Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats

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Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats Q1

Hariom Kumara, Bhupesh Sharmab,c,n a

CNS Research Lab., Department of Pharmacology, School of Pharmacy, Bharat Institute of Technology, Partapur Bypass, Meerut, Uttar Pradesh, India b Department of Pharmacology, Amity Institute of Pharmacy, Amity University, Sector-125, Noida, Uttar Pradesh, India c CNS Pharmacology, Conscience Research, Pocket F-233, B, Dilshad Garden, Delhi 110095, India

art i cle i nfo

ab st rac t

Article history:

Autism is a neurodevelopment disorder. One percent worldwide population suffers with

Accepted 29 October 2015

autism and males suffer more than females. Microglia plays an important role in neurodevelopment, neuropsychiatric and neurodegenerative disorders. The present study

Keywords:

has been designed to investigate the role of minocycline in prenatal valproic acid induced

Autism

autism in rats. Animals with prenatal valproic acid have reduced social interaction (three

Microglia inhibition

chamber social behaviour apparatus), spontaneous alteration (Y-Maze), exploratory activ-

Intestinal motility

ity (Hole board test), intestinal motility, serotonin levels (both in prefrontal cortex and

Mitochondrial complex

ileum) and prefrontal cortex mitochondrial complex activity (complexes I, II, IV). Further-

Blood brain barrier permeability

more, prenatal valproic acid treated animals have shown an increase in locomotion

Serotonin

(actophotometer), anxiety (elevated plus maze), brain oxidative stress (thiobarbituric acid reactive species, glutathione, catalase), nitrosative stress (nitrite/nitrate), inflammation (both in brain and ileum myeloperoxidase activity), calcium and blood brain barrier permeability. Treatment with minocycline significantly attenuated prenatal valproic acid induced reduction in social interaction, spontaneous alteration, exploratory activity intestinal motility, serotonin levels and prefrontal cortex mitochondrial complex activity. Furthermore, minocycline has also attenuated prenatal valproic acid induced increase in locomotion, anxiety, brain oxidative and nitrosative stress, inflammation, calcium and blood brain barrier permeability. Thus, it may be concluded that prenatal valproic acid has induced autistic behaviour, biochemistry and blood brain barrier impairment in animals, which were significantly attenuated by minocycline. Minocycline should be explored further for its therapeutic benefits in autism. & 2015 Published by Elsevier B.V.

Q2

n

Corresponding author at: Department of Pharmacology, Amity Institute of Pharmacy, Amity University, Sector-125, Noida, Uttar Pradesh, India. E-mail addresses: [email protected] (H. Kumar), [email protected], [email protected] (B. Sharma). http://dx.doi.org/10.1016/j.brainres.2015.10.052 0006-8993/& 2015 Published by Elsevier B.V.

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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1.

Introduction

Autism is a severe behavioural disorder characterized by pervasive impairments in social interactions, deficits in verbal and nonverbal communication along with stereotyped as well as repetitive patterns of behaviours and interests Q3 (DSM-V, 2013). The worldwide population prevalence of autism is about one percent and males have a higher rate of autism than females (Lai et al., 2014; Werling and Geschwind, 2015). Autistic individuals exhibits a variety of comorbid traits including seizures, anxiety, aggressive behaviour, gastrointestinal problems, motor deficits, abnormal sensory processing and sleep disturbances (Mazurek et al., 2013; Spencer et al., 2013; Hodge et al., 2014). Valproic acid (VPA) is a blocker of histone deacetylase widely used to treat epilepsy, bipolar disorders, and migraine; its administration during pregnancy increases the risk of autism in the child. Thus, prenatal VPA exposure has emerged as a rodent model of Autism (Kim et al., 2011). Prenatal valproic acid (Pre VPA) is one of the good models utilized for induction of experimental autism, because it shows broad spectrum autistic characteristics in rodents similar to humans suffering from autism (Schneider and Przewłocki, 2005). Microglia cells are known as resident macrophages of the CNS. These have an important role in neuro-development and neuro-degeneration disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (Kaushik and Basu, 2013). The chronic activation of microglia may result in up regulation of inflammatory cytokines (Kaushik and Basu, 2013). Minocycline is documented as possible inhibitor of microglia activation (Kumar et al., 2012; Zhu et al., 2014). Minocycline is a tetracycline antibiotic which used in psychiatry for its pleiotropic anti-inflammatory and neuroprotective action. Minocycline has pleiotropic mode of action in the brain as by modulation of retinoic acid signalling (Regen et al., 2015). Minocycline has been reported to modulate behaviour abnormalities, including social behaviour and locomotor activity (Zhu et al., 2014). In rodents, minocycline has shown the protection against apoptosis along with inflammation associated brain edema and blood brain barrier dysfunction of the early brain injury (Li et al., 2015). The role of minocycline in Pre VPA induced autism has not been studied till date. In the light of above, the present study has been undertaken to investigate the potential of minocycline in Pre VPA induced autism in rats.

2.

Results

Administration of 0.9% saline (on embryonic 12.5th day), CMC (0.5%, 10 ml/kg orally; once daily for 30 days) and minocycline dose 1 and 2 (25 and 50 mg/kg orally; once daily for 30 days) per se did not show any significant effect on any parameter in this study.

2.1.

Effect on locomotor activity

Pre VPA (500 mg/kg; intra-peritoneal, single administration) treatment has significantly increased the number of counts in 0–10 min and 10–20 min interval as compared to control animals (F7, 40 ¼ 263.651, po0.001), which is the indication of hyper-locomotor activity on actophotometer. Treatment with minocycline significantly attenuated Pre VPA induced increase in number of counts in 0–10 min and 10–20 min interval (F7, 40 ¼ 262.516, po0.001), which suggests reduction of Pre VPA induced hyper-locomotor activity (Table 1).

2.2.

Effect on social behaviour

2.2.1.

Sociability and sociability index

Control animals have spent more time in the stranger chamber as compared to empty chamber, during the sociability test on three chamber sociability apparatus, reflecting normal sociability. Pre VPA treatment has reduced time spent in stranger chamber (F7, 40 ¼13.824, po0.001) and increased time spent in the empty chamber (F7, 40 ¼21.905, po0.001), as compared to control animals, which shows lower sociability in Pre VPA treated animals. Administration of minocycline (doses 1 and 2) has significantly attenuated Pre VPA induced reduction in time spent in stranger chamber (F7, 40 ¼13.824, po0.001) followed by increased time spent in the empty chamber (F7, 40 ¼21.905, po0.001). Pre VPA treated animals, have shown reduction in the sociability index when compared to control animals (F 7, 40 ¼ 51.404, po0.001), which was significantly attenuated by minocycline (doses 1 and 2) (F7, 40 ¼51.404, po0.001) (Fig. 1).

