Protective effects of phosphodiesterase-1 (PDE1) and ATP sensitive potassium (KATP) channel...

Author's Accepted Manuscript

Protective effects of phosphodiesterase-1(PDE1) and ATP sensitive potassium (KATP)channel modulators against 3-nitropropionicacid induced behavioral and biochemicaltoxicities in experimental Huntington's dis-ease

Surbhi Gupta, Bhupesh Sharma

PII: S0014-2999(14)00238-6DOI: http://dx.doi.org/10.1016/j.ejphar.2014.03.032Reference: EJP69203

To appear in: European Journal of Pharmacology

Received date: 5 December 2013Revised date: 15 March 2014Accepted date: 24 March 2014

Cite this article as: Surbhi Gupta, Bhupesh Sharma, Protective effects ofphosphodiesterase-1 (PDE1) and ATP sensitive potassium (KATP) channelmodulators against 3-nitropropionic acid induced behavioral and biochemicaltoxicities in experimental Huntington's disease, European Journal of Pharmacol-ogy, http://dx.doi.org/10.1016/j.ejphar.2014.03.032

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Protective effects of phosphodiesterase-1 (PDE1) and ATP sensitive potassium (KATP)

channel modulators against 3-nitropropionic acid induced behavioral and biochemical

toxicities in experimental Huntington’s disease

Surbhi Gupta a and Bhupesh Sharma b, c, *

Neuropharmacology Lab, Department of Pharmacology, School of Pharmacy, BIT, India

Conscience Research, Delhi, India

a Research Student, Neuropharmacology Lab., Department of Pharmacology, School of

Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Uttar Pradesh, India

+91-992-7932746; [email protected]

b Associate Professor and Head, Department of Pharmacology, School of Pharmacy, Bharat

Institute of Technology, Partapur Bypass, Meerut, Uttar Pradesh, India


c Chief Consultant, CNS Pharmacology, Conscience Research, Pocket F-233, B, Dilshad Garden,

Delhi-110095 India.


* Corresponding Author: Dr. Bhupesh Sharma

Type of paper: Research article

Group: Behavioral Pharmacology

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Abstract

Huntington’s disease (HD), a devastating neurodegenerative disorder, is characterized by

weight loss, impairment of motor function, cognitive dysfunction, neuropsychiatric disturbances

and striatal damage. Phosphodiesterase-1 (PDE1) has been implicated in various neurological

diseases. Mitochondrial potassium channels in the brain take part in neuroprotection. This study

has been structured to investigate the role of vinpocetine, a selective PDE1 inhibitor as well as

nicorandil, selective ATP sensitive potassium (KATP) channel opener in 3-nitropropionic acid (3-

NP) induced HD symptoms in rats. Systemic administration of 3-NP significantly, reduced body

weight, impaired locomotion, grip strength and impaired cognition. 3-NP elicited marked

oxidative stress in the brain (enhanced malondialdehyde-MDA, reduced glutathione-GSH

content, superoxide dismutase-SOD and catalase-CAT), elevated brain acetylcholinesterase

activity and inflammation (myeloperoxidase-MPO), with marked nitrosative stress

(nitrite/nitrate) in the brain. 3-NP has also induced mitochondrial dysfunction (impaired

mitochondrial NADH dehydrogenase- complex I, succinate dehydrogenase-complex II and

cytochrome oxidase-complex IV activities in the striatum of the rat. Tetrabenazine was used as a

positive control. Treatment with vinpocetine, nicorandil and tetrabenazine ameliorated 3-NP

induced reduction in body weight, impaired locomotion, grip strength and impaired cognition.

Treatment with these drugs reduced brain striatum oxidative (MDA, GSH, SOD and CAT) and

nitrosative (nitrite/nitrate) stress, acetylcholinesterase activity, inflammation and mitochondrial

dysfunctions. These results indicate that vinpocetine, a selective PDE1 inhibitor and nicorandil, a

KATP channel opener have attenuated 3-NP induced experimental HD. Hence, pharmacological

modulation of PDE1 as well as KATP channels may be considered as potential research targets for

mitigation of HD.

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Keywords: 3-nitropropionic acid, vinpocetine, nicorandil, mitochondrial enzyme complexes,

myeloperoxidase, acetylcholinesterase

1. Introduction

Huntington's disease (HD) is a neurodegenerative disorder with an autosomal dominant

expression pattern and typically a late-onset appearance. HD is caused by the expansion of a

CAG (cytosine adenine guanine) repeat in the huntingtin gene. Expanded polyglutamine

facilitates formation of huntingtin protein aggregates, eventually leading to deposition of

cytoplasmic and intranuclear inclusion bodies containing huntingtin protein. It is a movement

disorder with a heterogeneous phenotype characterized by involuntary dance-like gait, motor

impairment, and cognitive, psychiatric deficits and bio energetic deficits (Bhateja et al., 2012).

Mitochondrial neurotoxin, 3-nitropropionic acid (3-NP), is an irreversible inhibitor of

mitochondrial complex II (Brouillet et al., 1993) that inhibits the activity of succinate

dehydrogenase, a key enzyme of oxidative energy production, and characteristically provokes

striatal lesions and motor deficits as demonstrated by bradykinesia and movement abnormalities,

which closely mimic the signs and symptoms associated with HD (Kumar and Kumar, 2008). 3-

NP-induced neurodegeneration has been widely used as an HD animal model because of its

symptom similarity with HD (Bhateja et al., 2012).

Vinpocetine, a selective PDE1 inhibitor improves neuronal plasticity (Medina, 2011),

learning and memory deficits (Deshmukh et al., 2009), in Parkinsonism (Zaitone et al., 2012)

and Alzheimer's disease (Pereira et al., 2000). It has prevented of neuronal cell damage, in

cholinergic transmission and ameliorated of oxidative and nitrosative stress (Deshmukh et al.,

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2009). It is also reported that PDE1 inhibitors may provide benefits in cognitive deficits (Patyar

et al., 2011; Deshmukh et al., 2009), oxidative stress (Herrera-Mundo and Sitges, 2013;

Deshmukh et al., 2009), inflammation (Zhao et al., 2011) and mitochondrial dysfunctions

(Pereira et al., 2000). Modulators of PDE1 have not been studied in HD. We hypothesized that

modulation of PDE1 may provide benefits in HD. Therefore, modulation of PDE1 deserves

investigations for its potential in HD.

Nicorandil, a KATP channel opener, is known to have protective effects on ischemic

injury in the brain (Kong et al., 2013). Opening of KATP channels has been demonstrated to exert

significant neuroprotection in in vivo and in vitro models of Parkinson's disease (Xie et al.,

2010). Alteration of brain mitochondrial KATP channels has been reported to cause exacerbating

brain injury (Li et al., 2013). Activated microglial cells are important effectors of demyelination

and neurodegeneration, by secreting cytokines and others neurotoxic agents and it has been

suggested that microglia expresses KATP channels and its pharmacological activation can provide

neuroprotective and anti-inflammatory effects (Virgili et al., 2011). As the role of KATP channels

in HD is not studied till now, so we hypothesized that these channels may have a role in brain

injury, mitochondrial enzyme complexes, neuroprotection and inflammation. Hence, modulation

of KATP channels deserves potential for their investigation in HD.

The vesicular monoamine transporter-2 (VMAT2) has been reported to predominantly

localize to monoaminergic brain regions of central nervous system, where it packages free

monoamines in the cytosol into small synaptic and dense core vesicles (Bernstein et al., 2012).

