Neuroprotective mechanism of Coenzyme Q10 (CoQ10) against PTZ induced kindling and associated cognitive dysfunction: Possible role of microglia inhibition

Pharmacological Reports 68 (2016) 1301–1311

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Original article

Neuroprotective mechanism of Coenzyme Q10 (CoQ10) against PTZ induced kindling and associated cognitive dysfunction: Possible role of microglia inhibition Manveen Bhardwaj, Anil Kumar* Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh, 160014, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 January 2016 Received in revised form 21 July 2016 Accepted 21 July 2016 Available online 22 July 2016

Background: Neuroinflammation, oxidative stress and mitochondrial dysfunction play a significant role to explain the pathophysiology of epilepsy. Neuroinflammation through microglia activation has been documented in epileptogenesis. Compounds which inhibit activation of glial cells have been suggested as one of the treatment approaches for the effective treatment of epilepsy. The present study has been designed to investigate the role of coenzyme Q10 and its interaction with minocycline (microglia inhibitor) against pentylenetetrazol (PTZ) induced kindling epilepsy. Methods: Laca mice received Coenzyme Q10 and minocycline for a period of 29 days. PTZ (40 mg/kg ip) injection has been given on alternate days. Various behavioural parameters (kindling score and elevated plus maze), biochemical parameters (lipid peroxidation, superoxide dismutase, reduced glutathione, catalase, nitrite and acetylcholinesterase) and mitochondrial enzyme complex activities of (I, II and IV) were assessed in the discrete areas of the brain. Results: Administration of a subconvulsive dose of PTZ (40 mg/kg) repeatedly increased significantly kindling score, oxidative damage and impaired mitochondrial enzyme complex activities (I, II and IV) and pro-inflammatory marker (TNF-a) as compared to naive animals. Coenzyme Q10 (10, 20 and 40 mg/kg) and minocycline (50 and 100 mg/kg) for a duration of 29 days significantly attenuated kindling score, reversed oxidative damage, TNF-a and restored mitochondrial enzyme complex activities (I, II and IV) as compared to control. Further, combinations of CoQ10 (10, 20 mg/kg) with minocycline (50 and 100 mg/kg) significantly modulate the protective effect of CoQ10 which was significant as compared to their effect per se in PTZ treated animals. Conclusion: The present study suggests the involvement of microglia inhibition in the protective effect of CoQ10 in PTZ induced kindling in mice. ã 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

Keywords: Kindling epilepsy Coenzyme Q10 Minocycline Mitochondrial Dysfunction Oxidative stress

Introduction Epilepsy is very complex neurological and known for its cognitive and behaviour impairments problem among general population [1]. The treatment and management of epilepsy are still considered as inadequate due to the side effects of the existing antiepileptic drugs as well as poor understanding of various biochemical and molecular aspects of its complex pathophysiological events. Therefore, to manage the epilepsy effectively, it is important to understand seizure-induced brain damage as well as neurobiology of seizure initiation. Kindling is an experimentally induced phenomenon to study the development of seizures.

* Corresponding author. E-mail address: [email protected] (A. Kumar).

Pathogenesis of epilepsy has been tried to explain with the help of various hypotheses particularly oxidative stress, mitochondrial dysfunction, neuroinflammation, excitotoxicity mechanism etc. Oxidative stress is emerging as a mechanism that plays an important role in the aetiology of seizure-induced neuronal death [2]. In humans, oxidative stress plays a crucial role in mitochondrial dysfunction and neuroinflammation induced brain damage during epileptic seizures. Mitochondrial dysfunction has been also identified as the potential cause of epileptic seizures. Mitochondrial oxidative phosphorylation provides ATP to neurons. Mitochondria participate in cellular Ca2+ homeostasis and generation of reactive oxygen species. Therefore, it has been proposed that mitochondrial dysfunction contribute seizure generation as well as strongly trigger neuronal cell death. Microglia cells play important functions in the developing brain by modulating neuronal apoptosis and participating in the pruning

http://dx.doi.org/10.1016/j.pharep.2016.07.005 1734-1140/ã 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

