Behavioural pharmacology

Pharmacological modulation of I1-imidazoline and α2-adrenoceptorsin sub acute brain ischemia induced vascular dementia

Surbhi Gupta 1, Bhupesh Sharma n

Department of Pharmacology, School of pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Uttar Pradesh, India

a r t i c l e i n f o

Article history:Received 16 April 2013Received in revised form30 November 2013Accepted 4 December 2013Available online 12 December 2013

Keywords:MoxonidineClonidineMorris water mazeAcetylcholinesteraseOxidative stressTTC staining

a b s t r a c t

Sub-acute brain ischemia is a risk factor for the development of vascular dementia (VaD). Sub-acute brainischemia induced VaD, participates in a negative role in impaired cognition. Imidazoline receptors arewidely expressed in the central nervous system. But the role of I1-imidazoline and α2-adrenoceptors inVaD are still unknown. The present study has been designed to investigate the role of selectiveI1-imidazoline receptor modulator; moxonidine as well as α2-adrenoceptor modulator; clonidine in sub-acute brain ischemia induced VaD in mice (n¼8). Permanent bilateral common carotid arteries ligation(2VO) technique was used to induce sub-acute brain ischemia in mice. Assessment of spatial learning andmemory was done by using Morris water maze. Brain damage was assessed as percent infarct, using TTCstaining of brain coronal sections. Oxidative stress was assessed by estimating brain malondialdehyde(MDA), catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD). Cholinergic status wasassessed by brain acetylcholinesterase (AChE) activity. 2VO animals have shown significant reduction inlearning and memory as well as brain CAT, GSH and SOD, with significant increase in brain infarct size,MDA and AChE activity. Whereas, administration of moxonidine and clonidine significantly attenuated2VO induced learning and memory deficits, brain damage, brain oxidative stress and higher AChEactivity. It may be concluded that 2VO induced sub-acute brain ischemia has elicited dementia, whichwas attenuated by moxonidine and clonidine. Thus, modulators of I1-imidazoline receptors may beexplored further for their benefits in sub-acute brain ischemia induced vascular dementia.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Vascular dementia (VaD), a second leading cause of dementia,is characterized by loss of memory, impaired cognition andvascular lesions in the brain. VaD is caused due to obstructionof blood supply to the brain, which leads to ischemia (Miyamotoet al., 2010). Cerebrovascular and cardiovascular diseases arestated as chief risk factors of dementia of various types (de laTorre, 2012). Sub-acute brain ischemia causes mitochondrialdysfunction (Guang and Du, 2006), inhibition of protein synthesis

(de la Torre, 2012), destruction of the delicate balance of endo-genous antioxidases (Cechetti et al., 2012) and reactive oxygenspecies (ROS), which ultimately leads to functional and histo-pathological alterations (Liu and Zhang, 2012).

The imidazoline receptors are available throughout the body andthey have been reported to possess various functions. Imidazolinereceptors are categorized into three main classes, I1, I2 and qI3-imidazoline receptor (Nechifor and Ciubotariu, 2012). I1 receptorbinding sites are located in the rostro-ventrolateral medulla and alsoin the hypothalamus region of the brain (Raasch et al., 2000).Imidazoline receptor binding sites have been shown to change inpsychiatric conditions such as depression and addiction, along withneurodegenerative disorders such as Alzheimer's disease (AD) andHD (Tyacke et al., 2012). These receptors are also involved in theregulation of behavioral disorders such as suicidal behavior, stress,anxiety, food intake etc. (Nechifor and Ciubotariu, 2012) as well as inthe regulation of vasomotor tone, inflammation, cell proliferation,depression, stress and pain (Nikolic and Agbaba, 2012).

Moxonidine is an imidazoline compound with selective ago-nistic activity at I1-imidazoline receptor (Masajtis-Zagajewskaet al., 2010). Moxonidine has been reported to improve memoryand thinking in the hypertensive human population (Ostroumovaet al., 2001). It has also influence on neuropsychological status andbrain perfusion in elderly patients with arterial hypertension

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Abbreviations: 2VO, bilateral common carotid arteries ligation or two vesselocclusion; AChE, acetylcholinesterase; AD, Alzheimer's disease; CAT, catalase;CCH, chronic cerebral hypo perfusion; CMC, carboxy-methylcellulose; CPCSEA,Committee for the Purpose of Control and Supervision of Experiments on Animals;DTNB, 5, 50-dithiobis (2-nitrobenzoic acid); ELT, Eescape latency time;GSH, glutathione; IAEC, Institutional animal ethics committees; MDA, malondial-dehyde; MWM, Morris water maze; NBT, nitrobluetetrazolium; PBS, phosphatedbuffered saline; ROS, Reactive oxygen species; SOD, superoxide dismutase; TBARS,thiobarbituric acid reactive substances; TPF, 1,3,5-triphenylformazan; TSTQ, timespent in target quadrant; TTC, 2-3-5-triphenylterazolium chloride; VaD, vasculardementia

n Corresponding author. Tel.: þ91 8791636281, þ91 9958219190.E-mail addresses: sharmaslab3@gmail.com (S. Gupta),

drbhupeshresearch@gmail.com, conscienceresearch@scientist.com (B. Sharma).1 Tel.: þ91 992 7932746.