2.2.2.

Social preference and social preference index

Control animals have spent more time in novel chamber as compared to the familiar chamber during the social preference test on three chamber sociability apparatus, reflecting normal social preference. Pre VPA treatment has reduced time spent in novel chamber (F7, 40 ¼73.216, po0.001) followed by increased time spent in the familiar chamber (F7, 40 ¼212.197, po0.001) compared to control animals, which suggests lower social preference in Pre VPA treated animals. Administration of minocycline (doses 1 and 2) has significantly attenuated Pre VPA induced reduction of time spent in novel chamber (F7, 40 ¼ 73.216, po0.001) followed by increased time spent in the familiar chamber (F7, 40 ¼ 212.197, po0.001) (Fig. 2). Pre VPA treated animals have shown reduction in the social preference index (F7, 40 ¼ 297.802, po0.001), when compared to control animals, which was significantly attenuated by minocycline (F7, 40 ¼297.802, po0.001) (doses 1 and 2). Thus the administration of minocycline has corrected the Pre VPA induced impairment in sociability as well as social preference.

2.3.

Effect on repetitive behaviour

Pre VPA treatment has significantly decreased the % spontaneous alteration as compared to control animals on Y-maze (F7, 40 ¼ 142.833, po0.001). This shows an increase in repetitive behaviour in Pre VPA treated animals. Treatment with minocycline significantly attenuated Pre VPA induced

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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Table 1 – Effect of various agents on repetitive behaviour, locomotor activity, anxiety and exploratory activity. Groups

Control Pre-saline CMC Mino D1 per se Mino D2 per se Pre VPA Pre VPAþMino D1 Pre VPAþMino D2

Repetitive behaviour on Y maze (% Spontaneous alteration)

Locomotor activity on actophotometer (number of counts)

Anxiety on elevated plus maze Exploratory activity on hole board apparatus

0–10 min

10–20 min

% Open arm entries

% Open arm time spent

Latency (s) Number of rearings

Number of hole pokes

7375.11 7174.97 7074.9 6574.55

310720.7 311721.77 312721.84 319722.33

13079.1 13379.31 13179.17 13979.73

70.8774.96 68.78274.814 70.8274.957 72.2975.06

35.4572.48 35.572.485 35.1672.46 33.0472.31

670.42 5.370.37 570.35 6.270.43

15.571.08 15.3771.07 15.571.08 14.1270.98

13.8770.97 13.570.945 13.7770.96 14.1270.98

6874.76

309721.63

13079.1

73.2675.12

35.0472.45

5.870.41

14.671.02

14.3771.00

2671.82n 4373.01#

680747.6n 447731.29#

285719.95n 162711.34#

30.97472.168n 46.16173.231#

17.7971.24n 21.571.50#

1571.05n 1270.84#

8.12570.56n 11.1270.77#

6.570.45n 9.2570.64#

336723.52#

143710.01#

65.3974.57#

30.7072.14#

7.870.5#

13.7570.96#

12.170.84#

6774.69

#

Results are mean7standard deviation; two way ANOVA followed by Bonferroni’s post-test. % Spontaneous alteration – n po0.001 vs % spontaneous alteration of control animals; # po0.001 vs % spontaneous alteration of Pre VPA treated animals. 0–10 min – n po0.001 vs number of counts by control animals; # po0.001 vs number of counts by Pre VPA treated animals. 10–20 min – n po0.001 vs number of counts by control animals; # po0.001 vs number of counts by Pre VPA treated animals. % Open arm entries – n po0.001 vs % open arm entries by control animals; #po0.001 vs % open arm entries by Pre VPA treated animals. % Open arm time spent – n po0.001 vs % open arm time spent by control animals; #po0.001 vs % open arm time spent by Pre VPA treated animals. Latency – n po0.001 vs latency of first poke of control animals; # po0.001 vs latency of first poke Pre VPA treated animals. Number of rearings – n po0.001 vs numbers of rearing by control animals; # po0.001 vs numbers of rearing by Pre VPA treated animals. Hole poke – n po0.001 vs numbers of hole poke by control animals; # po0.001 vs numbers of hole poke by Pre VPA treated animals. Pre VPA – prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2, CMC – carboxy-methyl cellulose.

Fig. 1 – Effect of various agents on sociability and sociability index on three chamber sociability apparatus. Results are mean7standard deviation, one way ANOVA followed by Bonferroni's post-test. (A) * po0.001 vs time spent in stranger chamber by control animals; # po0.001 vs time spent in stranger chamber by Pre VPA treated animals. (B) * po0.001 vs time spent in empty chamber by control animals; # po0.001 vs time spent in empty chamber by Pre VPA animals. (C) * po0.001 vs sociability index of control animals; # po0.001 vs sociability index of Pre VPA treated animals. Pre VPA – prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2.

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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Fig. 2 – Effect of various agents on social preference and social preference index on three chamber sociability apparatus. Results are mean7standard deviation, one way ANOVA followed by Bonferroni’s post-test. (A) n po0.001 vs time spent in familiar chamber by control animals; # po0.001 vs time spent in familiar chamber by Pre VPA treated animals. (B) n po0.001 vs time spent in novel chamber by control animals; # po0.001 vs time spent in novel chamber by Pre VPA treated animals. (C) n po0.001 vs social preference index of control animals; # po0.001 vs social preference index of Pre VPA treated animals. Pre VPA – prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2.

decrease in the % spontaneous alteration (F7, 40 ¼ 142.833, po0.001), (Table 1). Which suggest reduction of Pre VPA induced repetitive behaviour.

2.4.

Effect on anxiety

Control animals have shown more open arm time spent and less open arm entries in comparison of close arm, which shows the normal behaviour of animals on elevated plus maze. Pre VPA treatment has decreased the time spent in open arm (F7, 40 ¼ 123.111, po0.001), and number of open arm entries (F7, 40 ¼134.564, po0.001), as compared to control animals, which suggests induction of anxiety in Pre VPA treated animals. Treatment with minocycline significantly attenuated Pre VPA induced decrease of time spent in open arm (F7, 40 ¼123.111, po0.001) and number of open arm entries (F7, 40 ¼134.564, po0.001) (Table 1). Which suggest reduction of Pre VPA induced anxiety.

2.5.

