcumin was administered for 7 days from 19th day after confirming STZ induced dementia to observe its therapeutic effect. Biochemical parameters of oxidative stress and cholinergic function were estimated in brain on day 21. Key findings: The major finding of this study is that STZ (IC) caused a significant reduction in CBF along with memory impairment, cholinergic dysfunction and enhanced oxidative stress. Curcumin dose dependently improved CBF in STZ treated mice together with amelioration of memory impairment both in preventive and therapeutic manner. Significance: The present study clearly demonstrates the beneficial effects of curcumin, the dietary staple of India, on CBF, memory and oxidative stress which can be exploited for dementia associated with age related vascular and neurodegenerative disorders. © 2009 Elsevier Inc. All rights reserved.

Introduction The polyphenolic flavonoid, curcumin, found in turmeric (Curcuma longa), is a yellow curry spice with a long history of use in Indian diets and traditional herbal medicines (Ammon and Wahl 1991). Various in vitro and in vivo studies suggest that curcumin has potential antiinflammatory (Srimal and Dhawan 1973), antioxidant (Masuda et al. 1999), anti-protease (Sui et al. 1993) and anticancer properties (Kim et al. 1998). Further, curcumin regulates the expression of inflammatory enzymes, cytokines, adhesion molecules, and cell survival proteins (Goel et al. 2008). Moreover, curcumin is a potent free radical scavenger of superoxide anion, the hydroxyl radical and nitrogen dioxide (Unnikrishnan and Rao 1995). It protects the biomolecules in the brain from oxidative damage (Subramanian et al. 1994; Sreejayan and Rao 1994). In addition, the useful effects of curcumin in the treatment of memory dysfunction have been reported in animals (Dairam et al. 2007; Kuhad and Chopra 2007; Ishrat et al. 2009). Curcumin also reduces oxidative damage and amyloid pathology in a transgenic mouse model

⁎ Corresponding author. Division of Pharmacology, Central drug Research Institute, Lucknow (U.P.), India. Tel.: +91 522 2612411 18x4420; fax: +91 522 2623405. E-mail address: [email protected] (R. Shukla). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.11.007

of Alzheimer disease (AD) (Lim et al. 2001; Ono et al. 2004) and exerts a positive effect on neurogenesis and concentrations of brain-derived neurotrophic factor in the hippocampus (Xu et al. 2007; Wu et al. 2006). Recently it has been reported that curcumin also ameliorate impairment in insulin signaling and memory function in intracerebroventricular (ICV) streptozotocin (STZ) injected rats (Isik et al. 2009). Neuroprotective properties of curcumin are further supported by the observation that in India, where curcumin is an important ingredient of food and used as an herbal medicine, prevalence of AD in patients between 70 and 79 years of age is 4.4-fold less than that of the United States (Ganguli et al. 2000). Beneficial effects of curcumin are further backed by the epidemiologic evidences which show that increased consumption of curry is associated with better cognitive performance in nondemented subjects (Ng et al. 2006). Evidence suggest that AD and other types of dementia are associated with reduced cerebral blood flow (CBF) (Prohovnik et al. 1988; O'Brien et al. 1992; Wyper et al. 1993). This decline in CBF may be the result of the restricted flow due to vascular amyloidosis, oxidative stress and endothelial dysfunction. Cerebral microcirculatory impairment may initiate pathophysiological changes that play a role in the progression of AD (de la Torre and Mussivand 1993; Bush et al. 1993; Ajmani et al. 2000). Though beneficial effect of curcumin on memory functions has been reported earlier (Dairam et al. 2007;

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Therapeutic (posttreatment) group

Kuhad and Chopra 2007; Ishrat et al. 2009; Ganguli et al. 2000), its effect on CBF in models of memory deficit has not been investigated. Model of memory deficit induced by ICV injection of STZ in rodents is well accepted because it mimics various pathological aspects of AD like progressive deterioration of memory, cerebral glucose and energy metabolism (Nitsch and Hoyer 1991; Lannert and Hoyer 1998), oxidative stress (Sharma and Gupta 2001) and cholinergic dysfunction (Blokland and Jolles 1994; Plaschke and Hoyer 1993). Therefore we planned to investigate the effect of curcumin on CBF, memory impairment, oxidative stress and cholinergic dysfunction in intracerebral (IC) STZ induced model of memory impairment.