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of maturating synapses. Yet, the mechanisms by which microglia cells invade the brain and target maturating synapses at early postnatal developmental stages remain poorly understood. Growing body of evidences suggest that microglial activation known as ‘microgliosis’ occurs in the brain parenchyma of patients with recurrent seizure episodes (as well as reactive astrogliosis) [3]. Epileptogenesis increases the activation of glia cells which includes microglia and astrocytes. These activated microglia and astrocytes release cytokines included interleukin-1b (IL-1b) and tumour necrosis factor (TNF-a) [4]. Therefore, compounds with antiinflammatory properties or having an ability to inhibit glia activation may represent as one of the promising neuroprotective strategies to treat epilepsy and associated problems. CoQ10 is a lipid-soluble benzoquinone derivative. Structurally, it is similar to vitamin K. It resides in the inner mitochondrial membrane and is essential for Complexes I and II electron transfer activities during oxidative phosphorylation. CoQ10 plays a vital role in ATP production (oxidative phosphorylation). CoQ10 also has membrane-stabilizing properties and acts as an antioxidant in both mitochondrial and lipid membranes [5]. The neuroprotective effects of CoQ10 have been reported in multiple models of neurodegeneration, including HD and seizure [6,23]. However, the exact neuroprotective mechanism of CoQ10 is still not clear. Minocycline (Mino) is a small, highly lipophilic, secondgeneration tetracycline antibiotic. Minocycline has been known for its neuroprotective and anti-inflammatory properties [4]. Minocycline suppresses microglia activation and reduces proinflammatory cytokine (TNF-a) release in various neurological problems [7,8]. In addition, several recent studies demonstrated the anticonvulsant effects of minocycline against partial seizures and electrical or chemical-kindled seizures [9–11]. Minocycline has been demonstrated as effective in reducing seizures against amygdala kindling [24,25]. Both CoQ10 and minocycline have been well demonstrated to have neuroprotective effect. However, the exact neuroprotective mechanism in the epilepsy is still not fully understood. It still remains to be elucidated whether the mitochondrial restorative property of CoQ10 has any effect on oxidative damage or neuroinflammatory cascade of epilepsy. Similarly, microglia inhibiting property of minocycline has any influence on the neuroprotective actions of CoQ10. Therefore, the present study has been further extended to see the interaction of CoQ10 with minocycline with aim to explore any potential action against pentylenetetrazol (PTZ) induced kindling in mice. Materials and methods Animals In the study male laca mice (20–30 g) were used. The animals were housed under standard laboratory conditions with free access to food and water and maintained under a natural light and

dark cycle. Experimentation work was carried out between 09.00 and 15.00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) and it was carried out according to the guidelines of Committee for Control and Supervision of Experimentation on Animals (CPCSEA), Government of India on animal experimentation. Drugs and treatment schedule Animals were divided into different groups as per Table 1. The following drugs were used in the present study. PTZ dissolved in the normal saline and administered intraperitoneally on alternate days for a period of 29 days [12]. CoQ10 was suspended in sodium carboxy-methyl-cellulose (0.25% w/v) and minocycline was dissolved in the distilled water. Both CoQ10 and minocycline were administered daily as well as 30 min before PTZ treatment on an alternate day for a period of 29 days. Doses of coenzyme Q10 and minocycline were selected based on the previous documented studies of our own as well as previous literature [26,28,29]. Induction of kindled seizures and design of the experiment A subconvulsive dose of PTZ (40 mg/kg, ip) was administered on alternative days for a period of 29 days until an animal exhibited full motor seizures (Fig. 1). Plexiglass chamber (30  24  22 cm) was used to measure seizure intensity and was recorded for a period of 30 min. Racine scale was used to score the intensity of the seizure response which is as given below [13] 0 = No response; l = Sudden behavioural arrest and/or motionless staring; 2 = Facial jerking with muzzle or muzzle and eye; 3 = Neck jerks; 4 = Clonic seizure in a sitting position; 5 = Convulsions including clonic and/or tonic–clonic seizures while lying on the belly and/or pure tonic seizures 6 = Convulsions including clonic and/or tonic–clonic seizures while lying on the side and/or wild jumping [27]. Any mouse showing persistence convulsion on the first day was excluded from the study. Mice is said to achieve the kindled state when an exhibit three times clonic seizure in a sitting position i.e. stage 4. Elevated plus maze The elevated plus maze consists of two opposite white open arms (16  5 cm), crossed with two closed walls (16  5 cm) with 12 cm high walls. The arms were connected with a central square of dimensions 5  5 cm. The entire maze was placed 25 cm high above the ground. Acquisition of memory was tested on day 13. A mouse was placed individually at one end of the open arm facing away from the central square. The time taken by the animal to move from the open arm to the closed arm was recorded as the initial transfer latency (ITL). Animals were allowed to explore the maze for next 10 s after recording ITL. If an animal did not enter the

Table 1 Treatment Group. SNo.