Effect on exploratory activity

Control animals show exploratory movement towards a novel place which is a hole poke in hole board apparatus, which is the normal exploratory behaviour in animals. Pre VPA treatment has increased latency to first poke (F7, 40 ¼395.743, po0.001), along with decrease the number of rearing (F7, 40 ¼ 87.478, po0.001) as well as hole poking (F7, 40 ¼120.996, po0.001), as compared to control animals. Treatment with minocycline significantly attenuated Pre VPA induced increased latency to first poke (F7, 40 ¼ 395.743, po0.001) along with decrease the number of rearing (F7, 40 ¼87.478, po0.001) as well as hole poking (F7, 40 ¼ 120.996, po0.001) (Table 1). Which suggest increase of Pre VPA induced lower exploratory activity.

Fig. 3 – Effect of various agents on GIT motility measured as GIT transit index. Results are mean7standard deviation, one way ANOVA followed by Bonferroni's post-test. n po0.001 vs GIT transit index of control animals; # po0.001 vs GIT transit index of Pre VPA treated animals. Pre VPA – prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2.

2.6.

Effect on gastrointestinal tract (GIT) motility

Pre VPA treatment has decreased the GIT transit index as compared to control animals (F7, 16 ¼129.678, po0.001), which is the indication of reduction in GIT motility. Treatment with minocycline significantly attenuated Pre VPA induced reduction in GIT transit index (F7,

16 ¼129.678,

po0.001) (Fig. 3).

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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Fig. 4 – Effect of various agents on blood brain barrier permeability measured by Evans blue concentration in cerebellum. Results are mean7standard deviation, one way ANOVA followed by Bonferroni's post-test. n po0.001 vs concentration of Evans blue in cerebellum of control animals; # po0.001 vs concentration of Evans blue in cerebellum of Pre VPA treated animals. Pre VPA - prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2.

2.7. Effect of blood brain barrier permeability using Evans blue concentration and staining in the cerebellum Cerebellum of Pre VPA treated animals was stained by in vivo (intra-peritoneal) administration of Evans blue (qualitative estimation). Significantly higher concentration of Evans blue was found in the cerebellum of Pre VPA treated animals (F7, 16 ¼ 397.244, po0.001) (quantitative estimation). Treatment with minocycline, significantly attenuated Pre VPA induced staining and higher concentration of Evans blue in the cerebellum (F7, 16 ¼397.244, po0.001) (Figs. 4 and 5). Which suggest decrease of Pre VPA induced increase in blood brain barrier permeability.

2.8. Effect on brain oxidative stress (GSH, TBARS, and catalase activity), nitrosative stress (nitrite/nitrate), brain calcium levels, mitochondrial dysfunction (complexes I, II and IV in prefrontal cortex), inflammation (MPO in brain and ileum) and serotonin (prefrontal cortex and ileum) Administration of Pre VPA has increased oxidative stress [decrease brain GSH (F7, 16 ¼ 19.558, po0.001), brain catalase (F7, 16 ¼ 55.922, po0.001) and increase TBARS (F7, 16 ¼270.191, po0.001)], nitrosative stress [increase nitrite/nitrate (F7, 16 ¼ 55.242, po0.001)], brain calcium levels (F7, 16 ¼25.166, po0.001), mitochondrial dysfunction [decrease complex I (F7, 16 ¼ 45.746, po0.001), II (F7, 16 ¼480.659, po0.001) and IV (F7, 16 ¼ 460.161, po0.001) in prefrontal cortex], inflammation [increase MPO in brain (F 7, 16 ¼ 19.558, po0.001) and ileum (F7, 16 ¼ 19.558, po0.001)] and decreased serotonin levels [decrease prefrontal cortex (F7, 16 ¼ 19.558, po0.001) and ileum

Fig. 5 – Effect of various agents on blood brain barrier permeability measured by Evans blue staining in cerebellum. Pre VPA – prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2. Cerebellum of Pre VPA treated animal have shown higher staining compare with control animal, while Minocycline treatment have shown lower staining compare with Pre VPA animal. (F7, 16 ¼19.558, po0.001)], as compared to control animals. Treatment with minocycline significantly attenuated Pre VPA induced increase in oxidative stress [decrease brain GSH (F7, 16 ¼ 19.558, po0.001), brain catalase (F7, 16 ¼ 55.922, po0.001) and increase TBARS (F7, 16 ¼270.191, po0.001)], nitrosative stress [increase nitrite/nitrate (F7, 16 ¼55.242, po0.001)], brain calcium levels (F7, 16 ¼ 25.166, po0.001), mitochondrial dysfunction [decrease complex I (F7, 16 ¼45.746, po0.001), II (F7, 16 ¼ 480.659, po0.001) and IV (F7, 16 ¼460.161, po0.001) in prefrontal cortex], inflammation [increase MPO in brain (F7, 16 ¼ 19.558, po0.001) and ileum (F7, 16 ¼ 19.558, po0.001)] and decreased serotonin levels [decrease prefrontal cortex (F7, 16 ¼ 19.558, po0.001) and ileum (F7, 16 ¼ 19.558, po0.001)] (Tables 2 and 3).

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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Table 2 – Effect of various agents on brain oxidative stress, nitrosative stress, inflammation and calcium levels. Groups

Brain oxidative stress

Control Pre-saline CMC Mino D1 per se Mino D2 per se Pre VPA Pre VPAþMino D1 Pre VPAþMino D2

Brain nitrosative stress

Brain inflammation

Brain Calcium

GSH (μM/mg of protein)

TBARS (nM/mg protein)

Catalase (U/mg of protein)

Nitrate/nitrite (nM/ mg protein)

Brain MPO (U/mg of protein)

19.171.337 18.8171.316 19.0171.330 18.671.26 18.90571.323 13.0170.910n 14.9871.159#

4.870.336 4.670.322 4.5570.304 4.9170.360 4.6770.344 14.0770.984n 10.5670.711#

4.9570.346 4.6670.326 4.92570.344 4.6570.259 4.7670.310 2.43570.170n 3.2170.255#

12.870.896 12.3470.863 12.0170.840 12.7870.894 11.9670.847 20.171.336n 15.8771.169#

3.0270.211 2.92570.204 2.8670.200 3.2270.209 3.1070.183 11.6070.941n 8.4770.752#

63.89574.472 62.45574.371 62.01574.341 62.1274.348 60.6174.545 87.476.118n 73.175.601#

17.9971.291#

7.1170.462#

4.070.331#

13.0170.985#

5.1270.281#

67.174.627#

Results are mean7standard deviation; two way ANOVA followed by Bonferroni’s post-test. GSH – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. TBARS – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Catalase – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Nitrate/nitrite – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Brain MPO – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Calcium – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Pre VPA – prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2, CMC – carboxy-methyl cellulose.