To evaluate therapeutic potential of curcumin, STZ injected mice were subjected to Morris water maze test from the 14th to the 18th day after the 1st STZ injection. Then mice with memory deficit were divided into three groups. Two groups were treated with curcumin (25 or 50 mg/kg, PO) and the third group was given vehicle for 7 days (19th to 25th day). Mice were again subjected to Morris water maze test for five consecutive days starting from the 26th day to the 30th day (Fig. 2). In passive avoidance test curcumin (50 mg/kg, PO) was administered for 7 days from day 16 to see its effect post memory deficit (Fig. 3b).

Materials and methods

Tests employed for learning and memory functions

Animals

Morris water maze

Male Swiss albino mice (25–30 gm, 8 weeks old) were procured from the Laboratory Animal Division of Central Drug Research Institute, Lucknow. Mice were kept in a polyacrylic cage and maintained under standard housing conditions (room temperature 24–27 °C and humidity 60–65%) with a 12-h light and dark cycle. Food and water were available ad libitum but food was withdrawn 1 h prior to behavioral study. There were six to eight animals in each group. Experiments were performed according to internationally followed ethical standards and approved by Institutional Animal Ethics Committee (IAEC), Central Drug Research Institute, India.

The Morris water maze consisted of a large circular black pool of 120 cm diameter, 50 cm height, filled to a depth of 30 cm with water at 26 ± 2 °C. Four equally spaced points around the edge of the pool were designed as N (North), E (East), S (South) and W (West). A black colored round platform of 8 cm diameter was placed 1 cm below the surface of water in a constant position in the middle of the NE quadrant in the pool; the starting point was in the SW quadrant in all the trials. The water was colored with non-toxic black dye to hide the location of the submerged platform. The mice could climb on the platform to escape the necessity of swimming. Trials were given for 5 consecutive days in order to train mice in the Morris water maze. The mice were given a maximum time of 60 s (cut-off time) to find the hidden platform and were allowed to stay on it for 30 s (Chen et al. 2002). The experimenter put the mice that failed to locate the platform onto it. The animals were given a daily session of 3 trials per day. Latency time to reach the platform was recorded in each trial. Mean latency time of all three trials is shown in the results. A significant decrease in latency time as compared with the 1st session was considered as successful learning (Saxena et al. 2007).

Materials Curcumin was purchased from Kancor, India. The biochemicals like streptozotocin (STZ), chloral hydrate, sodium chloride (NaCl), sodium nitrate (NaNO2), sulphanilamide, phosphoric acid, napthaylamine diamine dihydrochloric acid, bovine serum albumin (BSA), acetylthiocholine iodide (AChI), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), 1,1,3,3-tetraethoxypropane (TEP), 2-thiobabituric acid (TBA), dichlorodihydrofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich, USA. Intracerebral (IC) administration of streptozocin STZ, dissolved in freshly prepared artificial CSF (aCSF) (Sharma and Gupta 2001) was injected (0.5 mg/kg/10 µl) intracerebrally (IC) in mice anesthetized with chloral hydrate (300 mg/kg, IP) (Haley and McCormick 1957). The same dose of STZ was repeated 48 h after the first dose. Experimental design and administration of curcumin The experimental groups were based on the time of administration of curcumin in relation to the memory deficit to evaluate its preventive and therapeutic potential against STZ induced dementia. Preventive (pretreatment) group To study preventive effect in Morris water maze test, different doses of curcumin (10, 20 and 50 mg/kg) were selected on the basis of earlier reports in mice (Eybl et al. 2004). These doses were administered for 21 days starting from first dose of STZ. For passive avoidance test, maximum effective dose of 50 mg/kg curcumin (PO) was selected on the basis of performance of mice at different doses in water maze test. Curcumin was suspended in 1.0% w/v gum acacia immediately before administration in a constant volume of 10 ml/kg body weight. The control, aCSF and STZ group received vehicle of curcumin for 21 days. Another group of animals was administered with curcumin (50 mg/kg, PO) for 21 days to study per se effect.