Treatment Group

Treatment (mg/kg)

1. 2. 3–5

Naive PTZ (40 mg/kg, ip) Coenzyme Q10 (10, 20 and 40 mg/kg)

6–7

Minocycline (50 and 100 mg/kg)

8.

Coenzyme Q10 (10 mg/kg) + Minocycline (50 mg/kg) Coenzyme Q10 (20 mg/kg) + Minocycline (100 mg/kg)

Treated with saline, Healthy animals PTZ was administered on alternate days for a period of 29 days. Coenzyme Q10 (10, 20 and 40 mg/kg) was administered daily for a period of 29 days and 30 min before PTZ treatment Minocycline (50 mg/kg and 100 mg/kg) was administered daily for a period of 29 days and 30 min before PTZ treatment This combination was administered daily for a period of 29 days and 30 min before PTZ treatment

9.

This combination was administered daily for a period of 29 days and 30 min before PTZ treatment

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Fig. 1. Experimental protocol of pentylenetetrazol (PTZ)-induced kindling.

enclosed arm within 90 s, same was guided to the enclosed arm and ITL was recorded as 90 s. Retention of memory was assessed by placing the mouse in an open arm on day 14th and 29th day of the ITL, termed as the first retention transfer latency (1 st RTL) and second retention transfer latency (2nd RTL), respectively [14,33,34] Biochemical estimations Animals were decapitated for behavioural assessments; brains were rinsed in isotonic saline and weighed. Each brain was divided into two equal halves and then hippocampus and cortex regions were isolated. One half was used for biochemical estimations and other one for mitochondrial complex enzyme estimations. 10% (w/ v) tissue homogenates were prepared for biochemical analysis in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000  g at 4  C for 15 min. Aliquots of supernatants were separated and used for biochemical estimations. A Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA) was used for biochemical and mitochondrial assessments. Measurement of lipid peroxidation Wills method was used for the quantitative measurement of lipid peroxidation in the brain. The amount of malondialdehyde (MDA), a measure of lipid peroxidation, was measured by reaction with thiobarbituric acid at 532 nm using the Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). Molar extinction coefficient of chromophore (1.56  105 M 1 cm 1) was used for calculations and it was expressed as nanomoles of MDA per milligram of protein[15]. Estimation of reduced glutathione (GSH) Ellman method was followed for the estimation of the GSH in the brain. Briefly, 1 ml of supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold-digested at 4  C for 1 h. The sample was centrifuged at 1200 rpm for 15 min at 4  C. To 1 ml of this supernatant, 2.7 ml of 0.1 M phosphate buffer (pH 8) and 0.2 ml of 5,5-dithiobis 2-nitrobenzoic acid (DTNB) were added. With the Perkin Elmer lambda 20 spectrophotometer yellow colour was read immediately at 412 nm. Molar extinction coefficient of

chromophore (1.36  l04 M 1 cm 1) was used for calculation of results and expressed as micromole GSH per milligram protein [16] Superoxide dismutase activity estimation (SOD) Kono method was followed for the assessment of SOD activity wherein the reduction of nitrobluetetrazolium (NBT) was inhibited by SOD and measured at 560 nm using the Perkin Elmer lambda 20 spectrophotometer. The reaction was initiated by the addition of the hydroxylamine hydrochloride to the mixture containing NBT and the sample. The results were expressed as unit/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 100% [17] Catalase estimation Catalase activity was assayed by the method of Luck, wherein breakdown of hydrogen peroxides (H2O2) is measured at 240 nm using the Perkin Elmer lambda 20 spectrophotometer. Briefly, the assay mixture consisted of 3 ml of H2O2 phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%) and at 240 nm change in absorbance was recorded. The results were expressed as micromole H2O2 decomposed per milligram of protein/min. Estimation of nitrite According to Green and his co-workers method the accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined with a colorimetric assay with Greiss reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid). Equal volumes of the supernatant and the Greiss reagent were mixed; the mixture was incubated for 10 min at room temperature. Perkin Elmer lambda 20 spectrophotometer was used to measure the absorbance at 540 nm. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and was expressed as micromole per litre [18] Estimation of acetylcholinesterase (AChE) activity AChE is a marker of extensive loss of cholinergic neurons in the forebrain. The AChE activity was assessed by Ellman method. The change in absorbance was measured for 2 min at 30 s interval at 412 nm using the Perkin-Elmer Lambda 20 spectrophotometer.