Table 3 – Effect of various agents on Prefrontal cortex mitochondrial enzyme complex (nicotinamide adenine dinucleotide– hydrogen dehydrogenase: Complex-I, succinate dehydrogenase: Complex-II and cytochrome oxidase: Complex-IV), serotonin (5HT), ileum myeloperoxidase activity (MPO) and 5HT levels. Groups

Prefrontal cortex Mitochondrial enzyme complexes (% of control)

Control Pre-saline CMC Mino D1 per se Mino D2 per se Pre VPA Pre VPAþMino D1 Pre VPAþMino D2

Complex-I

Complex-II

Complex-IV

10077.00 95.276.664 97.4576.821 95.976.713 98.176.867 50.473.528n 74.1275.18#

10077.432 97.376.818 9876.861 99.276.944 98.276.874 40.472.828n 70.474.928#

10077.121 98.676.902 96.9 76.783 98.6 76.902 96.3 76.741 58.474.088n 69.9974.899#

82.3275.662#

81.375.691#

84.275.894#

Ileum MPO (U/mg of protein)

Ileum 5HT (lg/g of tissue)

5.2270.364 5.1670.361 5.17570.361 5.6070.36785 5.070.35525 18.5371.297n 11.3270.6454#

0.99870.06986 0.96570.06755 0.93370.06531 0.94570.06804 0.97870.06993 0.42170.0326n 0.52170.04186#

5HT (l g/g of tissue)

0.60370.032 0.60170.032 0.59970.031 0.58470.031 0.63270.04361 0.12670.018n 0.24870.025# 0.47870.034#

7.2170.46375#

0.73270.05691#

Results are mean7standard deviation; two way ANOVA followed by Bonferroni’s post-test. Complex-I – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Complex-II – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Complex-IV – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. PFC 5HT – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Ileum MPO – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Ileum 5HT – n po0.001 vs control animals; # po0.001 vs Pre VPA treated animals. Pre VPA – prenatal valproic acid; Mino – minocycline; D1 – dose 1; D2 – dose 2, CMC – carboxy-methyl cellulose.

3.

Discussion

In the present study, prenatal administration of valproic acid has resulted in the reduction of social interaction, spontaneous alteration, exploratory activity, intestinal motility, serotonin levels (prefrontal cortex and ileum) and mitochondrial complex activity (complexes I, II, IV) along with an

increase in locomotion, anxiety, brain oxidative stress, nitrosative stress, inflammation (brain and ileum), calcium and blood brain barrier leakage. Pre VPA exposure has been well documented for behavioural, biochemical and pathological impairments in animals, similar to autistic individuals (Rinaldi et al., 2007; Chauhan et al., 2011; Kim et al., 2011; Sandhya et al., 2012; Ahn et al., 2014; de Theije et al., 2014). It is widely used for

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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induction of autism in rodents. There is no clear evidence of the molecular mechanisms by which VPA can trigger autism in humans or autistic features in the animal model. It has been reported that Pre VPA exposure results in various behaviour abnormalities such as reduction in social interaction (Kim et al., 2011), repetitive behaviour (Markram et al., 2008), exploratory activity (Sandhya et al. 2012) along with increase in locomotion (Sandhya et al. 2012) and anxiety (Schneider et al., 2006). Thus, our results are in parallel with previous findings. The emerging studies have suggested that overexpression of NMDA receptors, kinase calcium/calmodulin-dependent protein kinase II (Rinaldi et al., 2007), activation of glycogen synthase kinase 3 beta/beta-catenin pathway (Go et al., 2012) and microglial activation (Suzuki et al., 2013) may be involved in behaviour alteration in autism like condition. The examination of pathways and structures that may mediate these behavioural impairment, indicate a promising subject for future investigation of the aetiological triggers and molecular alterations associated with autism. Brain oxidative stress, nitrosative stress (Sandhya et al., 2012), mitochondrial dysfunction (Ahn et al., 2014) and inflammation (both in brain and ileum) (de Theije et al., 2014) have been reported in Pre VPA induced autism. Valproic acid may trigger the formation of reactive oxygen species (ROS) (Sandhya et al., 2012). This is known that, the neuronal cells at the early stage of development are more vulnerable to the effect of oxidative stress. ROS may interfere with the neurodevelopmental process by damaging lipids, proteins and DNA of the neuronal cells. Autistic individuals have shown neuro-inflammation and altered inflammatory responses, throughout their life. It has been reported that Pre VPA exposed animals have higher susceptibility for peripheral and neuronal inflammation along with microglial activation (Lucchina and Depino, 2014). VPA has been reported to result in mitochondrial ROS formation, lipid peroxidation, mitochondrial membrane potential collapse, mitochondrial swelling and finally the release of cytochrome c (Jafarian et al., 2013). Autism has been linked with gastrointestinal dysfunction, which may be due to structural defects in mucosa and muscle of the ileum and stomach (Kim et al., 2013). Reduced levels of serotonin (prefrontal cortex and ileum) have been reported in valproic acid treated animals (de Theije et al., 2014). Increase in intracellular calcium concentration is responsible for abnormal neuronal circuit functioning in autism (Rinaldi et al., 2008). It has been suggested that an increase in calcium levels may be due to upregulation of NMDA receptor and surface transient receptor potential 3 channels (Mizoguchi et al., 2014). In-vivo administration of Evans blue has shown significantly higher staining and concentration in the cerebellum of Pre VPA treated animals. We have tried to assess Pre VPA induced alteration in blood brain barrier permeability with the help of Evans blue. It appears from the results that there is some kind of correlation between the concentration and staining of Evans blue and other behavioural as well as biochemical parameters utilized in this study. We have not found any earlier report utilizing Evans blue to assess blood brain barrier permeability in autism and Pre VPA exposed animals. Blood brain barrier permeability change using Evans blue has been employed in various neurological conditions