Passive avoidance test The mice were subjected to the passive avoidance test by placing in a compartment with light at an intensity of 8 [scale from 0 to 10 (brightest)] in a computerized shuttle box with a software programme PACS 30 (Columbus Instruments, Ohio, USA). The light compartment was isolated from the dark compartment by an automated guillotine door. After an acclimatization period of 30 s, the guillotine door was opened and closed automatically after entry of the mouse into the dark compartment. The subject received a low-intensity foot shock (0.5 mA; 10 s) in the dark compartment. Infrared sensors monitored the transfer of the animal from one compartment to another, which was recorded as transfer latency time (TLT) in seconds. The 1st trial was for acquisition and retention was tested in a 2nd trial (1st retention) given 24 h after the 1st trial. The duration of a trial was 270 s. Further, 2nd, 3rd and 4th retention trials were given on alternate days to test retention in the STZ (IC) treated mice (Tota et al. 2009). The shock was not delivered in the retention trials to avoid reacquisition. The criterion for learning was taken as an increase in the TLT on retention (2nd or subsequent) trials as compared to acquisition (1st) trial. Spontaneous locomotor activity Each animal was observed for 10 min after a period of 30 min acclimatization in Optovarimax activity meter (Columbus Inc USA). Measurement of cerebral blood flow Cerebral blood flow (CBF) was measured by laser Doppler flowmetry (LDF 100, BIOPAC, USA). LDF qualitatively measures CBF

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in arbitrary blood perfusion units (BPU) (Tonnesen et al. 2005; Wienzek et al. 2007). The mice were anesthetized with chloral hydrate (300 mg/kg, i.p.) and a 0.5 mm diameter micro-fiber laser Doppler probe was fixed on the skull (6 mm lateral and 1 mm posterior of bregma) and CBF was monitored within cortical region (Stenman et al. 2007). CBF was measured continuously at abovementioned co-ordinates for a period of 10 min recording values after each 30 s and then average values of blood flow were calculated. Estimation of biochemical parameters Acetylcholinesterase (AChE), malondialdehyde (MDA), glutathione (GSH), nitrite (NO2) and reactive oxygen species (ROS) were measured in brain at the end of experiment.

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5 µM of DCF-DA and incubated in water bath at 37 °C for 15 min in the dark and subjected to fluorimetric estimation of ROS. Nitrite estimation Nitrite was estimated using Greiss reagent which served as an indicator of nitric oxide production. An amount of 100 µl 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 542 nm (Green et al. 1982). Nitrite concentration was calculated using a standard curve for sodium nitrite. Nitrite levels were expressed as percentage of control. Acetylcholinesterase assay in brain

Brain tissue preparation The mice were decapitated under ether anesthesia. The skull was cut open and the brain was exposed from its dorsal side. The whole brain was quickly removed and cleaned with chilled normal saline on the ice. A 10% (w/v) homogenate of brain samples (0.03 M sodium phosphate buffer, pH-7.4) was prepared by using an Ultra-Turrax T25 (USA) homogenizer at a speed of 9500 rpm. The homogenized tissue preparation was used to measure AChE, MDA, GSH and nitrite. Measurement of malondialdehyde (MDA) MDA, which is a measure of lipid peroxidation, was estimated spectrophotometrically by the method of Colado et al. 1997, using 1,1,3,3-tetraethoxypropane as standard. MDA is expressed as nanomoles per mg protein. To 500 µl of tissue homogenate in phosphate buffer (pH 7.4), 300 µl of 30% trichloroacetic acid (TCA), 150 µl of 5 N HCl and 300 µl of 2% w/v 2-thiobarbituric acid (TBA) were added. Then the mixture was heated for 15 min at 90 ° C and centrifuged at 12,000 g for 10 min. The pink colored supernatant was measured spectrophotometrically at 532 nm.