European Journal of Pharmacology 723 (2014) 80–90

(Martynov et al., 2002). Another clinical study has been reportedthat moxonidine, not in therapeutic doses, rather in low doses hasprovided learning, word recall and alertness, in hypertensivepatients (Wesnes et al., 1997). Modulators of I1-imidazoline recep-tors have not been studied in sub-acute brain ischemia inducedVaD. As I1-imidazoline receptors and their modulators have a rolein cognitive functions, inflammation and brain damage, so wehypothesized that modulation of I1-imidazoline receptors mayhave role in sub-acute brain ischemia induced VaD.

Clonidine is a mixed α2-adrenoceptor and I1-imidazolinereceptor agonist (Wang et al., 2005). It has been used as a pre-operative medication and sedative/anxiolytic drug (Hall et al.,2006). It has been reported as neuroprotective in cognitive deficits(Bardgett et al., 2008) and in the treatment of neuropsychiatricdisorders (Lemamy et al., 2012). Modulators of α2-adrenoceptorshave not been studied in sub-acute brain ischemia induced VaD.As α2-adrenoceptors and their modulators have an effect oncognition and neuroprotection, so we hypothesized that modula-tion of α2-adrenoceptors may provide benefits in sub-acute brainischemia induced VaD.

In the light of above, we have designed this study to investigatethe utility of moxonidine; a selective agonist of I1-imidazolinereceptors as well as clonidine; mixed α2-adrenoceptor agonist insub-acute brain ischemia induced VaD in mice.

2. Material and methods

2.1. Animals

Adult Swiss albino mice, weighing 20–25 g were employed inthe present study. The experimental protocol was approved byIAEC and the care of the animals was taken as per the guidelines ofthe CPCSEA, Ministry of Environment and Forest, Government ofIndia. All efforts were made to minimize animal suffering.

2.2. Drugs and chemicals

All chemicals were obtained from Sigma-Aldrich, USA; SiscoResearch Laboratory Pvt. Ltd., India; Unichem Laboratories, Indiaand Sun Pharma Pvt. Ltd., India. All drug solutions were freshlyprepared before use. Selection of doses and the dosing schedulewere based on previously published reports. Moxonidine andclonidine were suspended in 0.5% CMC and administered orallyto the animals. Moxonidine (30 and 60 mg kg�1) and clonidine(5 and 10 mg kg�1) were administered to rats thorough oral routerespectively (El-Ayoubi et al., 2002; Silva et al., 2004).

2.3. Bilateral common carotid arteries ligation or two vesselocclusion (2VO)

2VO rat model is a model of acute/subacute brain ischemia morethan a model of chronic hypoperfusion of the brain. Permanentbilateral common carotid arteries ligation was done as per thepreviously defined procedure (Azzubaidi1 et al., 2012). Two vesselocclusion (2VO) rat model of cerebral hypoperfusion has beenincreasingly used as a paradigm for neurodegenerative disorderswith permanent bilateral ligation of common carotid arteries (Farkaset al., 2007) creating a state of oligemia (cerebral hypoperfusion) thatin the long run leads to neurodegeneration predominantly topyramidal hippocampal neurons in charge of spatial (place) learningand memory (Nakazawa et al., 2004). Thus, 2VO may be utilized tocause a permanent and persistent cerebral hypoperfusion. Briefly,mice were anesthetized with chloral hydrate in dose of 400 mg/kg.A ventral midline skin incision was made in the neck area just abovethe sternal bone and after moderate tweezing of neck muscles, the

right carotid artery was identified and carefully separated from thevagus nerve. After that the carotid artery was doubly ligated with silksuture (4–0) just below the bifurcation into internal and externalcarotid arteries. After surgery, mice were placed under a heatinglamp for the prevention of hypothermia until full recovery fromgeneral anesthesia. The same surgical procedure was performed onthe left side for the ligation of the left common carotid artery after7 days of the first surgery. All surgical procedures were performedunder aseptic conditions. Sham-operated animals underwent thesame surgical procedure without carotid arteries ligation (Azzubaidi1et al., 2012).