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(Manaenko et al., 2011; Zeng et al., 2012; Liu et al., 2014). We have used this technique first time in prenatal valproic acid induced autistic condition in rats. In our preliminary studies we have compared the Pre VPA induced blood brain barrier permeability change with that of global ischaemia and reperfusion injury induced changes in blood brain permeability. These findings suggest that Evans blue extravasation may serve as an important qualitative and quantitative parameter to check blood brain barrier permeability alterations in experimental autism (refer to Supplementary material available with this article). Systemic inflammation has been reported in autism (de Theije et al., 2014), which may increase the blood brain barrier permeability (Stolp et al., 2005). Adhesion molecules modulate the signalling and the permeability of the blood brain barrier. Reduction in Adhesion molecules (sPECAM-1 and sP-selectin) (Onore et al., 2012), vascular endothelium activation (Yao et al., 2006) and decreased cerebral blood flow (Galuska et al., 2002) have been reported in autism. Valproic acid has been reported to cause ultra-structural changes in glial cells and nerve cells in brain, which may result in increased blood brain barrier permeability. Furthermore, astrocytes and neuronal lesions coexisted with a considerable damage to the elements of the blood brain barrier of the cerebellar structure in valproic acid treatment (SobaniecŁotowska and Lotowska, 2005; Sobaniec-Lotowska and Sobaniec 1999). In the present study, minocycline has significantly attenuated, Pre VPA induced reduction in social interaction, spontaneous alteration, exploratory activity, intestinal motility, serotonin level (prefrontal cortex and ileum) and mitochondrial complex activity (Complexes I, II, IV) along with increased locomotion, anxiety, brain oxidative stress, nitrosative stress, inflammation (brain and ileum), calcium levels and blood brain barrier leakage. This has been previously reported that minocycline may improve social behaviour (Zhu et al., 2014), exploratory activity and reduce rotation behaviour (Shan et al., 2011), locomotor activity (Zhu et al., 2014) and anxiety. These beneficial behaviour alterations by minocycline may be due to dendritic spine morphology (Bilousova et al., 2009) along with alteration in excitatory and inhibitory neurotransmission via calcium and tumour necrosis alpha (Ming et al., 2013; González et al., 2007). It has been reported that minocycline reduces the various inflammatory markers associated with microglia activation (Riazi et al., 2008). It has been reported that, minocycline reduces brain oxidative stress and nitrosative stress (Kumar et al., 2012; Monte et al., 2013) by its action on the nitric oxide pathway (Jiang et al., 2009), MAP kinase signalling and c-Jun N-terminal kinase signalling (Wilkins et al., 2004). Minocycline has shown anti-inflammatory activity via its inhibitory activity on nuclear factor-kappaB (NF-kappaB) (Nikodemova et al., 2006), which may help in exerting gastro protective effect (Asmari et al., 2014). It has been reported that minocycline may provide a protective role in mitochondrial dysfunction by restoration of mitochondrial complex activity (Kumar et al., 2012), inhibition of mitochondrial permeability and transition pore (Gieseler et al., 2009). Recently, minocycline has been reported to increase serotonin levels (Ahuja et al.,

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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2008), which may be due to the protection of serotonergic raphé neurons (Buller et al., 2012). Minocycline has been reported to inhibit glutamate-evoked elevation of the cytosolic calcium concentration in hippocampal neurons (González et al., 2007) and reduction of calcium retention capacity (Månsson et al., 2007). Minocycline has previously been reported to provide protection in stroke induced blood brain barrier disruption (Yenari et al., 2006). This protection may be due to minocycline dependent alteration in the MMP9 (Mishra et al., 2009) and nitric oxide pathway (Ryu and McLarnon, 2006). Zhao et al. (2007) have suggested that neuro-inflammation may be the important factor, responsible for disrupting the blood–brain barrier permeability and this condition was restored minocycline in animals (Zhao et al., 2007). Minocycline has shown to provide functional benefits in clinical study relevant to autism such as fragile X syndrome (Paribello et al., 2010; Utari et al., 2010). This study highlights the utility of minocycline with relevance to various behavioural, biochemical and blood brain barrier permeability alterations in autistic animals. On the basis of the results of this study and above discussion, it may be concluded that prenatal valproic acid has induced autism. Treatment with minocycline has recuperated pre natal valproic acid administration induced autistic behaviour, biochemistry and blood brain barrier leakage. This study highlights the utility of some important parameters (gastric motility, serotonin imbalance, mitochondrial dysfunction, and change in BBB permeability) in autistic condition which should be further investigated for the better understanding of developmental, molecular and cellular neurobiology of autism. Further, research is needed to identify the full potential of minocycline in autism condition.

4.

Experimental procedure

4.1.

Animals

In the present study, adult albino Wistar rats (Indian Veterinary Research Institute, Izatnagar, India) were allowed to mate together. Offspring were housed with their mother till weaning (postnatal day 20), with free access to water and standard laboratory pellet chow diet (Golden Feeds Ltd., New Delhi, India) along with the exposure to natural light and dark cycle. The experiments were conducted on the male offspring between 9.00 and 18.00 h. The offspring acclimatized to the laboratory condition, five days prior to behavioural study and 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 the 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 (Reg. no. 25/230/2011/AWD/CPCSEA).

4.2.

Drugs and chemicals

Minocycline was obtained from Micro Labs Limited India. Sodium valproate was obtained from Sun Pharma Pvt. Ltd., India. Lowry's reagent, 5,50 -dithiobis (2-nitrobenzoic acid)

(DTNB), Folin–Ciocalteu reagent, bovine serum albumin (BSA) and N-naphthylethylenediamine were purchased from Sigma Aldrich, India. 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, Evans blue was purchased from SISCO Research Laboratory Pvt. Limited, Mumbai, India. Hydrogen peroxide and pyridine were purchased from Rankem Laboratories Pvt. Ltd., India. Acetic acid, acetylcholine iodide, copper sulphate pentahydrate, disodium hydrogen phosphate, n-butanol, potassium chloride, potassium ferricyanide, sodium bicarbonate, sodium carboxymethyl-cellulose, sodium citrate, sodium dihydrogen phosphate, sodium dodecyl sulphate, sodium hydroxide, sodium phosphate monobasic, sodium phosphate dibasic, sodium tartarate, succinic acid, sucrose, trichloroacetic acid and zinc sulphate were purchased from CDH Laboratories Pvt. Ltd., India.

4.3.

Drug administration

All drug solutions were freshly prepared before use. The sodium salt of valproic acid was dissolved in 0.9% saline and given subcutaneously (500 mg/kg; on embryonic day 12.5th) to the animals (Markram et al., 2008). Minocycline was suspended in 0.5% carboxy-methylcellulose (CMC) and given orally (25 and 50 mg/kg; from postnatal day 21st to 50th) by oral gavage to the rats (Monte et al., 2013).

4.4.

Prenatal valproic acid (Pre VPA) induced autism

Pregnancy was determined by the presence of a vaginal plug on the embryonic 1st day. Sodium valproate was dissolved in 0.9% saline. Pregnant females on embryonic 12.5th day received sodium valproate (500 mg/kg; intra-peritoneal; single dose). Females were housed individually and allowed to raise their offspring until weaning (post-natal day 20) (Markram et al., 2008). Their male offspring were utilized for various assessments in present study.

4.5.