The brain homogenate in volume of 500 µl was mixed with 1% Triton X-100 (1% w/v in 0.03 M sodium phosphate buffer, pH-7) and centrifuged at 100,000 g at 4 °C in a Beckman Ultracentrifuge (LE 80, USA), using a fixed angle rotor (80 ti) for 60 min. Supernatant was collected and stored at 4 °C for AChE estimation by method of Ellman et al. (1961). The kinetic profile of enzyme activity was measured spectrophotometrically (Shimadzu, USA) at 412 nm with an interval of 15 s. One unit of AChE activity was defined as the number of micromoles (µmol) of acetylthiocholine iodide hydrolyzed per minute (min) per milligram (mg) of protein. The specific activity of AChE is expressed in micromoles/min/mg of protein. Protein estimation Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin (BSA) (1 mg/ml) as standard. Blood glucose estimation Glucose was measured by Accu-check sensor comfort glucose strips (Roche Diagnostic, India) in blood collected by tail prick.

Measurement of glutathione (GSH) Statistical analysis GSH was determined by its reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (Ellman's reagent) to yield a yellow chromophore which was measured spectrophotometrically (Ellman 1959). The brain homogenate was mixed with an equal amount of 10% trichloroacetic acid (TCA) and centrifuged (Remi cold centrifuge) at 2000 g for 10 min at 4 °C. The supernatant was used for GSH estimation. To 0.1 ml of processed tissue sample, 2 ml of phosphate buffer (pH 8.4), 0.5 ml of 5,5′-dithiobis (2Nitrobenzoic acid) (DTNB) and 0.4 ml of double-distilled water were added and the mixture was shaken vigorously on vortex. The absorbance was read at 412 nm within 15 min. Measurement of reactive oxygen species (ROS) by spectrofluorometry Determination of ROS was based on methods of LeBel et al. (1992) and Oyama et al. (1994) with slight modifications. The brain tissue was subjected to collagenase-D treatment in HEPES-buffered Hank's (HBH) solution (pH 7.4) containing CaCl2 (3 mM) at room temperature. Later, single cell suspension was centrifuged at 200 g for 5 min and resuspended in HBSS buffer for ROS estimation. Mitochondrial release of ROS was determined spectrofluorometrically, using the membranepermeable fluorescent dye dichlorodihydrofluorescein diacetate (DCF-DA). The formation of the fluorescent product DCF was monitored by a CARY Eclipse, fluorescence spectrometer with excitation wavelength of 488 nm and emission wavelength of 530 nm. DCF-DA, (5 µM) dissolved in dimethyl sulfoxide, enters the cells and loses its diacetate group and becomes DCFH by esterase action and gets oxidized to highly fluorescent DCF (Keller et al. 1998). Isolated 105 cells were treated with

The results are expressed as mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey test. Results Effect of curcumin on STZ (IC) induced memory deficit in Morris water maze test in mice Pretreatment group The effect of curcumin on spatial learning was evaluated using the Morris water maze test. As shown in Fig. 1a, STZ treated animals exhibited longer escape latencies throughout training sessions than the control and aCSF-treated groups [Control: F (4, 20) = 66.48, P b 0.001; aCSF: F (4, 20) = 38.3, P b 0.001]. Curcumin (20 and 50 mg/ kg, PO) treatment significantly decreased mean latency at the 4th and 5th sessions (retention) as compared to the 1st session (acquisition), while curcumin (10 mg/kg, PO) did not show the significant effect [10 mg/kg: F (4, 20) = 1.80, P N 0.05; 20 mg/kg: F (4, 30) = 5.58, P b 0.01 and 50 mg/kg: F (4, 25) = 9.3, P b 0.001] (Fig. 1b). Further, curcumin (50 mg/kg, PO) per se had no effect on cognition when compared to control (data not shown). Posttreatment group To evaluate therapeutic potential of curcumin, STZ injected mice were subjected to Morris water maze test (Fig. 2). Mice exhibited

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12.11, P b 0.001]. There was no significant change in mean latency to reach the platform in vehicle treated memory deficit group [F (4, 15) = 0.05, P N 0.05].

