Raloxifene potentiates the effect of fluoxetine against maximal electroshock induced seizures in mice

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Raloxifene Potentiates The Effect Of Fluoxetine Against Maximal Electroshock Induced Seizures In Mice Faheem Hyder Pottoo , Nahida Tabassum , Md. Noushad Javed , Shah Nigar , Shrestha Sharma , Md. Abul Barkat , Harshita , Md. Sabir Alam , Mohammad Azam Ansari , George E. Barreto , Ghulam Md Ashraf PII: DOI: Reference:

S0928-0987(20)30050-6 https://doi.org/10.1016/j.ejps.2020.105261 PHASCI 105261

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European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

1 November 2019 24 January 2020 7 February 2020

Please cite this article as: Faheem Hyder Pottoo , Nahida Tabassum , Md. Noushad Javed , Shah Nigar , Shrestha Sharma , Md. Abul Barkat , Harshita , Md. Sabir Alam , Mohammad Azam Ansari , George E. Barreto , Ghulam Md Ashraf , Raloxifene Potentiates The Effect Of Fluoxetine Against Maximal Electroshock Induced Seizures In Mice, European Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.ejps.2020.105261

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RALOXIFENE POTENTIATES THE EFFECT OF FLUOXETINE AGAINST MAXIMAL ELECTROSHOCK INDUCED SEIZURES IN MICE

Faheem Hyder Pottoo 1 *, Nahida Tabassum 2 *, Md. Noushad Javed 3 ,4 , Shah Nigar 2 , Shrestha Sharma 5 , Md. Abul Barkat 6 , Harshita 6 , Md. Sabir Alam 5 , Mohammad Azam Ansari 7 , George E. Barreto 8 ,9 * , Ghulam Md Ashraf 1 0 ,1 1 * 1

Department of Pharmacology, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, P.O.BOX, 1982, Dammam, 31441, Saudi Arabia 2 Department of Pharmaceutical Sciences, Faculty of Applied Sc. and Tech., University of Kashmir, Srinagar, India 3 Department of Pharmaceutics, School of Pharmaceutical Sciences and Research, Jamia Hamdard University, New Delhi, India 4 School of Pharmaceutical Sciences, Apeejay Stya University, Gurugram, Haryana, India 5 Department of Pharmacy, School of Medical and Allied Sciences, K.R.Mangalam University, Gurgaon, India 6 Department of Pharmaceutics, College of Pharmacy, University of Hafr Al Batin, Al Jamiah, Hafr Al Batin 39524, Saudi Arabia 7 Department of Epidemic Disease Research, Institute of Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, 31441, Dammam, Saudi Arabia 8 Deparment of Biological Sciences, University of Limerick, Limerick, Ireland 9 Health Research Institute, University of Limerick, Ireland 10 King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; 11 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia Running Title: Novel drug combination for treatment of electroshock induced convulsions. *

Corresponding authors:

Prof. Nahida Tabassum ([email protected]) Dr. Faheem Hyder Pottoo ([email protected]; [email protected]) Dr. Ghulam Md Ashraf ([email protected], [email protected]) Dr. George E. Barreto ([email protected])

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ABSTRACT The evidence to guide clinicians regarding rationale polytherapy with current antiepileptic drugs (AEDs) is lacking, and current practice recommendations are largely empirical. The excessive drug loading with combinatorial therapies of existing AEDs are associated with escalated neurotoxicity, and that emergence of pharmacoresistant seizures couldn’t be averted. In pursuit of judicious selection of novel AEDs in combinatorial therapies with mechanism based evidences, standardized dose of raloxifene, fluoxetine, bromocriptine and their low dose combinations, were experimentally tested for their impact on maximal electroshock (MES) induced tonic hind limb extension (THLE) in mice. Hippocampal neuropeptide Y (NPY) levels, oxidative stress and histopathological studies were undertaken. The results suggest the potentiating effect of 4mg/kg raloxifene on 14mg/kg fluoxetine against MES induced THLE, as otherwise monotherapy with 4mg/kg raloxifene was unable to produce an effect. The results also depicted better efficacy than carbamazepine (20 mg/kg), standard AED. Most profoundly, MES-induced significant (P<0.001) reduction in hippocampal NPY levels, that were escalated insignificantly with the duo-drug combination, suggesting some other mechanism in mitigation of electroshock induced seizures. These results were later corroborated with assays to assess oxidative stress and neuronal damage. In conclusion, the results demonstrated the propitious therapeutic benefit of duo-drug low dose combination of drugs; raloxifene and fluoxetine, with diverse mode of actions fetching greater effectiveness in the management of generalized tonic clonic seizures (GTCS).

