Behavioural Brain Research 171 (2006) 9–16

Research report

Coenzyme Q10 modulates cognitive impairment againstintracerebroventricular injection of

streptozotocin in rats

Tauheed Ishrat a, M. Badruzzaman Khan a, Md. Nasrul Hoda a, Seema Yousuf a,Muzamil Ahmad b, Mubeen A. Ansari c, Abdullah S. Ahmad b, Fakhrul Islam a,∗

a Neurotoxicology Laboratory, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110062, Indiab The Johns Hopkins University/School of Medicine, ACCM Department, Neuroresearch Division, 720 Rutland Avenue,

Traylor 806, Baltimore, MD 21205, USAc Pediatrics Medical University of South Carolina, South Carolina, USA

Received 24 November 2005; received in revised form 2 March 2006; accepted 9 March 2006Available online 18 April 2006

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Coenzyme Q10 (CoQ10), a peculiar lipophilic antioxidant, is an essential component of the mitochondrial electron-transport chain. It is involvedn the manufacturing of adenosine triphosphate (ATP) and has been linked with improving cognitive functions. The present study shows theeuroprotective effect of CoQ10 on cognitive impairments and oxidative damage in hippocampus and cerebral cortex of intracerebroventricular-treptozotocin (ICV-STZ) infused rats. Male Wistar rats (1-year old) were infused bilaterally with an ICV injection of STZ (1.5 mg/kg b.wt., inormal saline), while sham group received vehicle only. After 24 h, the rats were supplemented with CoQ10 (10 mg/kg b.wt. i.p.) for 3 weeks.he learning and memory tests were monitored 2 weeks after the lesioning. STZ-infused rats showed the loss of cognitive performance in Morrisater maze and passive avoidance tests. Three weeks after the lesioning, the rats were sacrificed for estimating the contents of thiobarbituric

cid reactive substances (TBARS), reduced glutathione (GSH), protein carbonyl (PC), ATP and the activities of glutathione peroxidase (GPx),lutathione reductase (GR), cholineacetyltransferase (ChAT) and acetylcholinesterase (AChE). Significant alteration in the markers of oxidativeamage (TBARS, GSH, PC, GPx and GR) and a decline in the level of ATP were observed in the hippocampus and cerebral cortex of ICV-TZ rat. A significant decrease in ChAT activity and a concomitant increase in AChE activity were observed in the hippocampus. However,upplementation with CoQ10 in STZ-infused rats reversed all the parameters significantly. Thus, the study demonstrates that CoQ10 may have aherapeutic importance in the treatment of Alzheimer’s type dementia.

2006 Elsevier B.V. All rights reserved.

eywords: Streptozotocin; Cognitive impairment; Coenzyme Q10; Oxidative stress; Lipid peroxidation; Alzheimer’s disease; Cholineacetyltransferase; Adenosineriphosphate

. Introduction

Alzheimer’s disease (AD) is a progressive and irreversibleeuropsychiatric disorder of the brain with a deadly outcomend unknown etiology. It is the leading cause of senile dementia,haracterized by neuronal degeneration and cognitive deteri-

∗ Corresponding author. Tel.: +91 11 2605 9688x5567; fax: +91 11 2605 9663;obile: +91 9818684535.

E-mail addresses: fislam2001@yahoo.co.in, fislam@jamiahamdard.ac.inF. Islam).

orations, especially in the elderly [14]. Oxidative stress, animbalance between free radicals and antioxidant system, playsa critical role in the pathogenesis of AD [16,8].

Because of its high sensitivity to stress-induced degenerativeconditions brain is targeted by different stressors. Oxygen radi-cals can attack on proteins, nucleic acids and lipid membranes,thereby disrupting cellular functions and integrity. Brain tissuecontains a large amount of polyunsaturated fatty acids whichare particularly vulnerable to free radical attacks [18]. Antioxi-dant enzymes like glutathione peroxidase (GPx) and glutathionereductase (GR) have a prominent role in the management ofoxidative stress within the cell; they convert superoxide radicals

166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2006.03.009

10 T. Ishrat et al. / Behavioural Brain Research 171 (2006) 9–16

and peroxides to innocuous forms, often with the concomitantoxidation of reduced glutathione (GSH), to its oxidized form(GSSG) and recycles GSSG to GSH to maintain the antioxidantpotential. Although other pathways may be involved in the cel-lular management of free radicals induced oxidative damage.Analysis of these antioxidant enzymes as well as contents ofGSH, lipid peroxidation and carbonyl group may yield a snap-shot of oxidative stress.