Monoamine homeostasis is highly regulated and dysfunction may play a role in HD and

neuropsychiatric disorders (Bernstein et al., 2012). Proper packaging of these monoamines,

particularly dopamine is critical to the function and survival of these neurons. It has already been

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documented that dopamine is essential for facilitating behavioral functions like motor

movements, learning and memory (Eisenhofer et al., 2004). Tetrabenazine (TBZ), an approved

drug by USFDA (Chen et al., 2012), has been used for the management of various movement

disorders including Huntington chorea (Gros and Schuldiner, 2010). The highest binding density

for TBZ is in the caudate nucleus, putamen, and nucleus accumbens, areas known to bear the

brunt of pathology in HD (Frank, 2010). TBZ, a selective VMAT2 inhibitor, depletes central

monoamines by reversibly binding to VMAT2 (Frank, 2010) and thus, depletes particularly,

dopamine, from presynaptic terminals (Scott, 2011). Therefore, dopamine depleting agents, like

TBZ, are reported to be used in the treatment of involuntary movements like chorea (Jankovic

and Orman, 1988).�We have used TBZ as positive control in the present study.

In the light of above, the present study has been undertaken to investigate the potential of

vinpocetine (a selective inhibitor of PDE1) as well as nicorandil (KATP channel opener) in 3-NP

induced experimental Huntington’s disease.

2. Material and methods

2.1 Animals

Albino Wistar rats have been widely used for the induction of HD symptoms by 3-

nitropropionic acid (Gupta and Sharma, 2014a; Shivasharan et al., 2013). Adult albino Wistar

rats (3-5 months old), of either sex, weighing 200-250g, (purchased from Indian Veterinary

Research Institute, Izatnagar, India), were employed in the present study and were housed in

animal house with free access to water and standard laboratory pellet chow diet (Kisan Feeds

Ltd, Mumbai, India). The animals were exposed to 12 h light and 12 h dark cycle. The

experiments were conducted between 9.00 and 18.00 h in a semi-sound-proof laboratory. The

animals were acclimatized to the laboratory condition five days prior to behavioral study and

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were maintained in the laboratory until the completion of the study. The protocol of the study

was duly approved by the Institutional Animal Ethics Committee (IAEC) and care of the animals

was taken as per the guidelines of the Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of

India.

2.2 Drugs and chemicals

Nicorandil was obtained from Torrent Pharmaceuticals, India. Vinpocetine and

tetrabenazine were obtained from Sun Pharma Pvt. Ltd., India. 3-NP, Lowry’s reagent, 5, 5`-

dithiobis (2-nitrobenzoic acid) (DTNB), Folin-Ciocalteu reagent, bovine serum albumin (BSA)

and N-naphthylethylenediamine were purchased from Sigma Aldrich, USA. 4-(2-hydroxyethyl) -

1-piperazineethanesulfonic acid (HEPES), ethylene glycol tetra acetic acid (EGTA), mannitol,

glycyl glycine buffer, nicotinamide adenine dinucleotide (NADH), nitrazobluetetrazolium (NBT)

and cytochrome-C was purchased from SISCO Research Laboratory Pvt. Ltd., India.

2.3 3-nitropropionic acid (3-NP) experimental model

3-NP was dissolved in 0.9% saline solution and was administered to rats alternatively for

28 days at a dose of 10 mg kg-1 through intraperitoneal route (Gupta and Sharma, 2014a;

Gopinath and Sudhandiran, 2012; Pandey et al., 2008). Weight, locomotor activity and grip

strength were measured before the initiation of 3-NP treatment (day 1). These parameters were

reassessed before exposure to Elevated plus maze-EPM (day 20 of the treatment).

2.4 Experimental protocol and Drug administration

All drug solutions were freshly prepared before use. Selection of doses and the dosing

schedule were based on previously published reports from other labs.

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It has been reported that intraperitoneal administration of 3-NP (10 mg kg-1 for 4 days)

caused significant body weight reduction, impaired motor function (locomotor activity,

movement pattern) and striatal lesions mimicking symptoms of HD (Pandey et al., 2008). After 4

doses 3-NP exhibited its effect on behavioural and biochemical parameters. Hence, we selected

the administration of treatment drugs from the 10th day onwards i.e. after 4 doses of 3-NP in rats.

All the drug treatments were started from the 10th day onwards (from the 10th day to 28th day)

daily. Saline; vehicle of 3-NP was administered to rats from 1st day to 28th day (on alternate

days) and CMC; vehicle of treatment drugs, were administered to rats from 10th onwards (10th

day to 28th day) daily till the end of the study (total 19 days).

Vinpocetine, nicorandil and tetrabenazine were suspended in 0.5%

carboxymethylcellulose (CMC). Vinpocetine (3 and 6 mg kg-1; orally) (Zaitone et al., 2012),

nicorandil (2 and 4 mg kg-1; orally) (Cavero et al., 1991) and tetrabenazine (3 mg kg-1; orally)

(Meyer et al., 2011) were administered to rats once daily, as exact drug administration in 3-NP

treated rats was also for 19 days, starting from 10th day of 3-NP treatment till the end of the study

(day 28). CMC (10 ml kg-1; orally), vinpocetine (3 and 6 mg kg-1; orally), nicorandil (2 and 4 mg

kg-1; orally) and tetrabenazine (3 mg kg-1; orally) per se were administered to rats for 19 days

once daily.

In total fourteen groups were employed in this study and total 112 animals were used.

Each group consisted of eight Albino Wistar rat.

2.4.1 Group I — Control group: Normal animals were exposed to acquisition trials and

retrieval trials on EPM and MWM.

2.4.2 Group II — Vehicle control (0.9% saline) group: Animals were administered with

0.9%w/v saline (10 ml kg-1 intraperitoneally) alternatively for 28 days and the treatment was

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continued during acquisition trials and retrieval trials on EPM and MWM. Acquisition trials on

EPM were performed from 21th and 22th day, followed by retrieval trial on the 23rd day. On

MWM acquisition trials on were performed from 24th to 27th day, followed by retrieval trial on

the 28th day.

2.4.3 Group III— Vehicle control (0.5% CMC) group: Animals were administered with

0.5%w/v carboxymethylcellulose (10 ml kg-1 orally; once daily) for 11 days and the treatment

was continued during acquisition trials and retrieval trials on EPM (12th to 14th day) and MWM

(15th to 19th day).

2.4.4 Group IV, V, VI, VII and VIII— Vinpocetine (dose 1 and dose 2), Nicorandil (dose 1

and dose 2) and Tetrabenazine (TBZ) per se: Animals were administered with vinpocetine (3

and 6 mg kg-1 orally; once daily), nicorandil (2 and 4 mg kg-1 orally; once daily) and TBZ (3 mg

kg-1 orally; once daily) for 11 days and the treatment was continued during acquisition trials and

retrieval trials on EPM (12th to 14th day) and MWM (15th to 19th day).

2.4.5 Group IX — 3-Nitropropionic acid (3-NP) treatment group: Animals were

administered with 3-NP (10 mg kg-1; intraperitoneally) on alternate days and the treatment was

continued during acquisition trials and retrieval trials on EPM and MWM. Acquisition trials on

EPM were performed from 21th and 22th day, followed by retrieval trial on the 23rd day. On

MWM acquisition trials on were performed from 24th to 27th day, followed by retrieval trial on

the 28th day on MWM.

2.4.6 Group X, XI, XII, XIII and XIV—3- NP and Vinpocetine (dose 1 and dose 2),

nicorandil (dose 1 and dose 2) and TBZ: Vinpocetine (3 and 6 mg kg-1), nicorandil (2 and 4

mg kg-1) and TBZ (3 mg kg-1) 1orally, once daily were administered to the 3-NP treated rats,

starting from 10th day of 3- NP treatment till the end of the study and the treatment was

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continued during acquisition trials and retrieval trials on EPM and MWM. The rest of the

procedure was same as in group IX.

2.5 Body weight

Body weight was recorded on the first day and at the end of treatment (on 20th day) before

exposure to EPM, to assess the percent change in body weight.

Percent change in BW = [(BW 1st day – BW 20th day) / BW 1st day] x 100

2.6 Behavioral assessment

Animals were tested for locomotor activity and grip strength before starting the 3-NP

treatment and at the end of treatment (on 20th day).