Effect of curcumin on STZ (IC) induced memory deficit in passive avoidance test in mice Pretreatment group The transfer latency time (TLT) significantly increased in the retention trials as compared to the acquisition trial in control [F (4, 25) = 8.95, P b 0.001] and aCSF-treated mice [F (4, 25) = 12.59, P b 0.001]. No significant increase in the TLT of the retention trials as compared to the acquisition trial was observed in the STZ treated mice [F (4, 45) = 0.98, P N 0.05]. However, curcumin treatment significantly reversed memory deficit and increased transfer latency in STZ injected mice [F (4, 20) = 7.90, P b 0.01]. There was no significant difference [F (3, 23) = 0.396, P N 0.05] in TLT of the 1st trial among the different groups (Fig. 3a).

Fig. 1. (a) STZ (IC) caused persistent memory impairment in mice as evidenced by no significant reduction in latency time during sessions 2–5 as compared to Session 1. Control and aCSF group showed significant reduction in latency time. Data values are expressed as mean latency time (s) ± S.E.M. ★Significant difference (★P b 0.001) in latency time in comparison to session 1. (b) Preventive effect of curcumin (10, 20 and 50 mg/kg, PO) on STZ (IC) induced memory deficit in Morris water maze test in mice. Data values are expressed as mean latency time (s) ± S.E.M. ★Significant difference ★ (★P b 0.01 and ★P b 0.001) in latency time in comparison to session 1.

Posttreatment group To study effect of curcumin post memory deficit, mice were injected with STZ and subjected to passive avoidance test on days 14 and 15. There was no significant change in TLT of the 1st retention trial as compared to acquisition trial (P N 0.05) (Fig. 3b). Curcumin (50 mg/ kg, PO) for 7 days caused a significant increase in TLT on 2nd and 3rd retention trials as compared to acquisition trial [F (3, 36) = 6.419, P b 0.01]. Whereas, no significant change was found in TLT of vehicle treated STZ group in all retention sessions as compared to acquisition trial [F (3, 28) = 1.839, P N 0.05].

longer escape latencies throughout training sessions [F (4, 75) = 1.73, P N 0.05] indicating memory impairment. Treatment with curcumin (25 or 50 mg/kg, PO) for 7 days from 19th day onwards dose dependently reversed STZ induced memory deficit. Curcumin (50 mg/kg, PO) significantly decreased mean latency at the 3rd, 4th and 5th retention sessions as compared to the 1st session [F (4, 15) =

Fig. 2. Effect of curcumin (25 and 50 mg/kg, PO) posttreatment on STZ (IC) induced memory deficit in Morris water maze in mice. Data values are expressed as mean ★ latency time (s) ± S.E.M. ★Significant difference (★P b 0.01 and ★P b 0.001) in latency time in comparison to session 1.

Fig. 3. (a) Preventive effect of curcumin (50 mg/kg, PO) on STZ (IC) induced memory deficit in passive avoidance in mice. Data values are expressed as mean transfer latency ★ time (s) ± S.E.M. ★Significant difference (★P b 0.01 and ★P b 0.001) in comparison to Acquisition trial. (b) Effect of curcumin (50 mg/kg, PO) post treatment on STZ (IC) induced memory deficit in passive avoidance in mice. Data values are expressed as mean transfer latency time (s) ± S.E.M. ★Significant difference (★P b 0.01) in comparison to Acquisition trial.