Deficiency of insulin affects numerous brain functionsincluding memory. Clinical studies have established a linkbetween AD and insulin resistance of the brain [17]. Insulin-receptor desensitization has been proposed as a cause for reducedenergy metabolism and cholinergic deficiency [6]. Impairedenergy metabolism in neurons may induce production ofincreased amount of free radicals [9,47]. Intracerebroventricularstreptozotocin (ICV-STZ) injection in rats’ brain causes desen-sitization of insulin receptors and biochemical changes, thusproviding a relevant model to sporadic dementia of Alzheimertype [21,22]. It causes reduced energy metabolism/oxidativestress leading to cognitive dysfunction by inhibiting the synthe-sis of adenosine triphosphate (ATP) and acetyl-CoA. This ulti-mately results into cholinergic deficiency supported by reducedcholineacetyltransferase (ChAT) activity in hippocampus and anincreased cholinesterase (ChE) activity in the brain of ICV-STZrats [7,20,37,41,44]. ICV-STZ also induces microglial activationand specific damage to myelinated tracts in the fornix throughgb

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sine 5-triphosphate (ATP) Bioluminescent assay kit, choline chloride, ethylenediamine tetraacetic acid (EDTA), sodium tetraphenyl borate, physostigmine(eserine sulfate), streptozotocin (STZ), acetyl-CoA and coenzyme Q10 (CoQ10)were purchased from Sigma–Aldrich, Chemicals Pvt. Ltd., India. [14C] acetyl-CoA was purchased from New England Nuclear (NEN), Boston, MA. Otherchemicals were of analytical reagent grade.

2.2. Animals

One-year-old Wistar male rats, weighing 480–520 g, were used in this study.They were kept in the Central Animal House of Hamdard University in colonycages at an ambient temperature of 25 ± 2 ◦C and relative humidity 45–55% with12 h light/dark cycles. They had free access to standard rodent pellet diet andwater ad libitum. The food was withdrawn 18–24 h before the surgical procedure.Experiments were conducted in accordance with the Animal Ethics Committeeof the University.

2.3. Experimental procedure

2.3.1. Surgical procedureThe animals were anesthetized with 400 mg/kg b.wt. chloral hydrates intra-

paritonially (i.p.) and placed on a stereotaxic frame and skull was exposed. Thestereotaxic coordinates for lateral ventricle [36] were measured accurately asanterio-posterior −0.8 mm, lateral 1.5 mm and dorso-ventral −4.0 mm relativeto bregma and ventral from dura with the tooth bar set at 0 mm. Through askull hole, a 28-gauge Hamilton® syringe of 10 �l attached to a micro-injectorunit and piston of the syringe was lowered manually into each lateral ventricle.The lesioned groups received a bilateral ICV injection of STZ (1.5 mg/kg, bodyweight in saline, 5 �l/injection site) with the same dose as in previous studies[7,37,38]. The sham groups underwent the same surgical procedures, but samevi

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eneration of oxidative stress, thereby disrupting connectionsetween the septum and hippocampus [42].

Since oxidative damage is implicated in the etiology of neuro-ogical complications, treatment with antioxidants has been useds a therapeutic approach in various types of neurodegenerativeisease [1,3]. Coenzyme Q10 (CoQ10), a vitamin-like lipophilicntioxidant compound, has been reported to improve cogni-ive functions [32], up-regulates mitochondrial function andacilitates the synthesis of ATP. CoQ10 significantly attenuatesntrastriatal malonate induced depletion of ATP and increasedactate in rat brain mitochondria [4]. Supplementation of CoQ10n rats increases endogenous CoQ10 content in the brain [25]nd affords protection as an antioxidant against generation ofree radicals as well as oxidative modifications of biomolecules5,28,40,43,45]. It can also regenerate other powerful antioxi-ants like alpha tocopherol [27].

CoQ10 may have a therapeutic role in the amelioration ofbove-mentioned complications via its antioxidant potential andodulation of energy generating properties. This study investi-

ates the post-treatment effects of CoQ10 therapy on cognitiveysfunction and biochemical alterations in hippocampus anderebral cortex of ICV-STZ rats.