2.6.1 Assessment of locomotor activity

Actophotometer (INCO, India) was used to assess locomotor activity. The apparatus was

placed in a darkened, sound attenuated and ventilated testing room during assessment. All

animals were placed individually in the activity cage for 3 min for making them habitual before

starting actual locomotor activity task for the next 5 min. Counts of basal activity of the animals

were noted. Total activity, including horizontal and vertical was expressed as counts per 5 min.

Counts/5 min is used as an index of locomotor activity. All the trials of locomotor activity were

completed between 09.00 and 18.00 h on 1st day and 28th day (Kumar et al., 2011).

2.6.2 Rota rod test

Rota rod experiments (Rota rod, Inco, India) were used to measure forelimb and hind

limb motor coordination (Kumar et al., 2011). Animals were placed individually on the rotating

rod with a diameter of 7 cm (speed 25 rpm). Rats were trained to use the Rota rod apparatus

during a 2 min trial (25 rpm) on the day before the first day of testing. The cut off time of

180 sec was fixed and each rat performed three separate trials at 5 min interval. Each trial was

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separated by a 5 min rest period to alleviate stress and fatigue. All the trials were completed

between 09.00 and 18.00 h on 1st day and 28th day. Falling time latency for each rat was recorded

by a trained observer blind to the experimental protocol (Yang et al., 2012).

2.6.3 Assessment of learning and memory using Elevated plus maze (EPM)

The apparatus consists of two opposing open arms (50 × 10 cm) perpendicular to two

enclosed arms (50 × 10 × 50 cm) that extend from a central platform (10 × 10 cm), elevated 65

cm above the floor. The maze was placed in the same position throughout the test in the

laboratory where extra maze cues were there to facilitate learning. The procedure and technique

were same as reported earlier by Haider et al., (2012). The test comprised of three days protocol,

first day was a training session while the next two days were considered as test sessions. In the

training session each rat was placed in the central square and allowed to explore the EPM for 10

min and then returned to the home cages. During test sessions cut off time was 5 min and time

spent in open arm was recorded. A significant decrease in time spent in open arm on subsequent

EPM exposure was taken as an index of successful memory retention. This is based on the idea

that during repetitive testing in EPM rat acquires information about the spatial environment and

avoids the elevated and open arms of the maze and prefers to stay in the closed arms where it

could be safe on the maze. Total time spent in the open arm measured on the first day served as

an index of learning and acquisition, whereas on the 2nd day it served as an index of retention of

learning task (memory) and on the 3rd day further served as the index of consolidation of

memory. Memory was measured by the degree to which the rat remembers and avoids the

elevated and open arms of the maze and prefers to stay in the closed arms (Gupta and Sharma,

2014a).

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2.6.4 Assessment of learning and memory by Morris water maze (MWM)

MWM is one of the most commonly used animal models to test memory. MWM

consisted of large circular pool (150 cm in diameter, 45 cm in height, filled to a depth of 30 cm

with water at 28°C). The water was made opaque with white colored dye. The tank was divided

into four equal quadrants with help of two threads, fixed at right angle to each other on the rim of

the pool. A submerged platform (10 cm²), painted white was placed inside the target quadrants of

this pool, 1 cm below surface of water. The position of the platform was kept unaltered

throughout the training session. Each animal was subjected to four consecutive trials on each day

with a gap of 5 min. The rat was gently placed in the water of the pool between quadrants, facing

the wall of the pool with the drop location changing for each trial, and allowed 120 sec to locate

a submerged platform. Then, it was allowed to stay on the platform for 20 sec. If it failed to find

the platform within 120 sec, it was guided gently onto platform and allowed to remain there for

20 sec. Day 4 escape latency time (ELT) to locate the hidden platform in water maze was noted

as an index of acquisition or learning. Daily starting positions were randomized and not repeated

on each day and quadrant 4 (Q4) was maintained as target quadrant in all acquisition trials. On

the fifth day, the platform was removed and rats were allowed to explore the pool for 120 sec.

Each rat was subjected to four such trials and each trial was started from different quadrant.

Mean time spent in all four quadrants i.e. Q1, Q2, Q3 and Q4 were recorded and the time spent

in the target quadrant i.e. Q4 in search of the missing platform provided an index of retrieval.

The experimenter was always standing in the same position. Care was taken that relative location

of water maze with respect to other objects in the laboratory serving, as prominent visual clues

were not disturbed during the total duration of study. All the trials were completed during the

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light cycle i.e. between 09.00 and 18.00 h (Morris, 1984; Sharma and Singh, 2008, 2011; Gupta

and Sharma, 2014b).

2.7 Dissection and homogenization

After the estimation of behavioral parameters, the animals were sacrificed by

decapitation. Brain striatum of each animal was isolated by putting on ice and weighed

individually. A 10% (w/v) tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4).

The homogenate was centrifuged at 10,000 g at 4°C for 15 min. Aliquots of supernatants were

separated and used for biochemical estimations (Gupta and Sharma, 2014a; Sharma and Singh,

2012, 2013).

2.8 Biochemical estimations

2.8.1 Assessment of striatum lipid peroxidation

Striatum thiobarbituric acid reactive substances (TBARS) were measured

spectrophotometerically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 532 nm

(Ohokawa et al., 1979; Sharma and Singh, 2011, 2013). The quantitative measurement of

TBARS, an index of lipid peroxidation in the striatum was performed. 0.2 ml of supernatant of

the homogenate was pipetted out in a test tube, followed by the addition of 0.2 ml of 8.1%

sodium dodecyl sulfate, 1.5 ml of 30% acetic acid (pH 3.5), 1.5 ml of 0.8% of thiobarbituric acid

and the volume was made up to 4 ml with distilled water. The test tubes were incubated for 1 h at

95°C, then cooled and added 1 ml of distilled water, followed by the addition of 5 ml of n-

butanol-pyridine mixture (15:1 v/v). The tubes were centrifuged at 4000 g for 10 min. The

absorbance of developing pink color was measured spectrophotometerically at 532 nm. A

standard calibration curve was prepared using 1-10 nM of 1, 1, 3, 3-tetra methoxy propane. The

TBARS value was expressed as nanomoles per mg of protein.

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2.8.2 Assessment of striatum nitrite/nitrate level

Striatum nitrite concentration was measured spectrophotometerically (UV-1800 ENG

240V; Shimadzu Coorporation, Japan) at 545 nm. Briefly, 400 �l of carbonate buffer (pH 9.0)

was added to 100 �l of striatum or standard sample followed by addition of small amounts (0.15

g) of copper-cadmium alloy. The tubes were incubated at room temperature for 1 h to reduce

nitrate to nitrite. The reaction was stopped by adding 100 �l of 0.35 M sodium hydroxide.

Following this, 400 �l of zinc sulfate solution (120 mM) was added to deproteinate the striatum

samples. The samples were allowed to stand for 10 min and then centrifuged at 4000 g for 10

min. Greiss reagent (250 �l of 1.0% sulphanilamide prepared in 3 N HCl and 250 �l of 0.1% N-

naphthylethylenediamine (prepared with water) was added to aliquots (500 �l) of clear

supernatant and striatum nitrite was measured spectrophotometerically at 545 nm. The standard

curve of sodium nitrite (5 to 50 �M) was plotted to calculate the concentration of striatum nitrite

(Sastry et al., 2002; Sharma and Singh, 2008, 2012).