2.4. Assessment of learning and memory by Morris water maze

We have exposed the animals to assess locomotor activity aswell as motor coordination before testing on Morris water maze(MWM). We have also noted the impairment in 2VO animals butthere was no significant difference was found to be presentbetween the 2VO animals and 2VOþdrug treated animals. Hence,we didn't show the data of locomotor activity and grip strength.Learning and memory was evaluated using Morris water maze, asper, previously published procedures (Sharma and Singh, 2010).

2.5. TTC staining

TTC staining was performed according to the previously pub-lished procedures (Gao et al., 2008).

2.6. Dissection and homogenization

Dissection and homogenization of brain were done accordingto the previously published procedures (Sharma and Singh, 2013).

2.7. Biochemical estimation of brain malondialdehyde (MDA),glutathione (GSH), acetylcholinesterase (AChE), catalase (CAT),superoxide dismutase (SOD) and protein

The brain MDA, GSH, AChE, CAT, SOD and protein were measuredspectrophotometerically (UV-1800 ENG 240V; Shimadzu Corporation,Japan) at 532 nm, 412 nm, 420 nm (Sharma et al., 2008b; Sharma andSingh, 2011a, 2012c), 240 nm (Aebi, 1984), 560 nm (Beauchamp andFridovich, 1971) and 750 nm (Singh et al., 2012) respectively usingpreviously defined procedures. (Supplementary material)

2.8. Experimental protocol

In the present study, total nine groups were employed and eachgroup was consisted of eight Swiss albino mice (Fig. 1). Initially 80animals were exposed to 2VO out of which some animals did notsurvive (10%), the remaining animals were randomly grouped intonine groups (as per the following protocol) so that each group wasconsisted of 8 mice. No significant difference in mortality rate wasfound between the groups.

Group I — Sham control: Sham surgery was performed on theanimals without ligating both carotid arteries with a 1-weekinterval. After that the animals were sutured back. Animalswere exposed to MWM, 26th day onwards. Acquisition trialswere performed from 26th to 29th day and retrieval trials wereperformed on the 30th day on MWM.Group II — Vehicle control group (CMC): Animals wereadministered 0.5% w/v sodium carboxy-methylcellulose i.e.CMC (10 ml kg�1 p.o., daily) for 24 days followed by exposureto MWM. Acquisition trials were performed from 20th to 23rdday and retrieval trials were performed on 24th day on MWM.

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Group III and IV —Moxonidine and clonidine per se: Animalswere administered moxonidine (60 mg kg�1 p.o., daily) andclonidine (10 mg kg�1 p.o., daily) for 24 days followed by expo-sure to MWM. Rest of the procedure was same as described ingroup II.Group V —2VO group: A permanent bilateral occlusion of thecommon carotid arteries with a 1-week interval between arteryocclusion was performed on the animals. Animals wereexposed to MWM, 26th day onwards. Acquisition trials wereperformed from 26th to 29th day and retrieval trials wereperformed on the 30th day on MWM.Group VI, VII, VIII and IX —2VO and moxonidine (dose1 anddose 2), clonidine (dose1 and dose 2): A permanent bilateralocclusion of the common carotid arteries with a 1-weekinterval between artery occlusion was performed on theanimals. Animals were administered with moxonidine(30 mg kg�1and 60 mg kg�1; p.o.) and clonidine (5 mg kg�1and10 mg kg�1; p.o.) for 24 days starting from the 7th day till endof study (day 30). Rest of the procedure was same asdescribed in group V.

2.9. Statistical analysis

Statistical analysis was done using SigmaStat v3.5. All resultswere expressed as mean7standard deviation. Data was statisti-cally analyzed using one-way analysis of variance (ANOVA) fol-lowed by Tukey's multiple range test. Po0.001 was considered tobe statistically significant.

3. Results

Sub-acute brain ischemia induced by 2VO impairs motorfunctions. Hence, there may be a possibility for modificationof ELT results. We have found no significant differencepresent between the sham control and 2VO animals (data notshown), which is in accordance to the (Shibata et al., 2007),where authors were not found any significant effect in motorfunctions.

3.1. Effect on escape latency time (ELT) and time spent in targetquadrant (TSTQ), using Morris water maze (MWM)

Sham control mice showed a downward tendency in their ELT.There was a noteworthy fall in day 4 ELT, when compared to day1 ELT of these mice (Fig. 2), reflecting normal learning capability. Onday 5, a considerable rise in TSTQ was noticed as compared to timespent in other quadrants (Fig. 3), reflecting normal retrieval as well.