. Materials and methods

.1. Chemicals and reagents

Oxidized (GSSG) and reduced (GSH) forms of glutathione, glu-athione reductase (GR), nicotinamide adenine dinucleotide phosphateeduced form (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), 5-5′-dithio-bis--nitrobenzoicacid (DTNB), thiobarbituric acid (TBA), trichloroacetic acidTCA) 2,4-dinitrophenyhydrazine (DNPH), acetylthiocholine iodide, adeno-

olume of saline was injected instead of STZ. After surgery, the rats were housedndividually and had access to food and water ad libitum.

.3.2. Experiment IExperiment I was carried out to evaluate the post-treatment effect of CoQ10

10 mg/kg b.wt., i.p. in corn oil) supplementation for 3 weeks on the con-ents of TBARS, GSH, PC and activity of antioxidant enzymes. The rats wereivided into four groups of 10 animals each. Group 1: sham operated vehiclereated control (S); group 2: sham operated and CoQ10 (10 mg/kg b.wt. i.p., inorn oil) treated (S + CoQ10); group 3: ICV-STZ infused and vehicle treatedesioned (L); group 4: ICV-STZ infused and CoQ10 (10 mg/kg b.wt.) treatedL + CoQ10).

.3.3. Experiment IIExperiment II was carried out to evaluate the post-treatment effect of CoQ10

10 mg/kg b.wt., i.p. in corn oil) supplementation for 3 weeks on the activity ofhAT and AChE in the hippocampus and on the content of ATP in the hippocam-us and cerebral cortex. The rats were divided into four groups as in experiment.

.4. Behavioral testing

The behavioral tests were started 2 weeks after lesioning. The experimentas performed between 9.00 a.m. and 4.00 p.m. in the laboratory at standardptimal conditions. All the tests were performed and analyzed by the subjectlind to the experiment.

.4.1. Passive avoidance testOn the day 14th and 15th of lesioning, the rats were tested for memory reten-

ion deficits using passive avoidance apparatus [39]. The apparatus consisted oftwo-compartment dark/light shuttle box. A guillotine door separated the two

ompartments. The dark compartment had a stainless steel shock grid floor.uring the acquisition trial, each rat was placed in the light chamber. After 60 sf habituation period, the guillotine door separating the light and dark chamberas opened, and the initial latency of animals to enter the dark chamber was

ecorded. Rats with an initial latency time of more than 60 s were excluded from

T. Ishrat et al. / Behavioural Brain Research 171 (2006) 9–16 11

further experiments. Immediately after the rat had entered the dark chamber,the guillotine door was closed and an electric foot shock (75 V, 0.2 mA, 50 Hz)was delivered to the floor grids with a stimulator for 3 s. Five seconds later, therat was removed from the dark chamber and returned to its home cage. After24 h, the retention latency time was measured in the same way as in the acqui-sition trial but foot shock was not delivered and the latency time was recordedto a maximum of 600 s. Short latencies indicate poor retention, compared tosignificantly longer latencies.

2.4.2. Morris water maze testAfter 24 h of passive avoidance test, spatial learning and memory of ani-

mals were tested in Morris water maze [34]. It consisted of a circular watertank (132 cm diameter and 60 cm height) that was partially filled with water(25 ± 2 ◦C). A non-toxic paint was used to render the water opaque. The poolwas divided virtually into four equal quadrants, labeled north–south–east–west.An escape platform (10 cm in diameter) was hidden 2 cm below the surface of thewater on a fixed location in one of the four quadrants of the pool. The platformremained in the same quadrant during the entire experiment. Before the trainingstarted, rats were allowed to swim freely into the pool for 60 s without platform.They were given four trials (once from each starting position) per session for 5days, each trial having a ceiling time of 60 s and a trial interval of approximately30 s. After climbing on to the platform, the animal remained there for 30 s beforethe commencement of the next trial. If rat failed to reach the escape platformwithin the maximum allowed time of 60 s, it was gently placed on the platformand allowed to remain there for the same interval of time. The time to reachthe platform (latency in s) was measured. Four hours after from the acquisitionphase, a probe test was conducted by removing the platform. Rats were allowedto swim freely into the pool for 60 s. The time spent in the target quadrant, whichhad previously contained the hidden platform was recorded. The time spent inthe target quadrant indicated the degree of memory consolidation taken placea

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culated as nmol GSH mg−1 protein, using a molar extinction coefficient of13.6 × 103 M−1 cm−1.