2.8.3 Assessment of striatum glutathione (GSH) level

The reduced GSH content in the striatum was estimated spectrophotometerically (UV-

1800 ENG 240V; Shimadzu Coorporation, Japan) at 412 nm. Briefly, the supernatant of the

homogenate was mixed with trichloroacetic acid (10% w/v) in 1:1 ratio. The tubes were

centrifuged at 1000 g for 10 min at 4°C. The supernatant obtained (0.5 ml) was mixed with 2 ml

of 0.3 M disodium hydrogen phosphate. Then 0.25 ml of 0.001 M freshly prepared DTNB

[DTNB dissolved in 1% w/v sodium citrate] was added and observance was noted

spectrophotometerically at 412 nm. A standard curve was plotted using 10-100 �M of the

reduced form of glutathione (Beutler et al., 1963; Sharma and Singh, 2011, 2013).

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2.8.4 Assessment of striatum superoxide dismutase (SOD) activity

Striatum SOD was assayed spectrophotometerically (UV-1800 ENG 240V; Shimadzu

Coorporation, Japan) as described by Beauchamp and Fridovich (1971). It was based on the

reduction of NBT to the water insoluble blue formation. The assay mixture contained 0.5 ml of

brain homogenate, 1 ml of 50 mM sodium carbonate, 0.4 ml of 24 �m NBT, and 0.2 ml of 0.1

mM ethylenediamine tetraacetic acid (EDTA). The reaction was initiated by adding 0.4 ml of 1

mM hydroxylamine hydrochloride. The developed blue color in the reaction was measured at

560 nm. Zero time absorbance was taken at 560 nm followed by recording the absorbance

reading every 30 sec for a period of 5 min at 25°C. The above- mentioned reaction mixtures

without the brain homogenate served as control. The rate of increase in absorbance units (A) per

min for the control and for the test sample(s) was determined and the percentage inhibition for

the test sample(s) was calculated by the following formula:

% inhibition = {[(�A560 nM/min) control - (�A560 nM/min) test] / (�A560 nM/min) control} x 100

Where, (A560 nM at 5 min and 30sec –A560 nM at 30 sec)/5 min = � A560 nM/min.

Units of the SOD activity were expressed as the amount of enzyme required to inhibit the

reduction of NBT by 50% and the activity was expressed as units per mg of protein (Gupta and

Sharma, 2014a).

2.8.5 Assessment of striatum catalase (CAT) activity

The activity of striatum catalase was determined spectrophotometerically (UV-1800 ENG

240V; Shimadzu Coorporation, Japan) at 240 nm by the method of Aebi (1984). Briefly, 1 ml of

the striatum homogenate was taken in a test tube and 1.9 ml of phosphate buffer (50 mM, pH

7.4) was added to it. The reaction was initiated by the addition of 1 ml of 30 mM H2O2. A

mixture of 2.9 ml of phosphate buffer and 1 ml of H2O2 without the striatum homogenate served

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as the blank. The decrease in absorbance due to the decomposition of H2O2 was recorded at 240

nm against the blank. Units of catalase were expressed as the amount of enzyme that decomposes

1 �M of H2O2 per min at 25°C and the activity was expressed in terms of units per milligram of

proteins (Gupta et al., 2014).

2.8.6 Assessment of striatum acetylcholinesterase activity

Striatum acetylcholinesterase activity was measured spectrophotometerically (UV-1800

ENG 240V; Shimadzu Coorporation, Japan) at 420 nm (Ellman et al., 1961; Sharma and Singh,

2008; Sharma and Singh, 2011). Briefly, this was measured in basis of the formation of yellow

color due to the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of

thiocholine from acetylthiocholine iodide in the presence of striatum cholinesterase was

measured using a spectrophotometer. 0.5 ml of clear supernatant liquid of the striatum

homogenate was pipetted out into a 25 ml volumetric flask and dilution was made with a freshly

prepared DTNB solution (10 mg DTNB in 100 ml of Sorenson phosphate buffer, pH 8.0). From

the volumetric flask, two 4 ml portions were pipetted out into two test tubes. Into one of the test

tube, 2 drops of eserine solution were added. 1 ml of substrate solution (75 mg of acetylcholine

iodide per 50 ml of distilled water) was pipetted out into both of the test tubes. The test tube

containing eserine was taken as blank and the change in absorbance per min of the test sample

was read spectrophotometerically at 420 nm. Acetylcholinesterase activity was calculated using

the following formula:

R = � O.D. x volume of Assay E x mg of protein

Where R = rate of enzyme activity in ‘n’ mole of acetylcholine iodide hydrolyzed/min/mg

protein

� O.D. = change in absorbance/min

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E = Extinction coefficient = 13600/M/cm.

2.8.7 Assessment of striatum myeloperoxidase (MPO)

MPO activity was measured using a modified spectroscopic method (UV-1800 ENG

240V; Shimadzu Coorporation, Japan) described by Bradley et al. (1982). Brain tissues were

homogenized in ice cold 50 mM potassium phosphate buffer (pH=6) containing 0.5% Hexadecyl

Trimethyl Ammonium Bromide. The homogenate was freezing then centrifuged at 11,000g for

20 min at 4°C. The supernatant (34 μl) was mixed with the same phosphate buffer (986 μl),

containing 0.167 mg/ml ortho-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The

change in absorbance at 460 nm was recorded by spectrophotometer. One unit of MPO activity

was defined as that consuming 1 nM of peroxide per min at 22°C (Gupta and Sharma, 2014a).

2.8.8 Assessment of striatum total protein

Striatum total protein was determined spectrophotometerically (UV-1800 ENG 240V;

Shimadzu Coorporation, Japan) at 750 nm according to the method described by Lowry et al.

(1951). The brain total protein was determined by using BSA as a standard. 0.15 ml of

supernatant of tissue homogenate was diluted to 1 ml, and then 5 ml of Lowry’s reagent was

added. The contents were mixed thoroughly and the mixture was allowed to stand for 15 min at

room temperature. Then 0.5 ml of Folin-Ciocalteu reagent (prepared by diluting 50 ml of Folin-

Ciocalteu reagent with 50 ml distilled water) was added and the contents were vortexed

vigorously and incubated at room temperature for 30 min. The standard curve was plotted using

0.2-2.4 mg/ml of BSA. The protein content was determined spectrophotometerically at 750 nm

(Gupta and Sharma, 2014b; Sharma and Singh, 2008, 2013).

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2.8.9 Isolation of rat brain striatum mitochondria and mitochondrial complex estimation

Striatum regions were homogenized in the isolation buffer with EGTA (215 mM

Mannitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES, 1 mM EGTA, and pH-7.2). Homogenate

was centrifuged at 13,000 g for 5 min at 4°C. Pellets were resuspended in the isolation buffer

with EGTA and spun again at 13,000 g for 5 min. The resulting supernatant was transferred to

new tubes and topped off with isolation buffer with EGTA and again spun at 13,000 g for

10 min. Pellets containing purified mitochondria were resuspended in the isolation buffer

without EGTA. Thus, rat brain mitochondria were isolated (Berman and Hastings, 1999).

2.8.9.1 Assessment of complex I (NADH dehydrogenase) activity

Complex I (NADH dehydrogenase activity) was measured spectrophotometrically (UV-

1800 ENG 240V; Shimadzu Coorporation, Japan). The method involves the catalytic oxidation

of NADH to NAD+ with subsequent reduction of cytochrome-C. The reaction mixture was

contained 0.2 M glycyl glycine buffer (prepared by dissolving 26.42 g in distilled water and

volume was made up to 1 liter with distilled water), pH 8.5, 6 mM NADH in 2 mM glycyl

glycine buffer and 10.5 mM cytochrome-C. The reaction was initiated by the addition of a

requisite amount of solubilized mitochondrial sample. The absorbance change at 550 nm was

followed for 2 min (King and Howard, 1967).

2.8.9.2 Assessment of complex II (succinate dehydrogenase-SDH) activity

SDH was measured spectrophotometrically (UV-1800 ENG 240V; Shimadzu

Coorporation, Japan). The method involves the oxidation of succinate by an artificial electron

acceptor, potassium ferricyanide. The reaction mixture was contained 0.2 M phosphate buffer pH

7.8, 1% BSA, 0.6 M succinic acid and 0.03 M potassium ferricyanide. The reaction was initiated

18��

by the addition of the striatum mitochondrial sample, and the absorbance change at 420 nm was

followed for 2 min (King, 1967).