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Spontaneous locomotor activity The spontaneous locomotor activity remained unaltered among different groups [Total: F (4, 10) = 2.107, P N 0.05, Ambulatory: F (4, 10) = 0.217, P N 0.05 and Vertical: F (4, 10) = 1.969, P N 0.05]. Effect of curcumin on cerebral blood flow (CBF) in STZ (IC) induced memory deficit mice CBF was measured by LDF and expressed in blood perfusion units (BPU). STZ administration significantly (P b 0.001) reduced CBF in comparison to control and aCSF groups. However, administration of aCSF by IC route had no significant (P N 0.05) effect on CBF in comparison to control. Curcumin (10, 20 and 50 mg/kg, PO) dose dependently restored CBF in STZ injected mice [F (3, 15) = 24.86, P b 0.001] (Fig. 4). Effect of curcumin on oxidative and nitrosative stress in STZ treated mice Malondialdehyde (MDA) level STZ treated group showed significant increase in MDA level (P b 0.001) in comparison to control and aCSF group. This increase in MDA was attenuated in the brain of curcumin fed mice (Fig. 5). MDA level in curcumin (20 and 50 mg/kg, PO) treated groups was significantly (P b 0.001) lower than the STZ treated group. Glutathione (GSH) level STZ caused marked (P b 0.001) depletion of GSH level compared with control and aCSF-treated mice. This reduction was significantly (P b 0.001) prevented by curcumin (20 and 50 mg/kg, PO) treatment in STZ injected mice (Fig. 6). Measurement of reactive oxygen species by spectrofluorometry The level of reactive oxygen species in the brain was measured on day 21 after the first dose of STZ. Production of ROS (%) was measured relative to control. Treatment with STZ increased ROS (194.13 ± 1.15%) production while preventive treatment with curcumin (50 mg/kg, PO) significantly (P b 0.05) reduced amount of ROS in STZ treated mice (Fig. 7). Effect of curcumin on STZ (IC) induced nitrosative stress Nitrite levels significantly (P b 0.001) elevated in the brain of STZ treated animals as compared to control. Curcumin (50 gm/kg, PO) administration significantly (P b 0.01) inhibited this increase in nitrite levels in STZ treated mice (Fig. 8).

Fig. 4. Effect of pretreatment of curcumin (10, 20 and 50 mg/kg, PO) on cerebral blood flow (CBF) in STZ (IC) induced memory deficit mice. Data values are expressed as mean blood perfusion unit (BPU) ± S.E.M. #Significant difference (#P b 0.001) in BPU as compared to control and aCSF group and ★significant difference (★P b 0.01 and ★ ★P b 0.001) in BPU as compared to the STZ group.

Fig. 5. Effect of pretreatment of curcumin (10, 20 and 50 mg/kg, PO) on MDA level. Data values are expressed as mean MDA level (nmol/mg protein) ± S.E.M. #Significant difference (#P b 0.001) in MDA level as compared to control and aCSF group and ★ significant difference (★P b 0.001) in MDA level as compared to the STZ group.

Effect of curcumin on STZ (IC) induced changes in acetylcholinesterase activity Acetylcholinesterase (AChE) activity significantly (P b 0.001) increased in STZ treated mice as compared with control and aCSF group. Curcumin (20 and 50 mg/kg, PO) significantly (P b 0.001) prevented rise in AChE activity in STZ treated mice (Fig. 9). Blood glucose There was no significant difference in blood glucose level (mg/dl) among control, aCSF, STZ and curcumin (10, 20 and 50 mg/kg) treated STZ groups [F (5, 30) = 0.245, P N 0.05]. Discussion The present study examined the effect of curcumin treatment on cerebral blood flow (CBF), memory impairment, oxidative stress and cholinergic dysfunction in intracerebral (IC) streptozotocin (STZ) induced model of memory impairment in mice. The intracerebroventricular (ICV) STZ rat model is an appropriate animal model used for study of sporadic Alzheimer type dementia (Nitsch and Hoyer 1991; Lannert and Hoyer 1998; Sharma and Gupta 2001; Agrawal et al. 2009). In the present study, STZ at a subdiabetogenic dose of 0.5 mg/kg was used. Twice administration of STZ at 48h apart in mice by IC route showed a persistent memory deficit in Morris water maze as well as

Fig. 6. Effect of pretreatment of curcumin (10, 20 and 50 mg/kg, PO) on GSH level Data values are expressed as mean GSH level (µg/mg protein) ± S.E.M. #Significant difference (#P b 0.001) in GSH level as compared to control and aCSF group and ★ significant difference (★P b 0.001) in GSH level as compared to the STZ group.