Assessment of social interaction test

Impairment in social interaction is the core feature of the autism (Restall and Magill-Evans, 1994; Byrge et al., 2015). In the present study, the social interaction was assessed by three chamber social behaviour apparatus (70 cm wide and 30 cm long and divided into three equal chambers) for rat as described by previous researchers with slight modification (Kim et al., 2011). Briefly, before starting first phase the rat being tested was placed in the central chamber and habituated for 5 min. The first phase was called sociability phase. In the sociability phase, immediately after 5 min habituation termination, a stranger rat was placed in the left chamber under a small wire cage with a radius of 5.5 cm. The right chamber wire cage was remained empty during sociability phase. During the sociability phase the left chamber was called stranger chamber while right chamber was called empty chamber. The testing rat was allowed to freely explore all the three chambers for 10 min. Directly after the termination of the first phase, the second phase was conducted for

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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10 min. This second phase was called social preference phase. In this phase, an unfamiliar rat from another control litter was placed in the right chamber with a wire cage. In this phase, left chamber was called familiar chamber while right chamber was called novel chamber. The time spent in the all three chambers by test animal was observed. The sociability was expressed as “Sociability Index”, (SI) which was defined as the ratio between the time spent by the test animal in stranger side to the empty side. On the other hand social preference was expressed as “Social Preference Index”, (SPI) which was defined as the ratio between the time spent by the test animal in the novel side to the familiar side. SI ¼

Time spent in stranger chamber Time spent in emptychamber

SPI ¼

4.6.

Time spent in novel chamber Time spent in familiar chamber

Assessment of repetitive behaviour

Repetitive behaviour has previously been reported in autism (Markram et al., 2008). Rats were tested in the spontaneous alteration paradigm where decrease in percentage spontaneous alternations was taken as an indicator of increase in repetitive behaviour (Markram et al., 2008; Merali et al., 2014). Rats were exposed individually into the start arm of a standard Y-maze and allowed to move freely for 8 min. The series of arm entries were observed to determine the number of spontaneous alternations. An alternation occurred if the rat entered the three different arms in succession. The following formula was used to calculate %Sponteneous alternations ¼

4.7.

Total alternations  100 Total arms entered
2

Assessment of locomotor activity

It has already been reported that valproic acid causes an increase in locomotor activity (Sandhya et al., 2012). Actophotometer (INCO, Ambala, India) was used to assess locomotor activity. The apparatus was placed in a darkened, sound attenuated and ventilated testing room during assessment. Locomotor activity was assessed by comparing the number of beam breaks across a 20-min session divided into two 10-min intervals by animals (Sandhya et al., 2012).

4.8.

Assessment of anxiety

Behavioural alteration in reference to anxiety has been reported in valproic acid induced autism (Sandhya et al., 2012).The anxiety behaviour was assessed by elevated plusmaze. The elevated plus-maze was made of wood and consisted of two opposite open arms (50  10 cm2), enclosed by 40-cm high walls. The maze was elevated 50 cm above the floor. Each rat was placed for 5 min in a pretest arena (45  45  45) prior to exposure to the maze. This step facilitates exploratory behaviour. An investigator sitting approximately 2 m apart from the apparatus, observed the rats.

Immediately after the pre-test, exposed rats were placed in the centre of the elevated plus-maze facing one of the open arms. During the 5-min test period, the following measurements were taken: the number of entries into the open and closed arms and the time spent in the open and closed arms. By these measures, the following variables were measured: % time spent in the open arms compared to time spent in both open and enclosed arms and % open arm entries compared to both open and enclosed arm entries. An entry was defined as entering into one of arm with all four paws, decrease in time spent and number of entries in open arm were considered as anxiety behaviour.

4.9.

Assessment of exploratory activity

Reduced exploration activity has been reported in valproic acid induced autism in animals (Sandhya et al., 2012). The exploratory activity was assessed using hole-board apparatus. The hole-board apparatus consisted of a grey Plexiglas platform (40 cm  40 cm) raised to a height of 15 cm from the floor of a grey box (40 cm  40 cm  40 cm). The grey platform consisted of 16 equivalent square compartments (12 peripheral and 4 central), each featuring a central circular hole (3 cm diameter). Test session was started by placing each animal in the centre of the platform and allowed to freely explore on the apparatus for 5 min. Platform consisted of 16 equivalent square compartments (12 peripheral and 4 central), each featuring a central circular hole (3 cm diameter). Latency of poke is, indirectly, while the number of rearing and holepoking (nose of the animal puts inside the hole) are directly, proportional to exploratory activity of animals. These parameters were measured during a 3-min time session.

4.10. test

Assessment of gastrointestinal tract (GIT) motility

Change in GIT motility has been reported in gestational exposure of valproic acid induced animal model of autism (Kim et al., 2013). Rats were fasted for 6 h and 1 ml of a semiliquid non-nutrient dye (Evans blue 50 mg/ml dissolved in 0.5% sodium carboxy-methylcellulose) was administrated via oral gavage. After 30 min, rats were euthanized by ether anaesthesia. The abdomen was cut off and the small intestine was carefully removed. Total length of the GIT was measured form pyloric sphincter to the ileocecal-junction and the distance travelled by the dye was recorded (Kim et al., 2013). The gastrointestinal transit index was calculated as GIT Transit index ¼

4.11.

Distance traveled by Evans blue  100 Total length of intestine

Dissection and homogenization

Animals were sacrificed by cervical dislocation, brains were removed then they were homogenized in phosphate buffer (pH 7.4) using Teflon Homogenizer. The homogenate was centrifuged at 3000 rpm for 15 min. The supernatant of the homogenate was separated out and then used for biochemical estimation as per the following methods.

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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4.12.

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Assessment of oxidative stress

Oxidative stress has been reported to contribute the aetiology of autism (Sandhya et al., 2012). In the present study, we have assessed brain lipid peroxidation, glutathione levels and catalase activity, as oxidative stress marker.

4.12.1.

Brain lipid peroxidation

Brain thiobarbituric acid reactive substances (TBARS) levels were measured spectrophotometrically (UV-1800 ENG 240V; Shimadzu Corporation, Japan) at 532 nm (Singh et al., 2015). 0.2 ml of supernatant of the homogenate was pipette out in a test tube, followed by the addition of 0.2 ml of 8.1% sodium dodecyl sulphate, 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 1C, then cooled followed by the addition of 1 ml of distilled water, and 5 ml of n-butanol–pyridine mixture (15:1 v/v). The tubes were centrifuged at 4000g for 10 min. The absorbance of developing pink colour was measured spectrophotometrically at 532 nm. A standard calibration curve was prepared using 1–10 nM of 1,1,3,3-tetra methoxy propane. The TBARS values were expressed as nanomoles per mg of protein.