Administration of CMC (0.5%, 10 ml/kg/day; p.o., 24 days),moxonidine (60 mg kg�1; p.o., 24 days) and clonidine (10 mg/kg/day; p.o., 24 days) per se did not show any substantial effect on ELTand TSTQ as compared to sham control mice. However, 2VO miceshowed a significant increase in day 4 ELT (29th day of 2VOsurgery) when compared to day 4 ELT of sham control animals(Fig. 2) indicating impairment of acquisition i.e. learning. Further-more, a significant decrease in day 5 TSTQ (30th day of 2VOsurgery) was noted, when compared to day 5 TSTQ of sham controlanimals (Fig. 3), indicating impairment of retrieval i.e. memory.

Administration of moxonidine (30 mg kg�1and 60 mg kg�1) andclonidine (5 mg kg�1and 10 mg kg�1), significantly prevented 2VOinduced rise in day 4 ELT, indicating attenuation of 2VO inducedimpairment of acquisition (Fig. 2). Treatment of these drugs alsoprevented 2VO induced decrease in day 5 TSTQ, indicating attenua-tion of 2VO induced impairment of memory (Fig. 3).

3.2. Effect on brain malondialdehyde (MDA)

2VO produced a significant increase in brain levels of MDA,compared to sham control animals (Fig. 4). Treatment withmoxonidine (dose 1 and dose 2) and clonidine (dose 1 and dose 2),significantly attenuated 2VO induced rise in MDA. Further, CMC,moxonidine and clonidine did not show any significant per se effecton MDA level (Fig. 4).

3.3. Effect on brain catalase (CAT)

2VO produced a significant reduction in brain levels of CAT,compared to sham control animals (Fig. 5). Treatment withmoxonidine (dose 1 and dose 2) and clonidine (dose 1 and dose 2),significantly attenuated 2VO induced reduction in CAT level. Further,

Fig. 1. Schematic representation of experimental protocol. MWM—Morris water maze; ELT—escape latency time (acquisition trials); TSTQ—time spent in the target quadrant(retrieval trial); CMC—carboxy-methylcellulose; 2VO—two-vessel occlusion; Mox—Moxonidine; Clo—Clonidine; D1—dose1; and D2—dose2.

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CMC, moxonidine and clonidine did not show any significant per seeffect on CAT level (Fig. 5).

3.4. Effect on brain glutathione (GSH) level

2VO produced a significant reduction in brain levels of GSH,compared to sham control animals (Fig. 6). Treatment withmoxonidine (dose 1 and dose 2) and clonidine (dose 1 and dose 2),significantly attenuated 2VO induced reduction in GSH level. Further,

CMC, moxonidine and clonidine did not show any significant per seeffect on GSH level (Fig. 6).

3.5. Effect on brain superoxide dismutase (SOD) activity

2VO produced a significant reduction in brain SOD activity,compared to sham control animals (Fig. 7). Treatment withmoxonidine (dose 1 and dose 2) and clonidine (dose 1 and dose 2),significantly attenuated 2VO induced reduction in SOD activity.

Fig. 3. Effect of various agents on mean time spent in the target quadrant (TSTQ) of animals, using Morris water maze. Results are expressed as mean7standard deviation;one way ANOVA followed by Tukey's multiple range test. F (8, 63)¼162.828; # Po0.001 versus mean time spent in other quadrants in respective groups; n Po0.001 versusmean time spent in target quadrant in sham control group; nn Po0.001 versus mean time spent in target quadrant in 2VO group. SC—sham control; CMC–carboxy-methylcellulose; Mox–moxonidine; Clo–Clonidine; 2VO–two vessel occlusion; D1–dose1; and D2–dose2.

Fig. 2. Effect of various agents on day 1 and day 4 escape latency time (ELT) of animals, using Morris water maze. Results are expressed as mean7standard deviation; oneway ANOVA followed by Tukey's multiple range test. F (8, 63)¼181.997; # Po0.001 versus day 1 ELT in respective group; n Po0.001 versus day 4 ELT of sham control group;nn Po0.001 versus day 4 ELT of 2VO group. SC—sham control; CMC—carboxy-methylcellulose; Mox—moxonidine; Clo—Clonidine; 2VO—two vessel occlusion; D1—dose1;and D2—dose2.

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Further, CMC, moxonidine and clonidine did not show any significantper se effect on SOD activity (Fig. 7).

3.6. Effect on brain acetyl cholinesterase (AChE) activity

2VO produced a considerable increase in brain AChE activity,compared to sham control animals. Treatment with moxonidine(dose 1 and dose 2) and clonidine (dose 1 and dose 2), significantlyattenuated 2VO induced rise in brain AChE activity. Further, CMC,

moxonidine and clonidine did not show any significant per seeffect on brain AChE activity (Fig. 8).