2.8.9.3 Assessment of complex IV (cytochrome oxidase) activity

Cytochrome oxidase activity was assayed in brain mitochondria. The assay mixture was

contained 0.3 mM reduced cytochrome-C in 75 mM phosphate buffer. The reaction was initiated

by the addition of the solubilized striatum mitochondrial sample, and absorbance change at 550

nm was measured spectrophotometrically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan)

for 2 min (Sottocasa et al., 1967).

3. Statistical analysis

Statistical analysis was done using SigmaStat v3.5. All results were expressed as mean ±

standard deviation. Data was statistically analyzed using one-way analysis of variance (ANOVA)

followed by Tukey's multiple range test. P<0.05 was considered to be statistically significant.

Individual F values for every parameter were also calculated.

4. Results

4.1 Results of behavioral studies

3-NP has been reported to cause a reduction in body weight, locomotor activity, motor

coordination and impaired learning and memory (Gupta and Sharma, 2014a; Colle et al., 2013;

Bhateja et al., 2012) in the HD animal model. Similar results are obtained in this study.

Administration of saline, CMC, vinpocetine (3 and 6 mg kg-1 orally; once daily for 19 days),

nicorandil (2 and 4 mg kg-1 orally; once daily for 19 days) and tetrabenazine (3 mg kg-1 orally;

once daily for 19 days) per se did not show any significant effect on the body weight (Fig. 1),

locomotor activity (Fig. 2), motor coordination (Fig. 3), transfer latency-TL (Fig. 4), total time

spent in target quadrant-TSOA (Fig. 5) on elevated plus maze-EPM, escape latency time-ELT

19��

(Fig. 6) and mean time spent in target quadrant-TSTQ (Fig. 7) on Morris water maze-MWM,

compared to control animals. Increased TL on day 1 and day 3 is considered as an index of

impairment of learning as well as decreased day 1 and day 3 TSOA is used for indexing

impairment of memory on EPM. In MWM, prolongation of day 4 ELT, as compared to control

animals is used to index impairment of learning and decrease in TSTQ on day 5, as compared to

control animals, is considered as impairment of memory. Whereas, 3-NP administration (10 mg

kg-1; intraperitoneally, alternatively for 28 days), significantly reduced the body weight

[P<0.001; F (13, 111) = 1637.808] (Fig. 1), locomotor activity [P<0.001; F (13, 111) = 157.161]

(Fig. 2), motor coordination [P<0.001; F (13, 111) = 174.827] (Fig. 3), with significant increase

in TL on day 1 and day 3 [P<0.001; F (13, 111) = 77.470] on EPM (Fig. 4), ELT on day 4

[P<0.001; F (13, 111) = 60.189] (Fig. 6) on MWM, as well as reduction in TSOA on day 1 and

day 3 [P<0.001; F (13, 111) = 171.218] (Fig. 5) on EPM and TSTQ on day 5 [P<0.001; F (13,

111) = 172.025] on MWM (Fig. 7), as compared to control animals. Treatment with vinpocetine

(3 and 6 mg kg-1, orally; once daily for 19 days), nicorandil (2 and 4 mg kg-1, orally; once daily

for 19 days) and tetrabenazine (3 mg kg-1, orally; once daily for 19 days) have significantly

attenuated 3-NP induced weight loss [P<0.001; F (13, 111) = 1637.808] (Fig. 1), reduced

locomotor activity [P<0.001; F (13, 111) = 157.161] (Fig. 2), motor coordination [P<0.001; F

(13, 111) = 174.827] (Fig. 3), increased day 1 and day 3 TL [P<0.001; F (13, 111) = 77.470] on

EPM (Fig. 4), ELT on day 4 [P<0.001; F (13, 111) = 60.189] (Fig. 6) on MWM, decreased

TSOA on day 1 and day 3 [P<0.001; F (13, 111) = 171.218] (Fig. 5) on EPM and TSTQ on day

5 [P<0.001; F (13, 111) = 172.025] on MWM (Fig. 7).

It is important to note that there is significant statistical difference in responses observed

in two different doses of vinpocetine (3 and 6 mg kg-1) and nicorandil (2 and 4 mg kg-1).

20��

Significant statistical difference is also observed in responses of vinpocetine (3 and 6 mg kg-1)

and nicorandil (2 and 4 mg kg-1) from TBZ (3 mg kg-1). Vinpocetine (6 mg kg-1) showed

statistically significant difference on all behavioral parameters such as, weight loss, motor

coordination, TL, TSOA, ELT and TSTQ, as compared to vinpocetine (3 mg kg-1). Similarly,

nicorandil (4 mg kg-1) showed statistically significant difference on all behavioral parameters as

compared to nicorandil (2 mg kg-1). Further, TBZ showed statistically significant difference on

all behavioral parameters, as compared to vinpocetine (dose 1 and dose 2) and nicorandil (dose 1

and dose 2).

4.2 Results of biochemical studies

It has been reported that 3-NP induces oxidative as well as nitrosative stress and

inflammation (Gupta and Sharma, 2014a; Bhateja et al., 2012), elevates levels of brain

acetylcholinesterase (Konagaya et al., 1992) as well as impairs the mitochondrial enzyme

complex activities like NADH dehydrogenase, succinate dehydrogenase and cytochrome oxidase

(Colle et al., 2013; Bhateja et al., 2012). 3-NP produced a considerable increase in level of brain

striatum thiobarbituric acid reactive substances-TBARS [P<0.001; F (13, 111) = 208.442]

(Table 1), nitrite/nitrate [P<0.001; F (13, 111) = 128.598] (Table 1), acetylcholinesterase

[P<0.001; F (13, 111) = 78.733] (Table 1) and myeloperoxidase-MPO [P<0.001; F (13, 111) =

1267.628], with significant reduction in glutathione-GSH [P<0.001; F (13, 111) = 225.995]

(Table 1), superoxide dismutase-SOD [P<0.001; F (13, 111) = 87.455] (Table 1), catalase-CAT

activities [P<0.001; F (13, 111) = 42.449] (Table 1), and mitochondrial enzyme complexes

[P<0.001; complex I- F (13, 111) = 81.926, complex II- F (13, 111) = 75.234, complex IV- F

(13, 111) = 89.506] (Fig. 8), as compared to control animals. Administration of saline, CMC,

vinpocetine (3 and 6 mg kg-1 orally; once daily for 19 days), nicorandil (2 and 4 mg kg-1 orally;

21��

once daily for 19 days) and tetrabenazine (3 mg kg-1 orally; once daily for 19 days) per se did not

show any significant effect on brain striatum TBARS, nitrite/nitrate, acetylcholinesterase, MPO,

GSH, SOD, CAT and mitochondrial enzyme complexes. Treatment with vinpocetine (3 and 6

mg kg-1, orally; once daily for 19 days), nicorandil (2 and 4 mg kg-1, orally; once daily for 19

days) and tetrabenazine (3 mg kg-1, orally; once daily for 19 days) have significantly attenuated

3-NP induced increased levels of brain striatum TBARS [P<0.001; F (13, 111) = 208.442]

(Table 1), nitrite/nitrate [P<0.001; F (13, 111) = 128.598] (Table 1), acetylcholinesterase

[P<0.001; F (13, 111) = 78.733] (Table 1) and MPO [P<0.001; F (13, 111) = 1267.628], along

with reduced GSH [P<0.001; F (13, 111) = 225.995] (Table 1), SOD [P<0.001; F (13, 111) =

87.455] (Table 1), CAT activities [P<0.001; F (13, 111) = 42.449] (Table 1), and mitochondrial

enzyme complexes [P<0.001; complex I- F (13, 111) = 81.926, complex II- F (13, 111) = 75.234,

complex IV- F (13, 111) = 89.506] (Fig. 8).