Keywords: Dopamine; Epilepsy; Generalized tonic clonic seizures; Neurodegeneration; Neurological disorders; Neuropeptide Y; Seizures; Serotonin. Abbreviations Used: AED: Antiepileptic drugs; BC: Bromocriptine; FT: Fluoxetine; GTCS: Generalized tonic clonic seizures; KA: Kainic Acid; MES: Maximal electroshock; NPY: Neuropeptide Y, OS: Oxidative stress; PILO: Pilocarpine, PWE: Patients with epilepsy; RF: Raloxifene; SE: Status Epilepticus, SRS: Spontaneously Recurring Seizures; THLE: Tonic hind limb extension; TLE: Temporal Lobe Epilepsy; BSA: Bovine serum albumin; PMS: Postmitochondrial supernatant; GSH: Reduced Glutathione; EDTA: ethylenediaminetetraacetic acid; GR: glutathione reductase; NADPH: reduced nicotinamide adenine dinucleotide phosphate

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1. INTRODUCTION Epilepsy is a life threatening chronic medical disorder or neuropathological condition, culminating in unpredictable, unprovoked recurrent seizures that influences diverse mental and physical functions (Goldenberg, 2010; Sarafroz et al., 2019). It escorts to neuronal debt particularly in the hippocampal regions cornu-ammonis (CA1, CA2, CA3) and dentate gyrus (DG) (Pottoo et al., 2016, 2014; Thom, 2014). Epilepsy globally affects in excess of 70 million people, about 90% of those are in developing nations (Singh and Trevick, 2016) and impacts quality of life badly (Pottoo et al., 2020) During a seizure, neurons blaze as many as 500 times a second, which is much faster than normal. Epileptic seizures sequel free radical generation and oxidative injury to cellular proteins, lipids and DNA, emanating in neuronal cell death (Devi et al., 2008; Nigar et al., 2016; Waldbaum and Patel, 2010). Although considerable advances eventuated on pharmacotherapy of epilepsy in the last century, (Firdaus et al., 2018; Husain et al., 2009; Sahu et al., 2017; Siddiqui et al., 2017, 2008) coincident with the use of bioinspired nanocomposites, nanoparticle conjugated AEDS with widespread applications (Alam et al., 2019; Javed et al., 2019; Mishra et al., 2019; Sharma et al., 2019), 35% of patients still remain unresponsive towards currently available anti-epileptic drugs (AEDs) (Do Val-da Silva et al., 2017). These refractory patients also encounter developmental delay in infancy, severe disability, morbidity in adult age, and that their mortality rate is 5–10 times that of the general population (Zhuo et al., 2017). The GTCS is the foremost seizure type in 20% of epilepsy patients (Goldenberg, 2010). Serotonergic system is critically involved in regulation of neurologic and psychiatric functions, the disruption of which escorts to various neurological disorders (Pottoo et al., 2019a). The retrospective observational study revealed that selective serotonin reuptake inhibitors (SSRIs) and serotonin-noradrenaline reuptake inhibitors (SNRIs) reduce seizure frequency (Ribot et al., 2017) which is in line with preclinical studies which reported anti-seizure effects of reboxetine and citalopram (SSRI) against KA–incited post–SE model for temporal lobe epilepsy (TLE) (Vermoesen et al., 2012). The safety of SSRIs and SNRIs in patients with epilepsy (PWE) is well reported (Kanner, 2016). 5- HT (conc. ≥100 microM) signals through presynaptic 5HT-1A receptors to reduce Ca2+ entry and glutamate release, detected as reduction (30-40%) in electrically evoked EPSPs (excitatory postsynaptic potentials) in CA1 pyramidal cells, the effect 3