2.5.4. Protein carbonyl (PC) assayPC content was measured by the method of Levine et al. [29]. The cytosol

(0.5 ml) was treated with an equal volume of 20% TCA for protein precipitation.After centrifugation, the pellet was resuspended in 0.5 ml of 10 mM DNPH in2 M HCl and kept in a dark place for 1 h by vortexing repeatedly at 10 mininterval. This mixture was treated with 0.5 ml of 20% TCA. After centrifugationat 11,000 × g at 4 ◦C for 3 min, the precipitate was extracted three times with0.5 ml of 10% TCA and dissolved in 2.0 ml of NaOH at 37 ◦C. Absorbance wasrecorded at 360 nm. PC level was expressed as nmol carbonyl mg−1 protein,using a molar extinction of coefficient 22 × 104 M−1 cm−1.

2.5.5. Glutathione peroxidase (GPx) activityGPx (EC 1.11.1.9) activity was measured at 37 ◦C by the coupled assay

method of Wheeler et al. [49] in which oxidation of GSH was coupled toNADPH oxidation, catalyzed by GR. The reaction mixture consisted of 0.1 mlH2O2 (0.2 mM), 0.1 ml GSH (1 mM), 1.4 unit of 0.1 ml GR, 0.1 ml NADPH(1.43 mM), 0.1 ml sodium azide (1 mM), 1.4 ml PB (0.1 M, pH 7.4) and 0.1 mlPMS. GPx activity was defined as nmol NADPH oxidized min−1 mg−1 protein,using a molar extinction coefficient of 6.22 × 103 M−1 cm−1.

2.5.6. Glutathione reductase (GR) activityGR (EC 1.6.4.2) activity was measured by the method of Carlberg and

Mannerviek [10]. The assay system consisted of 1.65 ml PB (0.1 M, pH 7.6),0.1 ml EDTA (0.5 mM), 0.05 ml GSSH (1 mM), 0.1 ml NADPH (0.1 mM) and0.1 ml PMS in a total volume of 2.0 ml. The enzyme activity was quantitatedat 25 ◦C by measuring the disappearance of NADPH at 340 nm, and calculatedas nmol NADPH oxidized min−1 mg−1 protein, using a molar extinction coeffi-c

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fter learning.

.5. Biochemical analysis

.5.1. Tissue preparationAfter 3 weeks of lesioning, animals were sacrificed and their brains were

aken out quickly to dissect hippocampus and cerebral cortex by using rat brainatrix in the light of rat brain atlas [36]. The dissected brain parts were homog-

nized in 10 mM phosphate buffer (PB, pH 7.4). The homogenate was used forstimation of lipid peroxidation in terms of TBARS content, and also centrifugedt 800 × g for 5 min at 4 ◦C to separate the nuclear debris. The supernatant wasurther centrifuged at 10,000 × g for 20 min at 4 ◦C to get the post-mitochondrialupernatant (PMS) which was used for estimation of reduced GSH, PC andntioxidant enzymes activity.

.5.2. TBARS contentThe method of Utley et al. [46], as modified by Islam et al. [23], was used

or estimation of lipid peroxidation. Homogenate (0.25 ml) was pipetted into15 × 100 mm test tube and incubated at 37 ◦C in a metabolic shaker for 1 h.n equal volume of homogenate was pipetted into a centrifuge tube and placed

t 0 ◦C and marked at 0 h incubation. After 1 h of incubation, 0.5 ml of 5%w/v) chilled TCA was added followed by 0.5 ml of 0.67% TBA (w/v) into eachest tube and centrifuge tube, and centrifuged at 1000 × g for 15 min. There-fter, supernatant was transferred to other test tubes and placed in a boilingater bath for 10 min. The absorbance of pink color produced was measured at35 nm. The TBARS content was calculated by using a molar extinction coef-cient of 1.56 × 105 M−1 cm−1 and expressed as nmol of TBARS formed per0 min−1 mg−1 of protein.

.5.3. Reduced glutathione (GSH) contentGSH content was determined by the method of Jollow et al. [24] with

light modification. PMS was mixed with 4.0% sulfosalisylic acid (w/v) in1:1 ratio (v/v). The samples were incubated at 4 ◦C for 1 h, and later cen-

rifuged at 1200 × g for 15 min at 4 ◦C. The assay mixture contained 0.1 mlf supernatant, 1.0 mM DTNB and 0.1 M PB (pH 7.4) in a total volumef 1.0 ml. The yellow color developed was read immediately at 412 nm inspectrophotometer (Shimadzu-1601, Japan). The GSH content was cal-

ient of 6.22 × 103 M−1 cm−1.