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Results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per min per mg protein.

at 13,000  g at 4  C for 10 min. Pellets containing pure mitochondria were resuspended in isolation buffer without EGTA [19]

Mitochondrial complex enzyme estimation

Nicotinamide adenine dinucleotide dehydrogenase (NADH) (complex I) activity King and Howard method was used for measurement of NADH dehydrogenase activity spectrophotometrically. The method involves catalytic oxidation of NADH to NAD+ with subsequent reduction of cytochrome c. The reaction mixture contained 0.2 M glycylglycine buffer (pH 8.5), 6 mM NADH in 2 mM glycylglycine buffer, and 10.5 mM cytochrome C. The reaction was initiated by the addition of a requisite amount of solubilised mitochondrial sample followed by an absorbance change at 550 nm for 2 min [20]

Isolation of rat brain mitochondria Berman and Hastings method was used for mitochondrial enzyme complex activities. In isolated buffer brains were homogenized, and homogenates were then centrifuged at 13,000  g for 5 min at 4  C. Pellets were re-suspended in isolation buffer with ethylene glycol tetra acetic acid (EGTA) and spun again at 13,000  g at 4  C for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun

Naive PTZ(40 mg/kg) CoQ10(10 mg/kg) CoQ10(20 mg/kg) CoQ10(40 mg/kg) Mino(50 mg/kg) Mino(100 mg/kg) CoQ10(10 mg/kg)+Mino(50 mg/kg) CoQ10(20 mg/kg)+Mino(100 mg/kg)

6

a

a

a a

4

b

a

a

b

b,c

b a

a

b

a

b

Seizure Score

b a

a

a

b

b

2

a

b,c,d,e

b,c

b,c,d,e,f b,c,d,e,f

b,c,d,e,f

b,c,d,e

b

b,c,d,e

b,c,d,e

b,c b,c

b,c,d,e

b,c

b,c

b,c,d,e

b,c,d,e b,c

b

a

b,c,d

b,c

b

b

b,c

b,c

b,c,d,e,f b,c,d,e,f b,c,d,e,f

b,c,d,e,f b,c,d,e,f b,c,d,e,f

0 1

3

5

7

9

11

13

15

17

19

21

23

25

27

29

DAY

-2 Fig. 2. Influence of minocycline on the protective effect of CoQ10 against pentylenetetrazol-induced kindling: Data are expressed as mean  SEM. ap < 0.05 as compared to Naive, bp < 0.05 as compared to PTZ, cp < 0.05 as compared to CoQ10 (10 mg/kg), dp < 0.05 as compared to CoQ10 (20 mg/kg), ep < 0.05 as compared to minocycline (50 mg/kg), f p < 0.05 as compared to minocycline (100 mg/kg).

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Succinate dehydrogenase (SDH) (complex II) activity King method was used to measure the SDH spectrophotometrically. The method involves the oxidation of succinate by an artificial electron acceptor, potassium ferricyanide. The reaction mixture 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 by the addition of the mitochondrial sample followed by an absorbance change at 420 nm for 2 min [21]

added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells. The enzyme reaction yielded a blue product that turned yellow when the stop solution was added. The intensity of the colour measured (using a microtiter plate reader read at 450 nm) was found to be in proportion to the amount of mice TNF-a bound in the initial steps. The sample values were then read off the standard curve.

Cytochrome oxidase (complex IV) assay Sottocasa method was used to assayed the cytochrome oxidase activity in brain mitochondria. The assay mixture contained 0.3 mM of reduced cytochrome C in 75 mM phosphate buffer. The reaction was started by the addition of solubilised mitochondrial sample, and absorbance change was recorded at 550 nm for 2 min [22]

Statistical analysis Graph Pad Prism (Graph Pad Software, San Diego, CA, USA) was used for all statistical analysis. Values are expressed as mean  SEM. The behavioural assessment data were analyzed by a repeated measures two-way analysis of variance (ANOVA) with drug-treated groups as between sessions and as within-subjects factors. The biochemical estimations were analyzed by one-way ANOVA. Post hoc comparisons between groups were made using Tukey’s test. The p < 0.05 was considered significant.