was replicated with 8-OH-DPAT (50 microM). However with the low conc. of 5- HT (10-50 microM) no such effect was surmountable (Schmitz et al., 1995). 5-HT3 receptor-mediates spontaneous increase in GABAergic IPSPs in CA1 pyramidal cells (Ropert and Guy, 1991). Evidences for dysregulation of dopaminergic system in epileptic disorders has also gained prominence. Epileptic patients with genetic variants for reduced dopaminergic activity were relatively more susceptible towards psychotropic adverse events of levetiracetam (Helmstaedter et al., 2013; Wood, 2012). The studies have emphasized the potential protective role of D2 agonist (Bromocriptine) not only in self-incited and drug refractory epilepsy, but also in TLE patients with pituitary prolactinomas (Bozzi and Borrelli, 2013). We have also reported the protective role of bromocriptine against pilocarpine (PILO)- induced SE and TLE (Pottoo et al., 2016). These findings are counter confirmed from animal studies who reported that D2R knockout (D2R-/-) mice are more prone to KA-induced excitotoxicity in the CA3 region compared to wild-type (WT) mice. D2R-mediated signaling in the CA3 region leads to Akt (Ser473) phosphorylation, which in turn phosphorylates-GSK-3β responsible for hippocampal granular cell survival. Thus, D2R-/mice exhibit inability to phosphorylate Akt (Ser473) and GSK-3β culminating in neuronal susceptibility to apoptosis (Dunleavy et al., 2013). Based on this, a hypothesis was inferred that treatment with fluoxetine (SSRI), or bromocriptine (Dopamine D2 agonist) would avert seizures. However, neurodegeneration being the intricate character of seizures, escorts to degenerate drug targets (the receptors for serotonergic and dopaminergic transmission) and/or alters the binding sites, leading to emergence of drug refractory epilepsies, as is seen with current AEDs. To counteract the problem, the combinatorial therapy of SSRI and/or Dopamine (D2) agonists with estrogens was proposed. Although estrogens are neuroprotective, their proconvulsant nature cannot be ignored, which overcomes the anti-convulsant benefits. The problem was circumvented with the use of selective estrogen receptor modulator (SERM), which modulate estrogen receptors in tissue specific manner, thereby also avoiding the risk of endometrial carcinoma associated with the use of estrogens. Apart from relying on exogenous agents, the therapeutic strategies aimed at surging innate immunity against seizures within the CNS, is the need of the hour. The NPY, is a natural obstacle against seizure continuance from perforant pathway (connectional course from entorhinal cortex to hippocampus) to dentate gyrus (Velísková and Velísek, 2007) and thus had emerged as putative target for anti-convulsant drug development. NPY is far dispersed across the CNS with effects 4