.5.7. Adenosine 5-triphosphate (ATP) levelMitochondria from hippocampus and cerebral cortex were isolated by the

ethod of Nagy et al. [35]. The isolated mitochondria (1 mg/ml of protein) wereesuspended in a reaction mixture containing 0.25 M sucrose, 1 mM MgCl2,0 mM HEPES and 1 mM EDTA. The suspension was briefly sonicated andentrifuged at 5000 × g for 5 min. The supernatant (100 �l) incubated withuciferin–luciferase (5 mg/ml) and the bioluminescence was measured in a Lumi-escence spectrometer microplate reader (Perkin-Elmer, S 50B). The resultsere expressed as pmol of ATP mg−1 protein.

.5.8. Cholineacetyltransferase (ChAT) activityThe activity of ChAT was assayed in the hippocampus, using the method of

onum [15]. The tissue was homogenized (5%, w/v) in ice cold PB (50 mM, pH.4). Triton X-100 (0.5%, v/v) was added to the homogenate to ensure enzymeelease. It was centrifuged at 12,000 × g at 4 ◦C and the supernatant was assayed.ubstrate mixture (125 �l) containing 0.4 mM [14C] acetyl-CoA, 300 mM NaCl,0 mM PB (pH 7.4), 8 mM choline chloride, 20 mM EDTA-2Na and 0.1 mMhysostigmine was added to enzyme solution (75 �l) in scintillation vial andhe mixture was incubated at 37 ◦C for 30 min. Thereafter, 4.5 ml of cold (4 ◦C)0 mM PB (pH 7.4) was added to stop the reaction. The [14C] ACh was extractedy adding 2.0 ml of acetonitrile containing 0.5% tetraphenylborate and 10 ml ofoluene scintillation fluid to the vial. The vials were shaken lightly and keptvernight for the separation of two layers before determining the radioactivityn a Backman Scintillation Counter (LKB Wallace-1410). Data were calculateds nmol acetylcholine h−1 mg−1 protein.

.5.9. Acetylcholinesterase (AChE) activityAChE activity was determined by a modified method of Ellman et al. [13].

riefly 2.6 ml of PB (0.1 M, pH 8.0), 40 �l of 0.075 M acetylthiocholine iodidend 0.1 ml of buffered Ellman’s reagent (DTNB 10 mM, NaHCO3 15 mM)ere mixed and allowed to incubate for 5 min at room temperature. Enzyme

ample (40 �l) was added and optical density was measured at 412 nm withinmin. AChE activity was expressed as nmol thiocholine formed min−1 mg−1

rotein.

12 T. Ishrat et al. / Behavioural Brain Research 171 (2006) 9–16

2.5.10. Protein contentProtein content was determined by the method of Lowry et al. [31], using

BSA as a standard.

2.6. Statistical analysis

Results are expressed as mean ± S.E. Statistical analysis of the data was doneby applying the analysis of variance (ANOVA), followed by Tukey’s test forbiochemical parameters and Mann–Whitney –test for behavioral observations.The p-value < 0.05 was considered statistically significant.

3. Results

3.1. Behavioral observations

3.1.1. Effect of CoQ10 on passive avoidance responseCoQ10 improved passive avoidance response in ICV-STZ

rats (Fig. 1). On day 14, the mean initial latency of L groupand S + CoQ10 group was not significantly different from thatof S group. On day 15, the mean retention latency was notaltered in S + CoQ10 group but decreased significantly (p < 0.05)in L group, as compared to S group. It was recovered signifi-cantly (p < 0.05) with CoQ10 treatment in L + CoQ10 group, ascompared to L group. The mean retention latency was signifi-cantly higher in L + CoQ10 group than in L group, indicating animproved acquisition or retention of memory.

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Fig. 2. (A) Effects of CoQ10 on the acquisition of spatial learning in ICV-STZrats in the Morris water maze. Values are expressed as mean ± S.E. of eightanimals. Swimming times of the four trials per day for 5 days for each groupare shown. Escape latencies required by rats to find the hidden platform along 5consecutive days. Average latency time (days 2–5) to find hidden platform wasprolonged in L group rats ((a) p < 0.05 L vs. S group). CoQ10 administrationsignificantly reversed STZ-induced learning deficits in L + CoQ10 group ((b)p < 0.05 L + CoQ10 vs. L group). (B) Effects of CoQ10 on the mean percentagetime spent in the target quadrant in which the platform had previously beenlocated during acquisition in ICV-STZ rats. Values are expressed as mean ± S.E.of eight animals. CoQ10 supplementation significantly inhibited STZ-inducedmemory deficits in L + CoQ10 group ((a) p < 0.05 L vs. S group; (b) p < 0.05L + CoQ10 vs. L group).

learning performance due to ICV-STZ infusion. The poorer per-formance of L group was significantly (p < 0.05) improved by thetreatment of CoQ10 in L + CoQ10 group. Furthermore, on day 5,STZ rats exhibited significantly poorer performance in L group,as compared to S group, which was improved significantly(p < 0.05) with the supplementation of CoQ10 in L + CoQ10group, as compared to L group.