We have observed that vinpocetine (6 mg kg-1) showed statistically significant difference

on all biochemical parameters such as, brain nitroso-oxidative stress, acetylcholinesterase

activity, inflammation as well as mitochondrial enzyme complexes, as compared to vinpocetine

(3 mg kg-1). Similarly, nicorandil (4 mg kg-1) showed statistically significant difference on all

biochemical parameters as compared to nicorandil (2 mg kg-1). Further, TBZ showed statistically

significant difference on all biochemical parameters, as compared to vinpocetine (dose 1 and

dose 2) and nicorandil (dose 1 and dose 2). Thus, there is dose dependent effect is observed on

all biochemical parameters on the treatment of different doses of all drugs.

5. Discussion

In the present study, we examined the effect of vinpocetine and nicorandil in 3-NP

induced experimental HD symptoms. Vinpocetine, nicorandil and TBZ have significantly

22��

attenuated 3-NP induced weight loss, reduced locomotion, impaired motor coordination as well

as learning and memory. These agents, in addition to the above effects, also significantly

attenuated brain striatum oxidative as well as nitrosative stress, increased acetylcholinesterase

activity, inflammation and impaired mitochondrial enzyme complexes (I, II and IV).

Results of this study suggest that higher dose of vinpocetine (6 mg kg-1) shows more

beneficial effect as compared to the lower dose of vinpocetine (3 mg kg-1). Lower dose of

vinpocetine showed less beneficial effect on weight loss, motor dysfunction, TL, TSOA on EPM,

ELT and TSTQ on MWM. Similarly, higher dose of nicorandil (4 mg kg-1) showed more

beneficial effect on all behavioral parameters, as compared to the lower dose of nicorandil (2 mg

kg-1). TBZ showed more beneficial effect, as compared to both doses of vinpocetine (dose 1 and

dose 2) as well as nicorandil (dose 1 and dose 2). Thus, we can say that there is dose dependent

effect is observed on the treatment of all drugs.

HD patients gradually suffer from weight loss and it has been reported that 3-NP used

for inducing HD symptoms, alters body weight of animals (Colle et al., 2013). Gait impairments

or perturbed locomotion is the characteristic feature of HD. It has already been reported that 3-

NP causes reduction in locomotor activity (Colle et al., 2013). Motor dysfunction has also been

reported in HD patients. 3-NP has been reported to cause reduction in motor coordination

(Bhateja et al., 2012). 3-NP model has been used to mimic two stage progressions of HD i.e.

hyperkinetic and hypokinetic, depending upon the time and doses administered, which allows

evaluating early and late phases of HD (Kumar and Kumar, 2008). Administration of 3-NP (10

mg kg-1 intraperitoneally) for more than four doses has been reported to produce the onset of

hypokinetic symptoms (Túnez et al., 2010).

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3-NP treated rats developed the motor system dysfunction, which was distinguished by

hypolocomotion in Actophotometer, and decreased motor performance in the Rota rod task. The

behavioral symptoms of HD are generally characterized by the loss of neurons, notably, the

medium-spiny GABAergic neurons in the caudate nucleus and putamen, which show a

progressive neuropathological change (Han et al., 2010). This neuronal degeneration has been

reported to be associated with motor dysfunction in 3-NP treated animals (Colle et al., 2013),

which can be reproduced after mitochondrial complex II inhibition. Additionally, deficiencies in

behavior and motor control are reminiscent of the loss of motor skills associated with increased

brain protein oxidation (Forster et al., 1996). Vinpocetine has been reported to improve

locomotor activity (Zaitone et al., 2012). TBZ has been reported to improve choreatic

movements because of alteration in striatal dopamine levels (Andersson et al., 2006). Similarly,

in our study vinpocetine, nicorandil and TBZ improved locomotion and motor coordination.

HD patients and mouse models show learning and memory impairment even before the

onset of motor symptoms. 3-NP administration significantly impaired the memory as observed in

EPM and MWM paradigm. It has been reported earlier that EPM may be utilized for assessment

of learning and memory, using TL as a parameter for acquisition and retention of memory

process on EPM in rats and mice (Haider et al., 2012). The prolongation of the TL on retention

testing in the EPM method has been considered as an indicator for impairment of learning and

memory. Furthermore, memory was quantified by transfer latency (the time taken by the rat to

move from the open arm to the enclosed arm) and anxiety was assessed by percent entries into

the open arms in EPM. The results of this study are in accordance with the previously published

reports (Bhateja et al., 2012; Colle et al., 2013).

24��

The neuropathology of HD is characterized by progressive loss of projection neurons in

cortex and striatum; striatal cholinergic inter neurons are relatively spared. Cholinergic

projections play important roles in hippocampal-dependent cognition. It has been documented

that cholinergic interneurons are capable of increased acetylcholine release, whereas, reduced

levels of extracellular striatal acetylcholine in HD may reflect abnormalities in the excitatory

innervation of cholinergic interneurons, which may have implications acetylcholine-dependent

processes that are altered in HD, including corticostriatal plasticity (Farrar et al., 2011). 3-NP has

also been reported to cause alteration in acetylcholinesterase activity (Bhateja et al., 2012). It has

been reported that 3-NP produces lesions in hippocampal CA1 and CA3 pyramidal neurons, the

area of brain that is associated with cognitive performance. It is known that the hippocampal

degeneration caused by mitochondrial dysfunction affects learning and memory, cognitive

functions (Przybyla-Zawislak et al., 2005). 3-NP administration produces a long-term

potentiation of the NMDA-mediated synaptic excitation (3-NP-LTP) in striatal spiny neurons

(Calabresi et al., 2001), with suppression of LTD expression in the sensorimotor striatum

(Dalbem et al., 2005). 3-NP-LTP has been reported to involve in augmenting intracellular

calcium and activation of the mitogen-activated protein kinase extracellular signal-regulated

kinase. Thus, 3-NP-LTP might play a key role in the regional and cell type-specific neuronal

death observed in HD (Calabresi et al., 2001).

It has been reported that PDE1 inhibitors such as, vinpocetine has the ability to prevent

hydrolysis of cAMP and/or cGMP and they can stimulate the cAMP/protein kinase A

(PKA)/cAMP element-binding protein (CREB) and cGMP/PKG/CREB pathway to enhance

synaptic transmission by increasing CREB phosphorylation and brain-derived neurotrophic

factor transcription (Sierksma et al., 2013). Vinpocetine has been reported to reduce significantly

25��

the increase in acetylcholinesterase activity (Deshmukh et al., 2009), thus, enhances spatial

memory through the modulation of cholinergic functions as well as inhibition of slow-

inactivating K+ currents (Patyar et al., 2011). Hippocampal KATP channels have been reported to

involve in learning and memory (Betourne et al., 2009) by preventing microglia activation

(Farkas et al., 2005). It has been reported that activation of KATP channels has an action on the

regulation of dopamine and glutamate release in the forebrain regions (Sun et al., 2010).

Vinpocetine, nicorandil and TBZ improves learning, memory, which may be due to modulation

of cholinergic functions, glutamate levels in the brain as well as prevention of microglia

activation.