3.7. Effect on brain damage

2VO produced a significant increase in brain damage as shown byhigher percentage of infarct size in 2VO animals (Figs. 9 and 10). 2VOhas resulted in a higher percentage of infarct size, when compared tosham control animals. This suggests that 2VO has induced brain

Fig. 4. Effect of various agents on brain malondialdehyde (MDA) level. Results are expressed as mean7standard deviation; one way ANOVA followed by Tukey's multiplerange test. F (8, 63)¼999.582; # Po0.001 versus sham control group; n Po0.001 versus 2VO group. SC—sham control; CMC—carboxy-methylcellulose; Mox—moxonidine;Clo—Clonidine; 2VO—two vessel occlusion; D1—dose1; D2—dose2; and TBARS—thiobarbituric acid reactive substances.

Fig. 5. Effect of various agents on brain catalase (CAT) activity. Results are expressed as mean7standard deviation; one way ANOVA followed by Tukey's multiple range test.F (8, 63)¼566.166; # Po0.001 versus sham control group; n Po0.001 versus 2VO group. SC—sham control; CMC—carboxy-methylcellulose; Mox—moxonidine; Clo—Clonidine; 2VO—two vessel occlusion; D1—dose1; D2—dose2; and CAT—catalase.

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damage inmice. Percentage infarct size has been significantly reducedby administration of moxonidine (dose 1 and dose 2) as well asclonidine (dose 1 and dose 2). This suggests attenuation of 2VOinduced brain damage by these agents (Figs. 9 and 10).

It is important to note here that, we have not observed anysignificant correlation between the doses. Though there was somebetter effect in all the parameters on higher doses of the treatmentbut during statistical analysis we were unable to find any sig-nificant dose dependent effect in any of the treatments.

4. Discussion

Impairment of learning and memory is the characteristicfeature of dementia like Alzheimer's disease (AD) and vasculardementia (VaD) (Sharma et al., 2008a, 2008bSharma and Singh,2012a, 2012b, 2012c); (Singh et al., 2012). Morris water maze(MWM) is the most extensively accepted model for testing learn-ing and memory abilities in rodents (Sharma and Singh, 2011a,2011b, 2013). The data of MWM test suggests a decrease in escape

Fig. 6. Effect of various agents on brain glutathione (GSH) level. Results are expressed as mean7standard deviation; one way ANOVA followed by Tukey's multiple rangetest. F (8, 63)¼303.005; # Po0.001 versus sham control group; v Po0.001 versus 2VO group. SC—sham control; CMC—carboxy-methylcellulose; Mox—moxonidine; Clo—Clonidine; 2VO—two vessel occlusion; D1—dose1; D2—dose2; and GSH—glutathione.

Fig. 7. Effect of various agents on brain superoxide dismutase (SOD) activity. Results are expressed as mean7standard deviation; one way ANOVA followed by Tukey'smultiple range test. F (8, 63)¼134.084; # Po0.001 versus sham control group; n Po0.001 versus 2VO group. SC–sham control; CMC–carboxy-methylcellulose; Mox–moxonidine; Clo–Clonidine; 2VO–two vessel occlusion; D1–dose1; D2–dose2; SOD–superoxide dismutase.

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latency time (ELT) on day 4 of acquisition trials in sham controlanimals that denotes normal learning. Furthermore, an increase inmean time spent in the target quadrant (TSTQ) in search of theplatform on day 5 of retrieval trial was observed in sham controlanimals, that denotes normal memory.

In the present study, 2VO (two-vessel occlusion) has resulted inimpairment of learning and memory of mice. It has also damagedthe brain of animals (high percent infarct). Furthermore, anincrease level of brain malondialdehyde (MDA) and acetylcholi-nesterase (AChE) activity, with significant reduction in the level of

brain catalase (CAT), glutathione (GSH) and superoxide dismutase(SOD) was observed in brain of 2VO animals.

Blokland (1995) has reported that the central cholinergicsystem is associated with intellectual abilities like learning andmemory. Nanri et al. (1998) have reported that the ability ofacetylcholine receptor binding and acetylcholinetransferase activ-ity was decreased in the brain of hypoperfused animals. Previousstudies suggested that AChE activity is enhanced in case ofoxidative stress (Sayan-Ozacmak et al., 2012). Presence of vascularpathology, such as arterial stiffness, arteriolosclerosis, endothelial

Fig. 8. Effect of various agents on brain acetylcholinesterase (AChE) level. Results are expressed as mean7standard deviation; one way ANOVA followed by Tukey's multiplerange test. F (8, 63)¼592.448; # Po0.001 versus sham control group; n Po0.001 versus 2VO group. SC—sham control; CMC—carboxy-methylcellulose; Mox—moxonidine;Clo—Clonidine; 2VO—two vessel occlusion; D1—dose1; D2—dose2; AChE—acetylcholinesterase.