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Fig. 7. Effect of pretreatment of curcumin (10, 20 and 50 mg/kg, PO) on % level of free radicals.#Significant difference (#P b 0.001) as compared to aCSF group and ★significant difference (★P b 0.05) as compared to the STZ group.

passive avoidance tests as evidenced by no reduction in escape latencies in Morris water maze and significantly reduced retention latencies in passive avoidance behavior. Interestingly, in passive avoidance test, we found that STZ administration affected memory recalling with no effect on memory acquisition and consolidation (Fig. 3b) whereas in the Morris water maze test, STZ treated animals exhibited acquisition deficit (Fig. 2) as evidenced in post treatment experiments. This phenomenon needs further exploration at different stages of memory. Further, there was a significant reduction in CBF in STZ treated mice as measured by laser Doppler flowmetry (LDF). To check validity of this method, we have also measured CBF within cortical region at 6 mm lateral and 1 mm posterior to bregma by LDF in normal mice (n = 30) and found no significant difference among them (data not shown). It suggests that LDF can be used to monitor change in CBF in different groups of animal. Moreover, intracerebral administration of aCSF had no significant effect on CBF measured at same site as in control, whereas STZ (IC) caused significant reduction in CBF. The decrease in CBF was associated with the impairment in memory functions following STZ administration. This finding is in conformity with many clinical studies showing alteration in cerebral microcirculation in patients with Alzheimer disease (AD) (Crawford 1996; Prohovnik et al. 1988; O'Brien et al. 1992; Wyper et al. 1993). Though the exact mechanism for this microcirculation impairment is not known, the probable reasons include oxidative stress and endothelial dysfunction causing restriction of blood flow to the brain (Ajmani et al. 2000). STZ caused oxidative stress as evidenced by significant increase in MDA level and decrease in GSH level. Further, there was a significant rise in reactive oxygen species (ROS) and nitrite levels in brain of STZ treated mice. This increase in

Fig. 8. Effect of pretreatment of curcumin (10, 20 and 50 mg/kg, PO) on nitrite level. # Significant difference (#P b 0.001) as compared to aCSF group and ★significant difference (★P b 0.01) as compared to the STZ group.

Fig. 9. Effect of pretreatment of curcumin (10, 20 and 50 mg/kg, PO) on AChE activity. Data values are expressed as mean AChE activity (µmol/min/mg protein)± S.E.M. #Significant difference (#P b 0.001) in AChE activity as compared to control and aCSF group and ★ significant difference (★P b 0.001) in AChE activity as compared to the STZ group.

oxidative stress may be due to hyperglycemic condition prevailing in brain following STZ. It has been reported that brain slices from ICV STZ rats show reduced glucose consumption from incubation medium as compared to control rats leading to hyperglycemic condition in brain (Pathan et al. 2006). Plaschke and Hoyer (1993) showed increased extracellular concentration of glucose in the brain of ICV STZ injected rats. This may cause increased nonenzymatic glycosylation of proteins and glucose auto-oxidation, resulting into formation of advanced glycation end-products. This results in oxidative stress and cellular damage (Ott et al. 1999). Oxidative stress, due to hyperglycemia, hyperlipidemia, hypertension and cigarette smoking, causes endothelial dysfunction (Cai and Harrison 2000). Besides increased free radicals, due to enhanced oxidative stress, there was an increase in level of nitrite. Hyperglycemia induces up regulation of iNOS and concomitant increase of superoxide production leads to the formation of peroxynitrite, a powerful pro-oxidant (Cosentino et al. 1997; Spitaler and Graier 2002; Ceriello et al. 2002), which further aggravates the oxidative stress and endothelial dysfunction. Therefore, in this model of memory impairment, both impaired glucose metabolism and oxidative stress may be responsible for endothelial dysfunction in cerebral vasculature. The impairment of endothelial function is accompanied with decreased cerebral perfusion which has been recently associated with dementia (Nash and Fillit 2006; Fisher et al. 2006). Pretreatment of curcumin, in STZ injected mice, improved spatial memory and condition avoidance memory as evidenced by improved performance in Morris water maze and step through passive avoidance test. Earlier studies in different memory impairment models (Frautschy et al. 2001; Dairam et al. 2007; Kuhad and Chopra 2007) and recently, Ishrat et al. (2009) in STZ induced model of memory deficit have reported preventive effect of curcumin. But the therapeutic effects of curcumin in STZ induced memory deficit mice have not been reported so far. In the present study curcumin not only exhibited preventive effect against memory decline but it also showed its potential against memory deficit induced by STZ in post memory deficit treatment. In passive avoidance test, administration of curcumin for one week (days 16–22) after STZ induced memory deficit (days 14–15) showed memory improvement without giving second acquisition trial. But vehicle treated group exhibited memory impairment even after reacquisition on day 23 (Fig. 3a). This indicates that STZ has no effect on acquisition of passive avoidance memory. In contrast, analysis of latency time in water maze test revealed that mice treated with STZ exhibited acquisition deficit. Mice with STZ induced memory deficit (days 14–18) were given one week treatment with curcumin (days 19– 25). These mice showed memory improvement from session 3 (Fig. 2)