On the other hand, CoQ10 supplementation showed no sig-nificant effect on the latency in S + CoQ10 group, as comparedto S group.

On the probe trial, with the platform removed, L group failedto remember the precise location of the platform, spending sig-nificantly (p < 0.05) less time in the training quadrant than S

.1.2. Effect of CoQ10 on performance in Morris wateraze taskThe change in escape latency was observed onto a hidden

latform produced by training trials (Fig. 2A). Although theatencies to reach the submerged platform decreased graduallyn all the groups during 5 days of training in Morris water mazeest, the mean latency (days 2–5) was significantly (p < 0.05) pro-onged in L group, as compared to S group, showing a poorer

ig. 1. Effect of CoQ10 on the acquisition latency (AL) and retention latencyRL) in passive avoidance test in ICV-STZ rats. The values are expressed asean ± S.E. of eight animals. Retention latency time was recorded to a maxi-um of 600 s. L group rats show significantly decreased latencies to enter the

ark side in the passive avoidance test ((a) p < 0.05 L vs. S group). CoQ10 treat-ent prolonged the latency to enter the dark compartment in L + CoQ10 group

(b) p < 0.05 L + CoQ10 vs. L group).

T. Ishrat et al. / Behavioural Brain Research 171 (2006) 9–16 13

and S + CoQ10-treated groups (Fig. 2B). The mean percent timespending in the target quadrant was increased by the adminis-tration of CoQ10 in L + CoQ10 group, as compared to L group.

3.2. Biochemical observations

3.2.1. Effect of CoQ10 on TBARS content in hippocampusand cerebral cortex

The effect of CoQ10 on TBARS content was mea-sured to demonstrate the oxidative damage on lipids in hip-pocampus and cerebral cortex of ICV-STZ rats. There wasno significant alteration in TBARS content in S + CoQ10group, while it was elevated significantly (p < 0.05) in Lgroup, as compared to S group (Fig. 3). The change inTBARS content was significantly (p < 0.05) reversed byCoQ10 treatment in L + CoQ10 group, as compared toL group.

3.2.2. Effect of CoQ10 on GSH in hippocampus andcerebral cortex

Protective effect of CoQ10 on GSH level in hippocampus andcerebral cortex was observed (Fig. 4). The level of GSH was notelevated significantly in S + CoQ10 group but it was depletedsignificantly (p < 0.05) in L group as compared to S group inhippocampus and cerebral cortex. The CoQ10 increased its levelsg

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Fig. 4. Effect of CoQ10 on GSH level in hippocampus and cerebral cortex ofICV-STZ rats. Values are expressed as mean ± S.E. of eight animals. STZ lead toa significant decrease in the content of GSH in L group as compared to S group((a) p < 0.05 L vs. S group). Administration of CoQ10 significantly restoredGSH level in L + CoQ10 group as compared to L group ((b) p < 0.05 L + CoQ10vs. L group).

3.2.4. Effect of CoQ10 on antioxidant enzymes activity inhippocampus and cerebral cortex

The activity of antioxidant enzymes (GPx and GR) inS + CoQ10 group was not attenuated significantly, compared toits control. But the activity of these enzymes decreased signif-icantly in L group (p < 0.05), compared to S group (Table 1).On the other hand, the activities were reversed significantly(p < 0.05) by CoQ10 administration in L + CoQ10 group, com-pared to L group.

3.2.5. Effect of CoQ10 on ATP level in hippocampus andcerebral cortex

The level of ATP was not elevated significantly in S + CoQ10but declined significantly (p < 0.05) in L group, as compared toS group. The CoQ10 treatment restored ATP level significantly(p < 0.05) in L + CoQ10 group, compared to L group (Fig. 6).

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ignificantly (p < 0.05) in L + CoQ10 group, as compared to Lroup (Fig. 2.).