Estimation of TNF-a activity The brain of first set of animals was used for the estimation of TNF-a levels in mice brain. The quantification of cytokine TNF-a was performed as per the instruction specified in BD Biosciences immune assay kit (BD opt EIA by BD Bioscience, San Jones, CA, USA). The mice TNF-a immunoassay was 4.5 h solid phase ELISA which employed sandwich enzyme immune assay technique. Monoclonal antibody specific for mice TNF-a had been pre-coated in the micro plate. When standards, control, and samples were pipette into the wells, any mice-TNF-a present was bound by the immobilized antibody. After washing away any unbound substance an enzyme linked polyclonal antibody specific for mice TNF-a was

Results Effect of CoQ10 and its modulation by minocycline on severity of seizure The sub convulsive dose of PTZ (40 mg/kg, on alternate days) for a period of 29 days gradual increased convulsive activity (seizure score) culminating in generalized clonic–tonic seizures as compared to the naive group. Twenty nine days treatment with CoQ10 (10–40 mg/kg) and minocycline (50 and 100 mg/kg) significantly

Naive PTZ(40 mg/kg) CoQ10(10 mg/kg) CoQ10(20 mg/kg) CoQ10(40mg/kg) Mino(50 mg/kg)

Elevated Plus Maze

CoQ10(10 mg/kg)+ Mino(50 mg/kg) Mino(100 mg/kg) CoQ10(20 mg/kg)+Mino(100 mg/kg)

80

a

Latency Time

60 a

a b

40

b b,c b,c

b

20

b,c

b,c,d

b,c,e b,c,e

b,c

b,c,d

b,c,e b,c,e

b,c, d,f

b,c,d,f

0

13th

14th

29th

DAY Fig. 3. Effect of minocycline on the protective effect of CoQ10 on elevated plus maze test in PTZ kindled mice: Data are expressed as mean  SEM. ap < 0.05 as compared to Naive, bp < 0.05 as compared to PTZ, cp < 0.05 as compared to CoQ10 (10 mg/kg), dp < 0.05 as compared to CoQ10 (20 mg/kg), ep < 0.05 as compared to minocycline (50 mg/kg), f p < 0.05 as compared to minocycline (100 mg/kg).

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attenuated severity of kindling as evidenced by reduced kindling score as compared to control (PTZ-kindled) group (Fig. 2). Further, combination of CoQ10 (10, 20 mg/kg) with minocycline (50 and 100 mg/kg) significantly reduced kindling score which was significant as compared to their effect per se in kindled animals (Fig. 2).

Effect of CoQ10 and its interaction with minocycline on the cognitive performance on kindled mice The repetitive administration of sub convulsive dose of PTZ (40 mg/kg, on alternate days) for a period of 29 days significantly impaired cognitive performance (increased initial acquisition

Naive PTZ(40 mg/kg) CoQ10(10 mg/kg) CoQ10(20 mg/kg) CoQ10(40 mg/kg) Mino(100 mg/kg) CoQ10(20 mg/kg)+Mino(100 mg/kg) CoQ10(10 mg/kg)+Mino(50 mg/kg)

a)

Mino(50 mg/kg)

MDA 1.0

a

a

nmol /mg pr

0.8

b

b

b

0.6

b,c

b,c

b,c

b,c b,c,d,e 0.4

b,c,d,e

b,c,d,e

b,c,d,e

b,c,d

b,c,d

b,c,d,e,f b,c,d,e,f

0.2

0.0

Cortex

b)

Hippocampus

GSH

c)

0.10

SOD 60 b,c,d,e,f

b,c,d,e,f b,c,d,e,f

b,c,d,e,f b,c,d

0.06

b,c

b,c

0.04

b,c,d

b,c,d,e b,c,d,e

b,c

b,c

b a

a

b,c,d,e b,c,d,e

b

0.02 0.00

Cortex

micromol/min/mg

micromol / mg pr

0.08

b,c,d 40

d)