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requiring reacquisition on day 26. However, no significant effect was observed in vehicle treated group even after reacquisition. No significant difference in locomotor activity was found among different groups. This excludes the possibility that the locomotor activity per se may have contributed to the changes in Morris water maze and passive avoidance behavior. Systemic administration of STZ induces diabetes which can lead to memory impairment (Kuhad and Chopra 2007) therefore we measured peripheral blood glucose level which remained unaffected by STZ (0.5 mg/kg, IC). This suggests that memory deficit is not linked to the peripheral glucose level in this model. Further, curcumin dose dependently improved CBF in STZ (IC) induced memory deficit mice. We found a dose dependent correlation of effect of curcumin on reduction in CBF and memory impairment. While, curcumin (20 and 50 mg/kg) improved both CBF and memory, the dose of 10 mg/kg had no effect on either parameter. This improvement in memory and CBF by curcumin has been attributed to its action as a potent antioxidant that results from its direct free radicals scavenging activity and ability to induce antioxidant enzymes (Sreejayan and Rao 1994). Curcumin produced a significant fall in MDA and increase in GSH levels indicating a decrease in oxidative stress in the brain of STZ treated mice. Our results also showed a decrease in ROS and nitrite level in curcumin treated mice. The reduction in nitrite level by curcumin may be due to its effect on inducible nitric oxide synthase (iNOS) expression as curcumin downregulates iNOS expression in vitro and in vivo (Chan et al. 1998; Begum et al. 2008). Beside oxidative stress, there is decreased activity of glycolytic enzymes in the STZ model of memory deficit resulting in reduction in acetylcholine level (Blokland and Jolles 1994; Weinstock et al. 2001; Plaschke and Hoyer 1993) which is intricately associated with cognition. Acetylcholine is degraded by the enzyme acetylcholinesterase (AChE) and AChE inhibitors are the most effective pharmacological approach for the symptomatic treatment of AD (Racchi et al. 2004). In the current study, we found increased AChE activity in the brain of STZ treated mice. This finding is in conformity with the previous studies showing increase in AChE expression (Lester-Coll et al. 2006) and activity following central STZ injection (Saxena et al. 2007; Tota et al. 2009; Agrawal et al. 2009). Further, it is reported that the activity of AChE increased in cerebral cortex of diabetic rats in which diabetes was induced by STZ (IP) (Sanchez-Chavez and Salceda 2000; Kuhad and Chopra 2007). Therefore, the changes in AChE activity may be due to, as discussed above, hyperglycemia like condition in brain produced by STZ administration. Curcumin, devoid of any inherent anti AChE activity (data not shown), dose dependently restored AChE activity in STZ treated mice. The restoration of AChE activity by curcumin may be due to amelioration of disturbed glucose metabolism and insulin signaling induced by STZ (Isik et al. 2009). Further, Kuhad and Chopra (2007) reported that curcumin decreased AChE activity in cerebral cortex of diabetic rats. Conclusion In conclusion, the present study corroborates many clinical findings that memory deficit is associated with impaired cerebral circulation as evidenced by decreased CBF following STZ. Curcumin treatment (pre and posttreatment) showed improvement in memory which may be due to its potent antioxidant action and improvement in cerebral circulation. Therefore, the use of curcumin as dietary supplement should be encouraged to ward off age-associated memory disorders like AD. Acknowledgements Authors HA and ST are grateful to Miss Sachi Bharti and Miss Gunjan Saxena for their assistance in the conducting experiments and

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