.2.3. Effect of CoQ10 on carbonyl content in hippocampusnd cerebral cortex

The carbonyl level was not altered significantly in S + CoQ10roup but increased significantly (p < 0.05) in L group, as com-ared to S group. The change in carbonyl content was signif-cantly (p < 0.05) reversed by CoQ10 treatment in L + CoQ10roup, as compared to L group (Fig. 5).

ig. 3. Effect of CoQ10 on TBARS content in hippocampus and cerebral cor-ex of ICV-STZ rats. Values are expressed as mean ± S.E. of eight animals.TZ lead to a significant increase in the content of TBARS in L group asompared to S group ((a) p < 0.05 L vs. S group). Administration of CoQ10ignificantly reduced TBARS content in L + CoQ10 group as compared to Lroup ((b) p < 0.05 L + CoQ10 vs. L group).

ig. 5. Effect of CoQ10 on carbonyl content in hippocampus and cerebral cortexf ICV-STZ rats. Values are expressed as mean ± S.E. of eight animals. STZ leado a significant increase in the content of carbonyl in L group as compared to

group ((a) p < 0.05 L vs. S group). Administration of CoQ10 significantlyeduced carbonyl content in L + CoQ10 group as compared to L group ((b)< 0.05 L + CoQ10 vs. L group).

14 T. Ishrat et al. / Behavioural Brain Research 171 (2006) 9–16

Table 1Effect of CoQ10 on the activity of antioxidant enzymes in hippocampus and cerebral cortex of ICV-STZ rats

Groups Hippocampus Cerebral Cortex

GPx (nmol NADPH oxidized min−1

mg−1 protein)GR (nmol NADPH oxidized min−1

mg−1 protein)GPx (nmol NADPH oxidized min−1

mg−1 protein)GR (nmol NADPH oxidized min−1

mg−1 protein)

S 56.51 ± 4.85 38.73 ± 4.12 127.63 ± 15.21 65.70 ± 5.76S + CoQ10 58.26 ± 6.55 (+3.09%) 37.86 ± 3.84 (−2.24%) 128.25 ± 12.36 (+0.04%) 68.45 ± 7.32 (+4.18%)L 36.70 ± 3.12a (−35.05%) 26.04 ± 2.13a (−32.76%) 88.92 ± 6.32a (−30.32%) 46.42 ± 4.24a (−29.34%)L + CoQ10 48.42 ± 3.62b (+32.20%) 33.96 ± 2.78b (+30.41%) 114.31 ± 9.31b (+28.55%) 62.84 ± 7.02b (+35.37%)

Values are expressed as mean ± S.E. of eight animals. STZ lead to significant alterations on the activity of antioxidant enzymes (GPx and GR) in L group as comparedto S group ((a) p < 0.05 L vs. S group). Administration of CoQ10 significantly attenuated activity of these enzymes in L + CoQ10 group as compared to L group ((b)p < 0.05 L + CoQ10 vs. L group). Values in percentage increase (+) or decrease (−) as compared to S or L group.

Fig. 6. Effect of CoQ10 on ATP level in hippocampus and cerebral cortex ofICV-STZ rats. Values are expressed as mean ± S.E. of eight animals. STZ leadto a significant depletion in ATP level in L group as compared to S group ((a)p < 0.05 L vs. S group). Administration of CoQ10 significantly restored its levelin L + CoQ10 group as compared to L group ((b) p < 0.05 L + CoQ10 vs. Lgroup).

3.2.6. Effect of CoQ10 on ChAT activity in hippocampusThe ChAT activity in the hippocampus was not attenu-

ated significantly in S + CoQ10 group but declined significantly(p < 0.05) in L group in comparison to S group. CoQ10 sig-

Table 2Effect of CoQ10 on the activity of ChAT and AChE in hippocampus of ICV-STZinfused rats

Groups ChAT activity(nmol acetycholine formed h−1

mg−1 protein)

AChE activity(nmol thiocholine formed min−1

mg−1 protein)

S 111.49 ± 5.90 1.52 ± 0.28S + CoQ10 120.87 ± 6.57 (+8.41%) 1.47 ± 0.36 (−3.28)L 46.75 ± 6.95a (−58%) 2.45 ± 0.24a (+61.18%)L + CoQ10 85.56 ± 3.81b (+84.78%) 1.66 ± 0.19b (−32.24%)

Values are expressed as mean ± S.E. of eight animals. STZ infusion produceda significant decrement on the activity of ChAT and a significant increment onthe activity of AChE in L group as compared to S group ((a) p < 0.05 L vs. Sgroup). Administration of CoQ10 significantly attenuates the activity of ChATand AChE in L + CoQ10 group as compared to L group ((b) p < 0.05 L + CoQ10vs. L group). Values in percentage increase (+) or decrease (−) as compared toS or L group.

nificantly (p < 0.05) increased the ChAT activity in L + CoQ10group, compared to L group (Table 2).