mediated via binding to Y1, Y2, Y4 and Y5 receptors (Yang et al., 2018). The antiepileptic effects of NPY incited via Y2 receptors, lead to reduced stimulation-induced EPSPs in DG and CA1, an in vitro study on human hippocampal slices (Ledri et al., 2015). The frequency of EEG seizures were reported longer in NPY knockout mice, compared to WT (wild type) in a mouse model of KA induced seizures (Baraban et al., 1997). Valproate (VPA) which suppresses partial and generalized seizures, was reported to elevated mRNA level of NPY as well as protein expression in localized region of both nucleus reticularis thalami (nRt) and hippocampus (Brill et al., 2006). The deregulation in production and disposal of ROS (reactive oxygen species) and RNS (reactive nitrogen species), culminates in oxidative/nitrosative stress often perceived with epileptogenesis (Nigar et al., 2016). In rodent electro-convulsion model, epileptogenesis events causes OS (oxidative stress) in neurons and astrocytes. Such events of OS during epileptogenesis result into generation of neuroinflammatory markers. Thus, therapeutic approach mitigating seizure induced OS could halt neuronal death (Khan et al., 2016). In lieu of above arguments, we hypothesized that combinatorial duo drug combination of raloxifene with fluoxetine and/or bromocriptine may intensify signaling through serotonergic and dopaminergic receptors, reduce oxidative stress, glutamate excitotoxicity and interfere with the process of epileptogenesis in mice model for electrical seizures. 2. MATERIALS AND METHODS 2.1 Animals Swiss albino mice (Central Animal House Facility of Indian Institute of Integrative Medicine, India) of body weight (25-35 g), age (10 –12 weeks) were grouped and sheltered in polypropylene caged, under ambient conditions of temperature (21 ± 1 °C) and relative humidity (55 ± 3 %). A natural light-dark rhythm was replicated. The experimental procedures were regulated by ethical guidelines from IAEC, Department of Pharma. Sc., University of Kashmir. The experimentation followed after approval [Approval No: F-IAEC(Pharm. Sc.) APPROVAL/ 2013/15]. 2.2 Drugs and Dosing Schedule The following drugs were used: raloxifene (Cipla Ltd. India), carbamazepine, fluoxetine (Sun Pharmaceuticals Ltd., India), and bromocriptine (Monarch Pharmaceuticals, India). All test substances were suspended in Tween 80 (2% aqueous solution) (Sigma, St. Louis, MO, USA) and

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delivered via oral gavage for 4 weeks prior to challenge with MES. Rationale for selection of dose of these test drugs were taken into consideration of human equivalent dose (HED) as well as literature-based evidences. HED of RF for osteoporosis is 8 mg/kg hence selected RF dose were rationalized to 4 and 8 mg/kg (Delmas et al., 2002). Subsequently, 22 mg/kg dose of FT is reported to prompt dematuration of granule cells and to elevate serotonergic modulation hence selected dose of FT were rationalized to 14 and 22 mg/kg (Kobayashi et al., 2010) Furthermore, 10 mg/kg dose of bromocriptine is reported to insulate MPTP- incited neurotoxicity hence selected dose of BC were rationalized to 6 and 10 mg/kg (Mohanasundari et al., 2006). 2.3 Experimental Design Mice were separated randomly in a group of ten with total thirteen experimental groups. The treatment groups were: Group I: vehicle control group (10 mg/kg), Group II: toxic control group (MES; 45 mA, 250 V, 0.2 s), Group III: Carbamazepine (CB; 20 mg/kg), Groups IV-V: Raloxifene (RF; 4 and 8 mg/kg), Groups VI-VII: Fluoxetine (FT; 14 and 22 mg/kg), Groups VIIIIX: Bromocriptine (BC; 6 and 10 mg/kg). Groups X-XII: Low dose duo-drug combinations with Raloxifene (RF), Fluoxetine (FT) Bromocriptine (BC) and Group-XIII: Triple drug combination of Raloxifene (RF), Fluoxetine (FT) and Bromocriptine (BC). The drugs and their low dose combinations were administered prophylactically, with oral gavage once daily for 4 weeks prior to challenge with MES for inducing Tonic Hind Limb Extension (THLE). 2.4 MES induced THLE Using auricular electrodes, electro-shocks were delivered for generation of electroconvulsions. Briefly, during electroshock the mice were restrained by hand, tied with ear clip, immediately released at the moment of stimulation and observed for seizure behavior throughout this period. For

estimating

the

electroconvulsive

threshold,

standardization was

conducted.