b

b a

a

Cortex

Hippocampus

e) Catalase

Nitrite

0.8

15

b,c 5 a

0

b,c,d,e,f

b,c,d,e,f

b,c,d

b,c,d

b,c,d,e

b,c,d,e b,c,d,e

b,c,d,e

b,c

b,c

b,c

b

b

a b

b b,c

b,c

0.4

b,c,d

b,c b,c,d,e b,c,d,e

b,c b,c,d,e

b,c,d

b,c,d,e

b,c,d,e,f

b,c,d,e,f

0.2

a

Cortex

micromol / L

micromol /min /mg

a

0.6

10

b,c

b,c

20

0

Hippocampus

b,c, d,e

b,c,d,e

b,c

b,c

b,c,d,e

b,c,d

b,c,d,e

Hippocampus

0.0

Cortex

Hippocampus

Fig. 4. (a–e): Effect of minocycline on the protective effect of CoQ10 on oxidative stress in hippocampus and cortex of PTZ kindled mice: Data are expressed as mean  SEM. a p < 0.05 as compared to Naive, bp < 0.05 as compared to PTZ, cp < 0.05 as compared to CoQ10 (10 mg/kg), dp < 0.05 as compared to CoQ10 (20 mg/kg), ep < 0.05 as compared to minocycline (50 mg/kg), fp < 0.05 as compared to minocycline (100 mg/kg).

M. Bhardwaj, A. Kumar / Pharmacological Reports 68 (2016) 1301–1311

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Effect of CoQ10 and minocycline on AChE levels in PTZ kindled mice

latency (IAL) on day 13) as compared to naive group. However, CoQ10 (10, 20 and 40 mg/kg) and minocycline (50 and 100 mg/kg) treatment did not produce any significant effect on acquisition latency time on day 13 as compared to their respective control (PTZ) group. In contrast, chronic treatment with CoQ10 (10– 40 mg/kg) and minocycline (50 and 100 mg/kg) significantly reduced 1st [retention transfer latency (RTL) (day 14)] and 2nd RTL (day 29) as compared to control (PTZ) treatment. Further, combination of CoQ10 (10 and 20 mg/kg) with minocycline (50 and 100 mg/kg) significantly produce their synergistic effect (reduced transfer latency time on day 14 and 29 respectively) as compared to their effect per se in PTZ kindled animals (Fig. 3).

Sub-convulsive dose of PTZ (40 mg/kg) repetitively significantly raised acetylcholinesterase levels in both hippocampus and cortex as compared to the naive group. 29 days treatment of CoQ10 (10, 20 and 40 mg/kg) and minocycline (50 and 100 mg/kg) significantly attenuated AChE level in both hippocampus and cortex as compared to PTZ treated animals. Further, combination of CoQ10 (10 and 20 mg/kg) with minocycline (50 and 100 mg/kg) significantly attenuated AChE enzyme activity both in hippocampus and cortex as compared to their effect per se in PTZ kindled animals (Fig. 5). Influence of CoQ10, minocycline and their interaction on mitochondrial enzyme complex activities (I to IV) in PTZ-kindled mice

Effect of CoQ10, minocycline and their interaction on oxidative damage in the PTZ induced kindling epilepsy

Chronic subconvulsive dose of PTZ significantly impaired mitochondrial enzyme complex (I, II and IV) activities as compared to the naive animals. However, 29 days treatment with CoQ10 (10, 20 and 40 mg/kg) and minocycline (50 and 100 mg/kg) significantly restored mitochondrial enzyme complex enzyme activities (I, II and IV) as compared to control (PTZ) group. Further, combination of CoQ10 (10 and 20 mg/kg) with minocycline (50 and 100 mg/kg) significantly restored mitochondrial enzyme complex enzyme activities (I, II and IV) as compared to their effect per se in PTZ kindled animals (Fig. 6).

Raised brain MDA, nitrite concentration and depleted GSH, SOD and catalase levels were observed with repeated administration of a sub convulsive dose of PTZ in both hippocampus and cortex as compared to the naive group. Twenty nine days chronic treatment of CoQ10 (10, 20 and 40 mg/kg) and minocycline (50 and 100 mg/ kg) significantly attenuated lipid peroxidation (MDA), nitrite concentration and restored GSH, SOD and catalase levels in both hippocampus and cortex as compared to the PTZ (control) group. Further, combination of CoQ10 (10 and 20 mg/kg) with minocycline (50 and 100 mg/kg) significantly produced their synergistic effect (restored GSH, SOD, catalase level and attenuated lipid peroxidation and nitrite concentration) in both hippocampus and cortex as compared to their effect per se in PTZ kindled mice (Fig. 4a–e).