The molecular mechanisms mediating neuronal death in HD involve oxidative stress and

nitrosative stress. Existing evidence indicates that excessive generation of free radicals might

contribute to the onset of symptoms in HD and other movement disorders (Mandavilli et al.,

2005). This effect can be related to reduction in specific endogenous antioxidant mechanisms,

such as a decrease in GSH levels and decreased activity of antioxidant defense enzymes,

including SOD and catalase. Considerable evidence supports that the oxidative process

significantly contributes to 3-NP toxicity (Bhateja et al., 2012; Colle et al., 2013). 3-NP

diminishes oxidative phosphorylation by interfering with the mitochondrial respiratory chain,

reducing the level of available ATP, and thus causing metabolic inhibition (Bhateja et al., 2012;

Colle et al., 2013). Furthermore, evidence indicates that the production of free radicals is a key

contributing factor in the pathology of HD (Mandavilli et al., 2005). Vinpocetine has been

reported to decrease 3-NP induced increase in reactive oxygen species (ROS) and lipid

peroxidation in the striatum-isolated nerve endings (synaptosomes) (Herrera-Mundo and Sitges,

2013) and ultimately offers neuroprotection (Solanki et al., 2011). Vinpocetine has been

26��

documented to reduce nitroso-oxidative stress (Deshmukh et al., 2009). It has been reported that

nicorandil shows antioxidative effects by inhibiting NADPH oxidase and eNOS uncoupling

(Serizawa et al., 2011) as well as decreasing ROS formation and TBARS levels, a lipid

peroxidation biomarker (Carreira et al., 2008). TBZ has been reported to attenuate oxidative

stress (Milusheva et al., 2003). In this study, vinpocetine, nicorandil and TBZ show antioxidant

effects; this may be due to reduction of lipid peroxidation and activation of the antioxidant

enzyme system.

Brain inflammation induces deregulations in programmed cell death (apoptosis) in HD

(Caballero and Coto-Montes, 2012). Inflammation has been implicated in various

neurodegenerative diseases such HD (Cleren et al., 2005). It has been documented that

inflammation has been induced after the onset of striatal degeneration (Bantubungi et al., 2005).

It has already been reported that 3-NP has also induced inflammation (MPO assay) (Bhateja et

al., 2012). 3-NP has been reported to increase expressions of pro-inflammatory mediators like

tumor necrosis factor-alpha, cyclooxygenase-2 and inducible nitric oxide synthase (Gopinath and

Sudhandiran, 2012). In this study, 3-NP also induced brain inflammation, which was estimated

by myeloperoxidase (MPO) assay. Vinpocetine has reported to exhibit an anti-inflammatory

effect by partly targeting NF-�B/AP-1 (Zhao et al., 2011). It has been reported to inhibit TNF-

alpha-induced NF-kappa B activation and the subsequent induction of proinflammatory

mediators, IL-1beta and macrophage inflammatory protein-2 (Jeon et al., 2011). KATP channel

openers have been reported to protect against inflammation in brain diseases. Nicorandil has

been reported to attenuate brain inflammation by inhibiting pro-inflammatory factor release such

as TNF-alpha (Zhao et al., 2013). In this study, vinpocetine, nicorandil and TBZ attenuated 3-NP

induced brain inflammation, which may be due to above reasons.

27��

Mitochondrial dysfunction has been implicated in HD pathogenesis. Mitochondrial

dysfunction is characterized by mitochondrial swelling, membrane fluidity, rupture, release of

cytochrome-C, and neuronal death, which may have a direct impact on membrane-based

processes such as fission-associated morphogenic changes, opening of the mitochondrial

permeability transition pore or oxidative phosphorylation at the complexes of the electron

transport chain, which further leads to HD (Bertoni et al., 2011). Disruption of the mitochondrial

enzyme complex activity is associated with ROS. 3-NP has also been reported to impair the

mitochondrial enzyme complex activities like NADH dehydrogenase, succinate dehydrogenase

and cytochrome oxidase (Colle et al., 2013). In the present study, 3-NP significantly impaired

mitochondrial enzyme complex activities (complexes I, II and IV) in the striatum. Vinpocetine

has been reported to reduce the decrease of mitochondrial inner membrane potential induced by

glutamate exposure. Pereira et al., (2000) have reported that vinpocetine block the inhibition of

the mitochondrial respiratory chain complexes II and IV. Nicorandil has been reported to restore

mitochondrial oxidative phosphorylation capacity (Ahmed and El-Maraghy, 2013). It has been

documented that nicorandil inhibits mitochondrial ultrastructural changes, apoptosis signaling

pathway, oxidative stress (Ahmed and El-Maraghy, 2013), as well as inhibition of release of

mitochondrial cytochrome-C and loss of mitochondrial member potential (Xie et al., 2010).

Nicorandil has also been reported to decrease mitochondrial swelling; calcium uptake and

mitochondrial calcium overload (Carreira et al., 2008). It has been suggested that the massive

elevation of extracellular noradrenaline under conditions of oxidative stress combined with

mitochondrial dysfunction may provide an additional source of highly reactive free radicals thus

initiating a self-amplifying cycle leading to neuronal degeneration. TBZ has been reported to

attenuate oxidative stress because it has effect on nonsynaptic release noradrenaline in response

28��

to oxidative stress (Milusheva et al., 2003). In this study, vinpocetine, nicorandil and TBZ show

mitochondrial protective effects, this may be due to various pathways.

3-NP model has been reported to reproduce various cognitive and behavioral aspects of

HD but other aspects such as, suicidal tendencies has not been possible with this model. 3-NP

induced HD model is also limited to the bioavailability of the toxic substance. If it gets

metabolized and eliminated, it stops to show an effect, enabling tissues to respond to the toxic

insult. Intense research is required to identify the molecular and biochemical mechanism of 3-

NP. 3-NP model, though considered being a very good model, that mimics the symptoms of HD,

but as 3-NP does not show complete pathophysiology of HD, thus future research with these

agents in genetic models is desirable. As this is the first study, which suggests the utility of these

agents in HD, further research in full-fledged genetic models is required to study the full

potential of these agents as potential therapies for human subjects suffering from HD.

Alternatively, studies using the 3-NP induction model could be designed to explore the specific

signalling pathways that have been shown to control on cognitive decline, mitochondrial

dysfunction, oxidative stress and neurodegeneration.

6. Conclusions

On the basis of the results of this study and above discussion, it may be concluded that 3-

NP has induced HD. Vinpocetine, nicorandil and TBZ treatment have recuperated 3-NP induced

HD in rats. Thus, modulators of PDE1, KATP channels and VMAT2 inhibitor may provide benefit

in HD, but further research is needed to identify the full potential of these agents in HD.

29��

7. Acknowledgements

Authors are thankful to Space age Research and Technical Foundation Charitable Trust

(SPRFCT), Bharat Institute of Technology, Meerut, India for providing all the necessary

facilities and funding to conduct this research work. We are also thankful to Dr. Nirmal Singh,

Associate Professor, Pharmacology Division, Department of Pharmaceutical Sciences and Drug

Research, Faculty of Medicine, Punjabi University, Patiala (Punjab), India, for his valuable

suggestions.

8. Conflict of Interest

None

9. Funding source

None

30��

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Table 1: Effect of various agents on brain oxidative stress, nitrite/nitrate and

acetylcholinesterase activity and myeloperoxidase of the animals

Group

TBARS, nM/mg

of protein (% of

control)

Brain nitrite/ nitrate,

μmol/mg of

protein (% of

control)

Brain GSH,

�M/mg of

protien (% of

control)

Brain SOD,

nU/mg protein (% of

control)

Brain CAT, U/mg

protein (% of

control)

Brain acetylcholinesterase, �M

of acetylcholine hydrolyzed/min/mg of

protein

Brain MPO (U/g of protein)