The

electroconvulsive threshold was evaluated in terms of current strength (in mA) denoted as CS 100, cardinal to induce THLE (tonic hind limb extension) in 100% of normal mice. Mice were segregated into four groups, each with 10 mice. They were given electroshocks of different intensities. An Intensity-response curve was plotted by counting the percentage of mice that convulsed. Subsequently, the model was standardized at alternating current 45 mA, Voltage 250 V for 0.2 sec. The prophylactic administration of drug(s) was continued orally once daily for a period of 4 weeks prior to challenge with MES (45 mA, 250 V, 0.2 s). The drug was contemplated 6

to hold antiseizure potential if it abated or terminated the extensor phase of electro-convulsions. Six mice were rapidly sacrificed after performing the behavioral studies from each group and proceeded for various biochemical and hippocampal NPY evaluations. The remaining four mice in each group were guarded under desirable environment for a time-period of 24 h, after which they were sacrificed for performing histological studies. The EEG recordings of mice were not engaged as current stimulation per se could incite significant artifacts in the EEG recordings that might distract from the epileptiform activity (Franco-Pérez et al., 2015). 2.5 MES induced neuronal damage MES (45 mA, 250 V, 0.2 s) stimulated neuronal injury was computed by histopathological appraisal of hippocampus 24hrs post MES challenge. The mice brains from each group were removed, washed in normal saline and preserved in formalin (10%) for fixation (to prevent post mortem decomposition). At 2.3 to 4.3 mm posterior region to the bregma 1, brains were slit coronally (thickness 8-μm) with the help of a microtome. The sections were mounted on glass slides. Two sets of slides were composed and stained with H/E (hematoxylin and eosin) to assess granular cell changes. The digital pictures were captured with light microscope from hippocampal CA1, CA2, CA3 and DG by pathologist heedless of drug regimen (40X for subregions and 4X for entire hippocampus). After seizures, the granular cell layer density was found to reduce, while remaining neurons were pyknotic or shrunken. The scrutiny of neurons in microscopic grid (dimension 1 cm2 ; 10x10 box) over CA1, CA2, CA3 and DG focused regions was concluded (computing was done with a microscopic magnification of 200X- to 400X- for every hippocampal subfield segment). As per standard practices neurons touching either inferior or right edges of grid were not taken into consideration. 2.6 Assessment of hippocampal NPY levels Immediately post behavioral monitoring of seizures, mice from all groups were euthanized, brains were separated and hippocampus isolated for estimation of NPY. The immunoassay protocolbased kit (Ray Biotech, Norcross, USA) were used for in vitro quantification of NPY levels, as per manufacturer instructions.

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2.7 Assessment of oxidative stress indices in the hippocampus After behavioral monitoring of seizures, mice were euthanized, brains were removed, hippocampus separated and homogenized in ice-cold phosphate buffer (pH: 7.4) followed by centrifugation at 3000 rpm for time period of 15 min at 4 °C for supernatant collection. 2.7.1 Protein Assessment Briefly, 0.2 ml of post-mitochondrial supernatant (10% w/v), MiliQ water (qs 1 ml) as well as 5 ml Copper Reagent were poured into a fresh graduated autoclaved glass tube. The incubation of samples was conducted for 10 min at room temperature. Folin’s reagent (1 ml) was poured into each sample. Lastly, the glass tubes were vortexed and incubated at ambient temperature for 30 min. Change of color was observed in comparison to the blank solution (BSA 0.1mg/ml) when maximum wavelength was set to determined 700 nm (Lowry et al., 1951). 2.7.2 Thiobarbituric acid reactive substances (TBARS) estimation Briefly, 0.1 ml of PMS (10% w/v) was poured into a freshly autoclaved aluminum covered graduated glass tube, succeeded by adding 1.0 ml each of trichloroacetic acid (10%) and thiobarbituric acid (0.67%). The samples were incubated for a period of 45 minutes in a water bath. Thereafter cooled, and subjected to 10 min centrifugation at 2500 rpm. After centrifugation, the supernatant solution was removed and evaluated spectrophotometrically by determining absorbance at 525 nm, compared with the blank solution. Using standard molar extinction coefficient 1.56 × 105 M-1 cm-1 and standard temperature 37oC, concentration of MDA i.e., nmol MDA formed per gm of wet tissue were used to estimate rate of Lipid Peroxidation (LPO) (Ohkawa et al., 1979). 2.7.3 Assay for Reduced Glutathione (GSH) Briefly, equal volume (1ml) of both PMS (10% w/v) and sulphosalicylic acid (4%w/v) reagents were poured into fresh autoclaved glass tubes, incubated at 4C for 1 hour, followed by centrifugation at 1200 rpm for 15 minutes. Post centrifugation, 0.4 ml supernatant solution was added 2.2 ml of PBS (0.1 M, PH 7.4) and 0.4 ml of Ellman's Reagent (4 mg/1ml). The UV spectrophotometric reading for flourished yellow (λmax : 412 nm) color were compared with that of blank solution. The concentration of GSH was estimated in terms of nmol of formed DTNB conjugate per mg of protein (Jollow et al., 1974). 8