Naive

PTZ(40mg/kg)

m icro m per m g of pro tein

Mino(50mg/kg)

Influence of CoQ10, minocycline and their interaction on TNF-a level in the hippocampus of PTZ-kindled mice Repetitive sub convulsive dose of PTZ significantly raised TNF-a level as compared to the naive group. Treatment with CoQ10

CoQ10(10mg/kg)

Mino(100mg/kg)

CoQ10(10mg/kg)+Mino(50mg/kg)

CoQ10(20mg/kg)+Mino(100mg/kg)

a

a b

b

b,c

b,c b,c,d

b,c,d

b,c,d,e

b,c

b,c

b,c,d

b,c,d

b,c,d,e

b,c,d,f

0.2

0.0

CoQ10(40mg/kg)

AChE

0.6

0.4

CoQ10(20mg/kg)

Cortex

b,c,d,f

Hippocampus

Fig. 5. Effect of minocycline on the protective effect of CoQ10 on AchE activity in hippocampus and cortex of PTZ kindled mice: Data are expressed as mean  SEM. ap < 0.05 as compared to Naive, bp < 0.05 as compared to PTZ, cp < 0.05 as compared to CoQ10 (10 mg/kg), dp < 0.05 as compared to CoQ10(20 mg/kg), ep < 0.05 as compared to minocycline (50 mg/kg), fp < 0.05 as compared to minocycline(100 mg/kg).

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n mol of substrate / min / mg pr

n mol of substrate / min / mg pr

Naive PTZ (40 mg/kg) CoQ10(10 mg/kg) CoQ10(20 mg/kg) CoQ10(40mg/kg) Mino(50mg/kg) Mino(100 mg/kg) CoQ10(10 mg/kg)+Mino(50 mg/kg) CoQ10(20 mg/kg)+Mino(10 0 mg/kg) Compl ex I 80

60 b,c ,d,e,f b,c,d

b,c,d,e

b,c,d

b,c,d,e b,c,d,e

40

b,c

b,c

b,c

b,c

b

20

b,c ,d,e,f

b,c,d,e

b

a

a

0 Cortex

Hippocampus

Compl ex II

100

b,c,d,e,f

80

b,c,d,e b,c,d

b,c,d

b,c

b,c

60

b,c,d b,c

b a

40

b,c,d,e,f b,c,d,e b,c,d b,c

b

a

20

0

Cortex

Hippocampus

n mol of substrate / min / mg pr

Complex IV 100

b,c,d,e,f

80

b,c,d,e,f b,c,d,e b,c,d

b,c,d 60

b,c

b,c

b b

b 40

b,c,d,e b,c,d

b,c,d b

a

a

20

0

Cortex

Hippocampus

Fig. 6. Effect of minocycline on the protective effect of CoQ10 on mitochondrial enzyme complex activity (I, II and IV) in hippocampus and cortex of PTZ kindled mice: Data are expressed as mean  SEM. ap < 0.05 as compared to Naive, bp < 0.05 as compared to PTZ, cp < 0.05 as compared to CoQ10 (10 mg/kg), dp < 0.05 as compared to CoQ10 (20 mg/ kg), ep < 0.05 as compared to minocycline (50 mg/kg), fp < 0.05 as compared to minocycline (100 mg/kg).

(10–40 mg/kg) and minocycline (50 and 100 mg/kg) for a period of 29 days significantly attenuated TNF-a level in the hippocampus as compared to control (PTZ kindled) group. Further, combination of

CoQ10 (10 and 20 mg/kg) with minocycline (50 and 100 mg/kg) significantly attenuated TNF-a levels in the hippocampus as compared to their effect per se in PTZ kindled animals (Fig. 7).

M. Bhardwaj, A. Kumar / Pharmacological Reports 68 (2016) 1301–1311

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Fig. 7. Effect of minocycline on the protective effect of CoQ10 on TNF-a in hippocampus of PTZ kindled mice: Data are expressed as mean  SEM. ap < 0.05 as compared to Naive, bp < 0.05 as compared to PTZ, cp < 0.05 as compared to CoQ10 (10 mg/kg), dp < 0.05 as compared to CoQ10 (20 mg/kg), ep < 0.05 as compared to minocycline (50 mg/kg), f p < 0.05 as compared to minocycline (100 mg/kg).