Fig. 9. Effect of various agents on brain infarct size. Results are expressed as mean7standard deviation; one way ANOVA followed by Tukey's multiple range test. F (4, 35)¼400.670; # Po0.001 versus sham control group; n Po0.001 versus 2VO group. Mox—moxonidine; Clo—Clonidine; 2VO—two vessel occlusion; D1—dose1; and D2—dose2.

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degeneration and blood–brain barrier dysfunction leads to chroniccerebral hypoperfusion (Kalaria et al., 2012). Thus, hypoperfusionrevealed significant impairment in brain energy metabolismwhich involves mitochondrial oxidative phosphorylation as wellas decreased concentrations of brain and plasma endogenousantioxidants (Horecký et al., 2011). Oxygen free radicals and lipidperoxidation are evidenced as capable of neuronal degenerationand death induced by sub-acute brain ischemia (Cechetti et al.,2012). Oxidative stress has been associated with the onset andprogression of dementias. Brain dementias display extensiveoxidative stress as indexed by protein oxidation, lipid peroxida-tion, free radical formation, DNA oxidation, and decreased anti-oxidants (Pocernich and Butterfield, 2012). Agents which interferewith free radical production and oxidative stress have long beenconsidered as one of the important approaches to slow downdementia progression (Pocernich and Butterfield, 2012).

Therefore in the present study to investigate the effect ofvarious agents in animal models of sub-acute brain ischemia, wehave assessed learning, memory, brain acetylcholinesterase activ-ity, brain oxidative status and brain damage, as all these para-meters are related to each other. All the results are emphasizing onthe occurrence of the vascular dementia condition by sub-acutebrain ischemia.

Sub-acute brain ischemia is believed to be a pathological cause inthe induction of VaD as well as AD (Daulatzai, 2012; Yao et al., 2012).Sub-acute brain ischemia was modeled by performing the perma-nent ligation of both common carotid arteries for studying thepathophysiology of VaD. While, performing the permanent ligationof bilateral common carotid arteries, one week interval was keptbetween the occlusion of both arteries to avoid abrupt reduction incerebral blood flow (Du et al., 2013). It has been reported thatthe cerebral blood flow falls too sharply and significantly after theligation of bilateral common carotid arteries, which results in thegeneration of inflammatory elements (Kitamura et al., 2012), which

have been reported to reduce the chances of survival up to a greaterextent and thus higher mortality rate. By keeping the differencebetween the arteries occlusion, reduction in mortality rate has beenachieved.

Previous reports have suggested that sub-acute brain ischemiacauses impairment in learning and memory, induction of oxidativestress as well as damage of the brain (Liu and Zhang, 2012). Resultsof various laboratories have shown that 2VO animals have shownelevated levels of brain malondialdehyde (MDA) (Chao et al., 2010)and brain acetylcholinesterase (AChE) activity (Duan et al., 2011),along with reduced levels of brain superoxide dismutase (SOD),brain catalase (CAT) (Castaño et al., 2013), brain glutathione (GSH)(Horecký et al., 2011). Thus, the results of this study are inaccordance with the previous reports from various labs.

Cerebral imidazoline receptors have been reported to improvemetabolic syndrome (Fellmann et al., 2013), stroke condition (Hanet al., 2013), baroreflex function (Wang et al., 2009), in drug addiction(Ciubotariu and Nechifor, 2012), in decreasing obesity (Chung et al.,2013), in treatment of depression, anxiety and post-traumatic stressdisorder (Halaris and Plietz, 2007), multiple sclerosis (Wang et al.,2011), inhibition of neuronal apoptosis (Montolio et al., 2012),protective against glutamate-induced neurotoxicity (Bakuridze et al.,2009), in control of hyperthermia (Rawls et al., 2007), antidipsogeniceffect (Andrade et al., 2003), in natriuresis, pancreatic insulin produc-tion (Nechifor, 2001).

Furthermore, in our study, moxonidine (a selective I1-imidazo-line receptor modulator) and clonidine (α2-adrenoceptor modu-lator) attenuated the impairment of learning and memory. Theyhave also attenuated brain damage of animals (reduced percentinfarct size). Administration of these drugs has also attenuated theincreased level of brain MDA and AChE activity, with significantreduction in the level of brain CAT, GSH and SOD induced by 2VO.

Moxonidine is involved in the brainstem control of the choli-nergic outflow (Haxhiu et al., 1998). Moxonidine has been shownto possess neuroprotective role in glutamate induced neurotoxi-city (Bakuridze and Gongadze, 2006). Ostroumova et al. (2001)have suggested that moxonidine significantly improves memoryand thinking. Long term treatment of moxonidine is also influ-enced neuropsychological status and brain perfusion in elderlypatients with arterial hypertension (Martynov et al., 2002). Fujitaet al. (2012) have suggested that moxonidine attenuates brainoxidative stress. Moxonidine has been reported to reduce theelevated levels of MDA and enhanced the levels of total glu-tathione, SOD and CAT (Yigiter et al., 2012). Thus, the results of thisstudy are in accordance with the previous reports.