2.7.4 Assay for Glutathione Peroxidase (GPx) Briefly, 0.1 ml of PMS (10 % w/v), 1.44 ml of phosphate buffer (0.1 M, PH 7.4), 0.1 ml EDTA (1.0 mM), 0.1 ml of sodium azide (1.0 mM), 0.05 ml of GR (1 eu/ml), 0.05 ml of GSH (1.0 mM), 0.1 ml of NADPH (0.2 mM) and 0.01 ml of Hydrogen peroxide (0.25 mM) were taken out into a freshly autoclaved graduated glass tube to obtain volume of 2.0 ml. In order to estimate enzymatic activity using standard molar extinction coefficient (6.22 × 10 3 M-1 cm-1), level of NADPH were spectrophotometrically estimated at λmax 340 nm for nmol of oxidized NADPH in 1 mg of protein sample each minute (Mohandas et al., 1984). 2.7.5 Assay for Glutathione Reductase (GR) Briefly, 0.1 ml of PMS (10% w/v) was taken out in a freshly autoclaved graduated glass tube and added with 1.65 ml of PBS (0.1 M, PH 7.6), 0.1 ml of EDTA (0.5 mM), 0.05 ml oxidized glutathione (1.0 mM) and 0.1 ml of NADPH (0.1 mM) to obtain a volume of 2.0 ml. In order to estimate enzymatic activity using standard molar extinction coefficient (6.22 × 103 M-1 cm-1), level of NADPH were spectrophotometrically estimated at λmax 340 nm for nmol of oxidized NADPH in1 mg of protein sample each minute. 2.8. Statistical analysis The quantitative data, NPY levels and biochemical estimations, were represented as Mean ± SEM and comparison were made by one-way Analysis of Variance (ANOVA) as well as Post Hoc Tukey Kramer’s multiple comparison test method. The comparison of Qualitative variables between the studied groups in case of MES induced THLE was done using Fischer’s exact test (Ratio of THLE / NO THLE). The values of different treated groups were compared with values of toxic control.

***

P< 0.001, ** P< 0.01, * P<0.05 were considered as extremely significant, highly

significant and significant in all cases, while value of P >0.05 was considered as nonsignificant (ns). The data was analyzed using SPSS - ver 1.6 (SPSS Inc., Chicago, IL, USA). 3. RESULTS 3.1 Impact of Raloxifene (RF), Fluoxetine (FT) and Bromocriptine (BC) in combinatorial therapy against MES Induced THLE. MES (45 mA, 250 V, 0.2 s) induced THLE in all mice, and the ratio of THLE/NO-THLE was 10/0. Monotherapy with RF (4 and 8 mg/kg) and BC (6 and 10 mg/kg) featured statistically 9