4.12.2.

Brain glutathione (GSH) levels

The reduced glutathione (GSH) levels in the brain was estimated spectrophotometrically (UV-1800 ENG 240V; Shimadzu Corporation, Japan) at 412 nm (Singh et al., 2015). Briefly, the supernatant of the homogenate was mixed with trichloroacetic acid (10% w/v) in 1:1 ratio. The tubes were centrifuged at 1000g for 10 min at 40 C. The supernatant obtained (0.5 ml) was mixed with 2 ml of 0.3 M disodiumhydrogen phosphate. Then 0.25 ml of 0.001 M freshly prepared 5,50 -dithiobis (2-nitrobenzoic acid) [DTNB dissolved in 1% w/v sodium citrate] was added and absorbance was noted spectrophotometrically at 412 nm. A standard curve was plotted using 10–100 μM of the reduced form of glutathione and results were expressed as micromoles of reduced glutathione per mg of protein.

4.12.3.

Brain catalase activity

Activity of brain catalase was determined spectrophotometrically (UV-1800 ENG 240V; Shimadzu Corporation, Japan) at 240 nm (Singh et al., 2015). Briefly, 1 ml of the brain 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 brain homogenate served 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 μM of H2O2 per min at 25 1C using molar extinction coefficient of 43.6 M
1 cm
1 and the activity was expressed in terms of units per milligram of proteins.

4.13.

Brain nitrite/nitrate levels

Brain nitrite concentration was measured spectrophotometrically (UV-1800 ENG 240V; Shimadzu Corporation, Japan) at 545 nm (Singh et al., 2015). Briefly, 400 μl of carbonate buffer (pH 9.0) was added to 100 μl of brain or standard sample followed by addition of small amounts (0.15 g) of coppercadmium alloy. The tubes were incubated at room temperature for 1 h to reduce nitrate to nitrite. The reaction was stopped by adding 100 μl of 0.35 M sodium hydroxide. Following this, 400 μl of zinc sulphate solution (120 mM) was added to deproteinate the samples. The samples were allowed to stand for 10 min and then centrifuged at 4000g for 10 min. Greiss reagent (250 μl of 1.0% sulphanialamide prepared in 3 N HCl and 250 μl of 0.1% N-naphthylethylenediamine prepared with water) was added to aliquots (500 μl) of clear supernatant and brain nitrite was measured spectrophotometrically at 545 nm. The standard curve of sodium nitrite (5– 50 μM) was plotted to calculate the concentration of brain nitrite.

4.14.

Assessment of brain total protein

The brain total protein was determined spectrophotometrically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) at 750 nm (Singh et al., 2015). The brain total protein was determined by using bovine serum albumin (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 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 spectrophotometrically at 750 nm. Protein concentration was expressed as mg/ml of supernatant.

4.15.

Assessment of myeloperoxidase activity (MPO)

Inflammation in brain and intestine has been reported in autism (de Theije et al., 2014, El-Ansary et al., 2014). MPO activity in the brain and ileum was assessed by the odiansidine method (Kobata et al., 2007). Briefly, tissues were homogenized in 10 volumes 50 mmol/l phosphate buffer containing 0.5% hexadecyl trimethyl ammonium bromide (HTAB; pH 6.0). The homogenized samples were subjected to freezing and thawing three times and centrifuged at 10,000g for 10 min at 4 1C and then the resultant supernatant was used for assay sample. After the addition of 1.9 ml of 10 mM phosphate buffer (pH 6.0) and 145 μl of 1.5 mol/l odianisidine hydrochloride, containing 0.0005% w/v hydrogen peroxide to the supernatant, changes in the absorbance at 450 nm of each sample were recorded on the spectrophotometer (UV-1800 ENG 240V; Shimadzu Corporation, Japan). Sample MPO activity was obtained from the slope of the reaction curve based on the following equation: Specific activity (μmol H2O2 /min/mg protein)¼ΔOD per min/ΔOD per μmol H2O2  mg protein.

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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4.16.

Assessment of calcium levels in brain

An imbalance in intracellular calcium signalling has been reported in valproic acid induced autism (Rinaldi et al., 2008). The calcium levels were estimated using an Auto analyser (RMSBCA 201) using commercially available kit (Excel Diagnostics Pvt. Ltd. Hyderabad, India) at 570 nm. The calcium levels were measured in the basis of the formation of purple coloured complex due to the reaction with alkaline and ortho-cresolphathalin. The rate of absorbance was proportional to the calcium concentration present in the sample.

4.17.

Assessments of mitochondrial complex dysfunction

Mitochondrial dysfunction has been implicated in autism pathogenesis. Previously it has been reported that levels of different mitochondrial complexes get impaired in the frontal cortex of autistic brain (Chauhan et al., 2011).

4.17.1.

Isolation of rat brain prefrontal cortex mitochondria

Prefrontal cortex was 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 13,000g for 5 min at 4 1C. Pellets were suspended in the isolation buffer with EGTA and spun again at 13,000g for 5 min. The resulting supernatant was transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000g for 10 min. Pellets containing purified mitochondria were re-suspended in the isolation buffer without EGTA (Singh et al., 2015).

4.17.2. Assessment of nicotinamide adenine dinucleotide– hydrogen dehydrogenase (Complex-I) activity Complex-I was measured spectrophotometrically (UV-1800 ENG 240V; Shimadzu Corporation, Japan). The method involves the catalytic oxidation of NADH to NADþ with subsequent reduction of cytochrome-c. The reaction mixture was contained 0.2 M glycyl glycine buffer, 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 prefrontal cortex mitochondrial sample. The absorbance change at 550 nm was followed for 2 min (Singh et al., 2015)

4.17.3. Assessment of succinate dehydrogenase (Complex-II) activity Complex-II was measured spectrophotometrically (UV-1800 ENG 240V; Shimadzu Corporation, 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 by the addition of the prefrontal cortex mitochondrial sample, and the absorbance change at 420 nm was followed for 2 min (Singh et al., 2015)

4.17.4. Assessment of cytochrome oxidase (Complex-IV) activity Complex-IV activity was assayed in brain mitochondria. The assay mixture was contained 0.3 mM reduced cytochrome-c

11

in 75 mM phosphate buffer. The reaction was initiated by the addition of the solubilized mitochondrial sample, and absorbance change at 550 nm was measured spectrophotometrically (UV-1800 ENG 240V; Shimadzu Coorporation, Japan) for 2 min (Singh et al., 2015).