Beneficial effects of moxonidine on coronary and systemic hemo-dynamics, metabolic markers of ischemia and neurohumoral para-meters in patients with essential hypertension were observed byMitrovic et al., 2001. Low non-hypotensive doses of moxonidine havebeen reported to decrease sympathetic activity and interfere withorgan damage (Dikow et al., 2004). Moxonidine has been reported toincrease the blood flow in cerebral ischemia (Doggrell, 2001). Cseteand Papp (2000) have reported the beneficial effect of moxonidine inimpaired cerebral blood flow in acute ischemic conditions. Tsutsuiet al. (2009) have reported the reno-protective effect of moxonidine inprevention of post-ischemic renal injury (ischemic acute kidneyinjury). Moxonidine also improves ischemic tolerance of the sparedmyocardium in myocardial infarction induced hypertrophic conditions(Van Kerckhoven et al., 2002). Selective agonistic modulation of I1receptors enhanced the phosphorylation of mitogen-activated proteinkinase, via the phosphatidylcholine-specific phospholipase C pathway(Zhang and Abdel-Rahman, 2005). Moxonidine after binding to theI1-imidazoline receptor activates adenosine A2A receptors (Harrazet al., 2012). It has been reported to inhibit voltage-dependent Caþ2

current (Kim et al., 2011), inhibition of NMDA and AMPA/kainatereceptors (Wang et al., 2007), effects on downstream Akt and p38

Fig. 10. Coronal sections stained with TTC, demonstrating ischemic brain injuryand protective effect of various agents. Representative photographs of brainsections stained with 2% 2, 3, 5-triphenyltetrazolium chloride (TTC). White patchesshow the infraction. There was a significant increase in infract size in 2VO group,which was reduced by moxonidine as well as clonidine. VO—two vessel occlusion;Mox—moxonidine; Clo—Clonidine; D1—dose1; and D2—dose2.

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MAPK (Aceros et al., 2011), upregulation of protein expression ofboth GABAA and GABAB receptors (Peng et al., 2011) and phos-phatidylcholine-selective phospholipase C (PC-PLC) activity (Zhangand Abdel-Rahman, 2005), activation of extracellular signal-regulatedkinase (ERK) as well as JNK (Li et al., 2006). For the first time we arereporting the beneficial effect of moxonidine in subacute brainischemia induced VaD. This beneficial effect may be utilizing variouspathways for providing its benefits; future research should be targetedto find the exact mechanism of this agent in VaD.

It has been reported that clonidine is effective in cognitive deficits(Schneider et al., 2010). Chao et al. (2000) have reported thatα2-adrenoceptors imidazolines like clonidine, attenuates hypoxia-induced damage to brain by acting as a free radical scavenger.Karmen (2003) has reported that clonidine inhibits lipid peroxidationand activates antioxidant enzymes. Clonidine has been reported toelevate the level of glutathione (Wang et al., 1992). Hence, results ofthis study are in parallel to previously published reports.

Clonidine produces extensive effects in mammalian tissues viaimidazoline recognition sites (or receptors) or α2-adrenoceptors(He et al., 2012). Imidazolines have been reported to exertinhibitory effect on cholinergic gastric contractions (Zádori et al.,2013) as well as in an inhibition of insulin secretion (Scheltdorfand Mest, 2002), regulation of high blood pressure, in the treat-ment of neuropsychiatric disorders (Lemamy et al., 2012) andmammalian platelet aggregation (Yokota et al., 2013).

Clonidine binds both α2-adrenergic and I1-imidazoline receptors.When clonidine binds α2-adrenoceptors, Gi signaling pathways areactivated that reduce cAMP levels by inhibiting adenylyl cyclase(Osborne, 1991). The neuroprotective effect of clonidine during ische-mia may be ascribed to sensitization of central sympathetic activityand a reduced release of glutamate thereby reducing NMDA receptoractivation and neuronal damage (Jellish et al., 2005).

Brede et al. (2011) have reported that pretreatment with cloni-dine did not protect from ischemic brain damage. But according toMilot and Plamondon (2011), pre-testing administration of theα2-adrenoceptor agonist clonidine attenuated ischemia-inducedworking memory impairments. It has been reported thatα2-adrenoceptor activation shortly after perinatal hypoxia-ischemiacan promote neural recovery (Dean et al., 2008). The neuroprotec-tive effect of clonidine (α2-adrenoceptor imidazoline agonist) hasbeen reported in near complete cerebral ischemia (Jellish et al.,2005). Zhang (2004) has reported the neuroprotective properties ofclonidine preconditioning in cerebral ischemic conditions.