C 100 ± 7 100 ± 6.5 100 ± 5 100 ±

7.4 100 ± 8 3.4 ± 0.26 0.026 ± 0.001

S 94 ± 6.58 94 ± 6.11 98 ± 4.9 97 ± 7.17

96 ± 7.68 3.5 ± 0.27

0.025 ± 0.001

CMC 97 ± 6.79 99 ± 6.43 97 ± 4.85

99 ± 7.32

94 ± 7.52 3.3 ± 0.26

0.026 ± 0.001

VD1 98 ± 6.86 97 ± 6.30 96 ± 4.8 98 ± 7.25

97 ± 7.76 3.5 ± 0.27

0.024 ± 0.001

VD2 97 ± 6.79 96 ± 6.24 98 ± 4.9 96 ± 7.10

96 ± 7.68 3.3 ± 0.26

0.026 ± 0.001

ND1 96 ± 6.72 98 ± 6.37 97 ± 4.85

97 ± 7.17

98 ± 7.84 3.5 ± 0.27

0.025 ± 0.001

ND2 97 ± 6.79 99 ± 6.43 98 ± 4.9 99 ± 7.32

96 ± 7.68 3.4 ± 0.26

0.024 ± 0.001

T 98 ± 6.86 91 ± 5.91 97 ± 4.85

98 ± 7.25

99 ± 7.92 3.6 ± 0.28

0.024 ± 0.001

HD 259 ± 18.13a

201 ± 13.06a

34 ± 1.7a

41 ± 3.03a

51 ± 4.08a 6.7 ± 0.52a

0.154 ± 0.007a

HD + VD1

189 ± 13.23b

152 ± 9.88b

51 ± 2.55b

52 ± 3.84b

67 ± 5.36b 5.7 ± 0.45b

0.125 ± 0.006b

HD + VD2

143 ± 10.01b,c

116 ± 7.54b,c

64 ± 3.2b,c

64 ± 4.73b,c

79 ± 6.32b,c 4.1 ± 0.32b,c

0.082 ± 0.004b,c

HD + ND1

179 ± 12.53b,d

145 ± 9.42b,d

56 ± 2.8b,d

59 ± 4.36b,d

62 ± 4.96b,d 5.3 ± 0.41b,d

0.118 ± 0.005b,d

HD + ND2

152 ± 10.64b,d

123 ± 7.99b,d

69 ± 3.45b,c,d

71 ± 5.25b,c,d

75 ± 6b,d 4.3 ± 0.33b,d

0.075 ± 0.003b,c,d

HD + T

123 ± 8.61b,c,d,e

105 ± 6.82b,c,d,e

76 ± 3.8b,c,d,e

90 ± 6.66b,c,d,e

85 ± 6.8b,c,d,e

4.1 ± 0.32b,c,d,e

0.051 ± 0.002b,c,d,e

41��

n= 8; results are expressed as mean ± standard deviation; one way ANOVA followed by Tukey's

multiple range test. a P<0.001 versus control group; b P<0.001 versus 3-NP treated group; c

P<0.001 versus 3-NP treated VD1 group; d P<0.001 versus 3-NP treated VD2 group; e P<0.001

versus 3-NP treated ND2 group

Brain TBARS– F (13, 111) = 208.442; Brain nitrite/nitrate– F (13, 111) = 128.598; Brain GSH–

F (13, 111) = 225.995; Brain SOD– F (13, 111) = 87.455; Brain CAT– F (13, 111) = 42.449;

Brain acetylcholinesterase– F (13, 111) = 78.733; Brain MPO– F (13, 111) = 1267.628

TBARS- thiobarbituric acid reactive substances; GSH- glutathione; SOD- superoxide dismutase;

CAT- catalase; MPO- myeloperoxidase; C-control; S- saline; CMC- carboxy-methyl cellulose;

V- vinpocetine; N- nicorandil; T- tetrabenazine; HD- 3-nitropropionic acid; D1- dose 1; D2-

dose 2

42��

Figure legends

Fig. 1: Effect of various agents on body weight


multiple range test. F (13, 111) = 1637.808, # P<0.001 versus control group; * P<0.001 versus 3-

NP treated group; � P<0.001 versus 3-NP treated VD1 group; � P<0.001 versus 3-NP treated

VD2 group; P<0.001 versus 3-NP treated ND2 group. C-control; S- saline; CMC- carboxy-

methyl cellulose; V- vinpocetine; N- nicorandil; T- tetrabenazine; HD- 3-nitropropionic acid;

D1- dose 1; D2- dose 2

Fig. 2: Effect of various agents on locomotor activity


multiple range test. F (13, 111) = 157.161; # P<0.001 versus control group; * P<0.001 versus 3-





43��

Fig. 3: Effect of various agents on motor coordination�


multiple range test. F (13, 111) = 174.827; # P<0.001 versus control group; * P<0.001 versus 3-





Fig. 4: Effect of various agents on transfer latency (TL) of animals, using Elevated plus

maze


multiple range test. F (13, 111) = 77.470; # P<0.001 versus day 1 TL in all groups; * P<0.001

versus day 3 TL of all 3-NP treated groups; � P<0.001 versus day 3 TL of respective day of 3-NP

treated VD1 group; � P<0.001 versus day 3 TL of respective day of 3-NP treated VD2 group;

P<0.001 versus day 3 TL of respective day of 3-NP treated ND2 group. C-control; S- saline;

CMC- carboxy-methyl cellulose; V- vinpocetine; N- nicorandil; T- tetrabenazine; HD- 3-

nitropropionic acid; D1- dose 1; D2- dose 2

44��

Fig. 5: Effect of various agents on total time spent in open arm (TSOA) of animals, using

Elevated plus maze�


multiple range test. F (13, 111) = 171.218, # P<0.001 versus day 1 TSOA in all groups; * P<0.001

versus day 3 TSOA of all 3-NP treated groups; � P<0.001 versus day 3 TSOA of respective day

of 3-NP treated VD1 group; � P<0.001 versus day 3 TSOA of respective day of 3-NP treated

VD2 group; P<0.001 versus day 3 TSOA of respective day of 3-NP treated ND2 group. C-

control; S- saline; CMC- carboxy-methyl cellulose; V- vinpocetine; N- nicorandil; T-

tetrabenazine; HD- 3-nitropropionic acid; D1- dose 1; D2- dose 2

Fig. 6: Effect of various agents on escape latency time (ELT) of animals, using Morris

water maze (MWM)


multiple range test. F (13, 111) = 60.189; # P<0.001 versus day 1 ELT in respective group; *

P<0.001 versus day 4 ELT of control group; � P<0.001 versus day 4 ELT of 3-NP treated VD1

group; � P<0.001 versus day 4 ELT of 3-NP treated VD2 group; P<0.001 versus day 4 ELT of

3-NP treated ND2 group. C-control; S- saline; CMC- carboxy-methyl cellulose; V- vinpocetine;

N- nicorandil; T- tetrabenazine; HD- 3-nitropropionic acid; D1- dose 1; D2- dose 2

45��

Fig. 7: Effect of various agents on mean time spent in the target quadrant (TSTQ) of

animals, using Morris water maze�


multiple range test. F (13, 111) = 172.025, # P<0.05 versus mean time spent in other quadrants in

control; * P<0.001 versus mean time spent in target quadrant in control group; � P<0.001 versus

mean time spent in target quadrant in 3-NP treated VD1 group; � P<0.001 versus mean time

spent in target quadrant in 3-NP treated VD2 group; P<0.001 versus mean time spent in target

quadrant in 3-NP treated ND2 group. C-control; S- saline; CMC- carboxy-methyl cellulose; V-

vinpocetine; N- nicorandil; T- tetrabenazine; HD- 3-nitropropionic acid; D1- dose 1; D2- dose 2

Fig. 8: Effect of various agents on striatal mitochondrial enzyme complex I, II and IV


multiple range test. Brain mitochondrial complex I- F (13, 111) = 81.926, complex II- F (13,

111) = 75.234, complex IV- F (13, 111) = 89.506, # P<0.001 versus control group; * P<0.001

versus 3-NP treated group; � P<0.001 versus 3-NP treated VD1 group; � P<0.001 versus 3-NP

treated VD2 group; P<0.001 versus 3-NP treated ND2 group. C-control; S- saline; CMC-

carboxy-methyl cellulose; V- vinpocetine; N- nicorandil; T- tetrabenazine; HD- 3-nitropropionic

acid; D1- dose 1; D2- dose 2

Figure1

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