4.18. levels

Assessment of prefrontal cortex and ileum serotonin

The prefrontal cortex and ileum serotonin was estimated as previously published method (Abdel-Salam et al., 2012) with slightly modification. Briefly, animals were sacrificed by decapitation method. Prefrontal cortex and ileum tissues were isolated then washed with ice-cold saline solution (0.9% NaCl) and weigh. The frozen tissues were homogenized in cold 0.1 N-perchloric acids to give a final concentration of 10% w/v for the serotonin assays. A homogenized sample were centrifuged at 20,000g for 20 min at 4 1C, supernatant pass through a 0.22 mm membrane filter and was used for serotonin estimated using high performance liquid chromatography (HPLC) system, equipped with a quaternary pump (Water system). Separation was achieved on ODS-reversed phase column. The mobile phase consisted of 0.1 M potassium phosphate buffer/methanol 97/3 (v/v) with pH adjustment at 4.05 and was delivered at a flow rate of 1 ml/min. UV detection was performed at 270 nm, and the injection volume was 20 ml. The concentration of serotonin was determined by external standard method using peak areas. Serial dilutions of standards were injected, and their peak areas were determined. A linear standard curve was constructed by plotting peak areas versus the corresponding concentrations. The concentrations in samples were obtained from the curve. The serotonin levels were expressed in mg/g wet wt. of the tissue. Quantification was made by comparing peak heights of the samples to the corresponding standard curve.

4.19.

Assessment of blood brain barrier (BBB) permeability

The permeability of the BBB was determined by measuring the amount of Evans blue dye in brain as per previously described method (Manaenko et al., 2011). Briefly, 4% of Evans blue was administered intra-peritoneal at a dose of 4 ml/kg body weight and was allowed to circulate for 2 h prior to measurement. The anaesthetised animals were perfused transcardial with saline to wash away any remaining dye in the blood vessels prior to sample collection. Some of the brain samples were used for qualitative Evans blue estimation by macroscopic method. Other samples were utilized for quantitative Evans blue estimation by the spectroscopic method. The samples those utilize for quantitation, was weighed. Briefly, cerebellum was weighed. The Evans blue was extracted by homogenizing the sample in 3.5 ml of 0.1 mol/l phosphate buffered saline at pH 7.4. Proteins were precipitated by the addition of 6 ml of 60% trichloroacetic acid. The mixture was then vortexes for 2 min and cooled for 30 min. The sample was centrifuged for 40 min at 4000 rpm to pellet the brain tissue. Absorption of the supernatant was measured at a wavelength of 610 nm using a spectrophotometer (UV-1800 ENG 240V; Shimadzu Corporation, Japan). The

Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 Fig. 6 – Schematic representation of experimental protocol. E – embryonic day; P – postnatal day; Pre VPA – prenatal valproic 1459 acid; Mino – Minocycline; D1 – dose 1; D2 – dose 2. 1460 1461 biochemical parameters in between post-natal 43rd and content of Evans blue was measured as μg of Evans blue /g of 1462 50th day. cerebellum tissue using a standardized curve. 1463 1464 4.20. Experimental protocol 4.20.7. Group VII – Pre VPAþminocycline dose 1 group 1465 Offspring of pre-natal-12.5th day sodium valproate treated 1466 In the present study pregnant females were isolated, few of female rats, received minocycline (25 mg/kg, per oral) from 1467 them were injected with valproic acid or saline, whereas few post-natal 21st day, were exposed to behaviour and biochem1468 have not received any treatment. Male offspring were thus ical parameters in between post-natal 43 rd and 50th day. 1469 received were distributed in to eight groups as per the 1470 following protocol, where each group was consisted of six 4.20.8. Group VIII – Pre VPAþminocycline dose 2 group 1471 animals (Fig. 6). Offspring of pre-natal-12.5th day sodium valproate treated 1472 female rats, received minocycline (50 mg/kg, per oral) from 1473 4.20.1. Group I – control group post-natal 21st day, were exposed to behaviour and biochem1474 Male offspring of untreated pregnant females were exposed ical parameters in between post-natal 43 rd and 50th day. 1475 to behaviour and biochemical parameters in between post1476 natal 43rd and 50th day. 4.21. Statistical analysis 1477 1478 4.20.2. Group II – 0.9% saline treated group Statistical analysis was done using Sigma Stat v3.5. All results 1479 Pregnant female received 0.9% saline on embryonic 12.5th were expressed as mean7standard deviation. Data for all 1480 day and their male offspring were exposed to behaviour and parameters were statistically analysed by one-way ANOVA 1481 biochemical parameters in between post-natal 43rd and followed by Bonferroni’s post-test. po0.05 was considered to 1482 50th day. be statistically significant. 1483 1484 4.20.3. Group III – 0.5% sodium carboxymethyl cellulose 1485 Conflict of interest (CMC) 1486 Male offspring of untreated pregnant females have received 1487 None. CMC from post-natal 21st day to till the end of the study. 1488 These animals were exposed to behaviour and biochemical 1489 parameters in between post-natal 43rd and 50th day. 1490 Role of funding source 1491 4.20.4. Group IV – minocycline dose 1 per se 1492 None. Male offspring of untreated pregnant females have received 1493 minocycline (25 mg/kg, per oral, daily) from post-natal 21st 1494 day and they were exposed to behaviour and biochemical Uncited reference Q4 1495 parameters in between post-natal 43rd and 50th day. 1496 1497 American Psychiatric Association (2013). 4.20.5. Group V – minocycline dose 2 per se 1498 Male offspring of untreated pregnant females have received 1499 minocycline (50 mg/kg, per oral) from post-natal 21st day and Acknowledgments 1500 they were exposed to behaviour and biochemical parameters 1501 in between post-natal 43rd and 50th day. Authors are thankful to Dr. Nirmal Singh, Associate Professor, 1502 Pharmacology Division, Department of Pharmaceutical 1503 Sciences and Drug Research, Faculty of Medicine, Punjabi 1504 4.20.6. Group VI – Pre VPA treated group University, Patiala (Punjab), India, for his valuable sugges1505 Pregnant female received sodium valproate (500 mg/kg, intra1506 peritoneal, single administration) on embryonic 12.5th day tions. We are also thankful to Prof. J.S.P Rai, Director General, 1507 and their male offspring were exposed to behaviour and Bharat Institute of Technology, Meerut, India and Space age Please cite this article as: Kumar, H., Sharma, B., Minocycline ameliorates prenatal valproic acid induced autistic behaviour, biochemistry and blood brain barrier impairments in rats. Brain Research (2015), http://dx.doi.org/10.1016/j. brainres.2015.10.052

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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.

Appendix A.

Supplementary material

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.10.052.

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