Riekkinen and Riekkinen (1999) have reported that clonidineenhanced a spatial working memory and verbal fluency. Memoryin combined lesioned rats was restored when cholinomim-etic therapy was administered in combination with low doses ofclonidine (Santucci et al., 1991). Filos et al. (2012) have reportedthe beneficial effect of clonidine pretreatment in hemorrhagicshock and oxidative stress. The antihypoxic effect of clonidine wasreported directly in the lipid phase of membranes (Karmen, 2003).Castrejón-Sosa et al. (2002) have reported that augmented levelsof hydroxyl free radicals were blocked by clonidine.

Jiménez-Rivera et al. (2012) have reported that clonidinereduces the amplitude of glutamate-induced excitatory postsy-naptic currents. It has been reported that clonidine activatesprotein kinase C intracellular pathway (Jiménez-Rivera et al.,2012). Clonidine has been reported to show interaction betweencalcium channels, μ-receptors and α2-receptors (Ouyang et al.,2012). Kawahito et al. (2011) have been reported to inhibit KATP

channel activity in vascular smooth muscle cells. Clonidine hasbeen reported to produce an increase in ETA receptor expression inthe brain (Lavhale et al., 2010). Thus, clonidine may be providingits beneficial effect through multiple pathways. But as this is thefirst study, further research exploring the exact mechanistic path-ways in subacute brain ischemia induced VaD are required.

Cognitive impairment associated with modifications ofI1-imidazoline receptors is independent by modification of bloodpressure. It is important to note here that we have used the doses,which are much less than the minimal antihypertensive doses ofthese agents. Previous reports have also suggested the benefits ofthese agents at sub-therapeutic doses (Bakuridze et al. 2009;Briaud et al., 2005). Moxonidine has shown has provided learning,word recall and alertness, in hypertensive patients in low doses(Wesnes et al., 1997). Clonidine has shown neuroprotection (Jellishet al., 2005) and effective in cognitive deficits in low doses(Schneider et al., 2010). Hence, neuroprotective effects of theseagents are independent of blood pressure control.

There is possibility of a link between the inhibition of sympa-thetic tone, the cerebral perfusion and the prevention/reduction ofhypoperfusion may be the effect of moxonidine and clonidine oncerebral blood vessels. It has already been reported that moxoni-dine and clonidine may reduce vascular resistance (Rothbergeret al., 2010; Fenton et al., 2006). Moxonidine and clonidine havealso been reported to increase cerebral norepinephrine levels (NE)(Meana et al., 1997) and increased levels of NE produced anincrease in cerebral perfusion pressure (Kurihara et al., 1989).

Alternatively, studies using the subacute brain ischemia induc-tion model could be designed to explore the specific signalingpathways that have been shown to control on cognitive decline,oxidative stress and neurodegeneration. This study did not exam-ine cerebral blood flow. More extensive histological assessmentscould be done to quantify neuron cell death. Further studies couldbe aimed at quantifying the surviving neurons.

5. Conclusions

On the basis of the results of this study and above discussion, itmay be concluded that sub-acute brain ischemia is responsible forthe induction of vascular dementia. Agonistic modulation ofI1-imidazoline and subtype α2-adrenoceptors attenuated sub-acute brain ischemia induced vascular dementia in mice. There-fore, they may be considered as a beneficial therapeutic agent forthe management of chronic cerebral hypoperfusion induced vas-cular dementia. As this is the first report which suggests thebeneficial effect of modulation of I1-imidazoline and subtypeα2-adrenoceptors in sub-acute brain ischemia induced vasculardementia, further studies are required to explore the full ther-apeutic potential of I1-imidazoline receptors in vascular dementia.

Acknowledgments

Authors are thankful to Space age Research & TechnicalFoundation Charitable Trust (SPRFCT) and Prof. V. K. Sharma,Director, School of Pharmacy, Bharat Institute of Technology,Meerut, India for providing all the necessary facilities and fundingto conduct this research. We are also thankful to Dr. Nirmal Singh,Associate Professor, Pharmacology Division, Department of Phar-maceutical Sciences & Drug Research, Faculty of Medicine, PunjabiUniversity, Patiala (Punjab), India, for his valuable suggestions andguidance.

Appendix A. Supplementary material

Supplementary data associated with this article can be found in theonline version at http://dx.doi.org/10.1016/j.ejphar.2013.12.003.

S. Gupta, B. Sharma / European Journal of Pharmacology 723 (2014) 80–9088

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