Discussion Epilepsy is a complex neurological disorder with inadequate treatment due to side effects and drug interaction of the existing antiepileptic drugs. Pathogenesis of epilepsy is still not clearly understood in order to provide adequate and satisfactory treatment. Kindling is an experimentally induced phenomenon, frequently used to study the mechanism of epileptogenesis. Epilepsy has also been known to be associated with several other neuropsychiatric problems including cognitive dysfunction which further make the problems more complicated and complex. In the present study, sub-convulsive dose of PTZ on alternate days for a period of 29 days produced kindling epilepsy (as evidenced by an increase in kindling score), oxidative damage (raised lipid

peroxidation, nitric concentration, depletion of reduced glutathione and catalase activity), impaired mitochondrial enzyme complex activities (I to IV) and raised neuroinflammation (TNF-a) in the kindled brain. Oxidative stress causes free radicals generation which contributed to the development of the seizures. Oxidative stress further damages cellular macromolecules like mitochondria and endoplasmic reticulum which ultimately leads to cause the cell death. Mitochondrial dysfunction and neuroinflammation have been well reported to play a critical role in the pathophysiology of epilepsy [30]. However, neuroinflammation theory of epilepsy is still not clear. However, it is still not clear whether epilepsy causes neuroinflammation or vice versa. Coenzyme Q10 is a potent antioxidant, membrane stabilizer and an integral cofactor in the mitochondrial respiratory chain,

Fig. 8. Schematic diagram showing the mechanism of action of CoQ10 and Minocycline.

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facilitating the generation of adenosine triphosphate as a major energy source. It is a potent antioxidant to mitochondrial often designated as the mitochondrial restorer. CoQ10 is an essential cofactor of the electron transport chain. It accumulates in the mitochondria and restores the loss in mitochondrial transmembrane potential which results reduction of mitochondrial ROS generation and thus protect the mitochondria and cellular components from the oxidative damage. It is normally produced throughout the body and it can cross the blood brain barrier. CoQ10 has also been well demonstrated to have neuroprotective properties including anticonvulsant like effect [23]. In spite of having demonstrated neuroprotective effects, its exact mechanism of action has not been properly understood. In the present study, CoQ10 treatment for a period of 29 days significantly attenuated kindling score, oxidative damage, neuroinflammation (TNF-a) and restored mitochondrial enzyme complex activities (I–IV) in discrete areas of the brain suggesting its multiple target actions at various cellular and molecular cascades of complex epilepsy pathogenesis responsible for its neuroprotective effect. Minocycline, known anti-inflammatory agent and has been demonstrated to produce its neuroprotectantive effects in several experimental models of neurodegeneration including an antiepileptic effect. It has been well reported that microglia activation occurs during the progression of the seizures [32]. Besides, microglia activation has been well reported to contribute the neuroinflammatory theory of neurological problems including epilepsy [24]. Minocycline has been well documented to prevent microglia activation [31]. Inflammation is one of the mechanisms in seizure generation [30]. TNF-a is an inflammatory mediator in CNS. However, there is still a controversy on the role of TNF-a in seizure. Minocycline has been reported to attenuate TNF-a receptor gene expression. In the present study, minocycline treatment for a period of 29 days significantly attenuated kindling score, oxidative damage, alerted mitochondrial enzyme complex activities (I to IV) and raised neuroinflammation (TNF-a) in discrete areas of the brain suggesting its potential neuroprotective effects due to its action at multiple site against kindling epilepsy suggesting its role in epileptogenesis. It seems that both CoQ10 and minocycline might produce their respective neuroprotective effect by virtue of their action on multiple sites in epilepsy pathogenesis. Therefore, study has been further extended to elucidate pharmacological interaction of CoQ10 with minocycline. Combination studies of CoQ10 with minocycline further produce their synergistic effect suggesting that both drugs might share common mechanism of action. However, it is remained to be elucidated that how or at what level these drugs share their common mechanism in producing neuroprotective their gross neuroprotective effect. Further, patients with epilepsy often experience psychiatric comorbidities especially depression, cognitive dysfunction and anxiety like problems. These problems significantly impact on the quality of life of the patients. In the present study, PTZ induced kindling significantly impaired cognitive performance in an elevated performance task which is used commonly for spatial long term memory in addition to anxiety assessment. Chronic treatment of CoQ10, minocycline as well as their combinations (CoQ10 with minocycline) significantly attenuated their cognitive dysfunction as evidenced from shortening of both 1st RTL and 2nd RTL suggesting their potential effect on cognitive performance. Further, synergistic effect on cognitive performance is further supported and strengthened by their reduced acetylcholinesterase enzyme activities in cortex and hippocampus against kindling induced cognitive dysfunction. It seems that CoQ10 with minocycline treatment might also have some potential therapeutic effect against PTZ induced cognitive dysfunction. The schematic diagram of the present study is present in Fig. 8.

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