3.2.7. Effect of CoQ10 on AChE activity in hippocampusThe AChE activity in the hippocampus was not attenuated

significantly in S + CoQ10 group but increased significantly(p < 0.05) in L group, compared to S group. The CoQ10 sig-nificantly (p < 0.05) attenuated its activity in L + CoQ10 group,compared to L group (Table 2).

4. Discussion

The present study on the neuroprotective effect of CoQ10 oncognitive dysfunctions and biochemical alterations in rats, hasshown that CoQ10 significantly reversed the cognitive deficitsand biochemical alterations seen in hippocampus and cerebralcortex induced by ICV-STZ; this has often been attributed toan essential cofactor of the electron transport chain as well as apotent free radical scavenger in lipid and mitochondrial mem-branes [4,5,43].

Lipids and proteins, the major structural and functional com-ponents of the cell membrane, are the target of oxidative modi-fication by free radicals in neurodegenerative disorders. Exten-sive evidence exists on lipid peroxidation and protein oxidationleading to loss of membrane integrity, an important factor inadGlsamcbIlkpwaof[

cceleration of aging and age-related neurodegenerative disor-ers [30]. Lipid peroxidation may enhance due to depletion ofSH content in the brain, which is often considered as the first

ine of defense as an endogenous antioxidant against oxidativetress. Evidence has been presented that the neuronal defensegainst H2O2, which is the most toxic molecule to the brain, isediated primarily by the glutathione system [12]. In the pro-

ess, GSH is oxidized to GSSG which is reconverted to GSHy the action of enzyme GR, thus maintaining the pool of GSH.n conjunction with the reductant NADPH, GSH can reduceipid peroxides and other free radicals too. Oxidant radicals arenown to inactivate GR and GPx [19], as also observed in theresent and some earlier studies [41,48]. Prophylactic treatmentith CoQ10 significantly reversed the impact of oxidative alter-

tions seen in ICV-STZ rats; this shows the antioxidant potentialf CoQ10. Previous studies confirm that CoQ10 is a power-ul antioxidant that scavenges free radical induced damages5,28,40,45].

T. Ishrat et al. / Behavioural Brain Research 171 (2006) 9–16 15

Glucose is the principal source for energy production in thebrain, and an undisturbed glucose metabolism is pivotally sig-nificant for normal functioning of the brain and to conserve thecellular energy in the form of ATP. Moreover, adequate cellularfunctions such as learning, memory and cognition, are closelydependent on the integrity of the cellular energy metabolism[26]. Our results show that the depleted level of ATP in ICV-STZgroup via reducing the glucose energy metabolism in brain wassignificantly restored by CoQ10 supplementation. This showedthat the application of CoQ10 significantly attenuated malonate-induced depletion in the mitochondrial ATP level as earlierreported by Beal et al. [4]. CoQ10 has a modulatory effect on theessential cofactor of electron-transport chain in mitochondria forthe production of ATP.

Acetylcholine (ACh) is necessary for memory formation andretrieval. Its synthesis depends on the availability of acetyl Co-A,provided by the breakdown of glucose and insulin, which con-trols the activity of ChAT, a synthesizing enzyme for ACh. Tra-ditionally, ChAT activity has been considered as the best markerof cholinergic function, and the cognitive deficits is related tothe hippocampal ChAT activity. Blokland and Jolles [7] havereported a decreased ChAT activity in the hippocampus of ICV-STZ infused rats. Our study also show significantly decreasedChAT activity in the lesioned group. CoQ10 supplementationsignificantly restored ChAT activity in ICV-STZ infused rats.On the other hand, activity of AChE, a hydrolyzing enzymefwtiprd

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In a nutshell, present findings indicate that ICV-STZ treat-ment causes learning and memory deficits and biochemicalalterations in hippocampus and cerebral cortex in rats proba-bly by generating free radicals and depleting the ATP level byaltering its synthesis. CoQ10 supplementation improves learn-ing and memory deficits possibly by inhibiting the oxidativestress and improving the level of ATP.

Acknowledgments

The authors thank the Department of Ayurveda, Yoga &Naturopathy, Unani, Siddha and Homoeopathy (AYUSH), Min-istry of Health and Family Welfare, Government of India, NewDelhi, for financial assistance.

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