insignificant effects to elevate threshold towards electroshock induced convulsions/THLE. However, FT (14 and 22 mg/kg) showed significant (p<0.05 and p<0.001) dose dependent reduction in the ratio of THLE/NO-THLE (5/5 and 1/9). The combined dose of RF (4 mg/kg) and FT (14 mg/kg) exhibited statistically significant (P<0.001) decrease in ratio of THLE/NO-THLE (1/9). However RF (4 mg/kg) rendered null benefits when being combined with BC (6 mg/kg) and same patterns of inadequate efficiency was obtained with combination of FT (14 mg/kg) and BC (6 mg/kg). In an attempt to explore more effective combination, triple drug combinations were attempted with rationalized selection of RF (4 mg/kg), FT (14 mg/kg) and BC (6 mg/kg) which established their potential role to decrease ratio of THLE/NO-THLE (3/7) in a statistically significant manner (p<0.01). It is worth mentioning that addition of low dose of BC (6 mg/kg) to duo-drug combination of RF (4 mg/kg) with FT (14 mg/kg) seems to reduce protective effect of duo-drug combination. It is further important to figure out that duo-drug combination of RF (4 mg/kg) with FT (14 mg/kg) are as much effective as of higher dose of FT (22 mg/kg). However, selection of single monotherapy at high dose level in the treatment of epilepsy bears limited success in terms of high chances for drug resistance, adverse drug reactions as well as toxicity. Subsequently, carbamazepine, standard AED exhibited significant (p<0.01) reduction in the ratio of THLE/NO-THLE (3/7), however protective was less so than duo-drug combination. In lieu of above arguments, combination of lower doses of FT with RF, in clinical management of epilepsy, seems to be more promising and judicious approach (Fig. 1). 3.2 Impact of Raloxifene (RF), Fluoxetine (FT) and Bromocriptine (BC) in combinatorial therapy on hippocampal NPY levels. MES (45 mA, 250 V, 0.2 s) induced a statistically significant (p<0.001) reduction in NPY levels in comparison to normal control. Nevertheless, test drugs and their combinations increased NPY levels insignificantly, indicating some other mechanism in the mitigation of electroshock-induced convulsions in mice (Fig. 2). 3.3 Impact of Raloxifene (RF), Fluoxetine (FT) and Bromocriptine (BC) in combinatorial therapy on hippocampal oxidative stress indices. a. Effect on Protein Levels (g/dl) MES (45 mA, 250 V, 0.2 s) induced significant reduction (p<0.001; 53.88%) in protein levels in comparison to normal control (100%). Administration of FT (14 and 22 mg/kg) significantly 10

(p<0.05; 72.82 and 74.51%) resisted abatement in protein levels compared to toxic control. Duodrug combination of RF (4 mg/kg) with FT (14 mg/kg) significantly (p<0.001; 94.66%) resisted alleviation in protein levels. The triple drug combination of RF (4 mg/kg) with FT (14 mg/kg) and BC (6 mg/kg) also significantly (p<0.01; 78.16%) resisted reduction in protein levels, an effect similar to standard AED i.e carbamazepine (p<0.01;78.64%) was surmountable with triple drug combination. However, the effect of carbamazepine was less so than duo-drug combination. Other drug(s) and their combinations rendered insignificant resistance towards such change (Fig. 3). b. Impact on Lipid Peroxidation levels (nmol MDA /g of wet tissue) MES (45 mA, 250 V, 0.2 s) compelled significant (p<0.001; 236.50%) increase in hippocampal MDA levels compared to normal control (100%). Administration of monotherapy with test drug, FT (14 and 22 mg/kg), significantly (p<0.05; 160.23 and 152.64%) reduced MDA levels compared to toxic control. Duo-drug combination of RF (4 mg/kg) with FT (14 mg/kg) significantly (p<0.001; 109.89%) reduced MDA levels. Also, triple drug combination of RF (4 mg/kg) with FT (14 mg/kg) and BC (6 mg/kg) significantly (p<0.01; 146.49%) reduced MDA levels, an effect similar to carbamazepine (p<0.01;140.83%) was surmountable with triple drug combination. However, the effect of carbamazepine was less so than duo-drug combination. Other drugs and drug combinations didn’t reveal reduction of MDA levels significantly (Fig. 4A). c. Impact on Glutathione reduced (GSH) levels (nmol of DTNB conjugate formed/ mg of protein) MES (45 mA, 250 V, 0.2 s) significantly (p<0.001; 48.52%) decreased GSH levels in comparison to normal control (100%). Administration of monotherapy with FT (14 and 22 mg/kg) depicted significant (p<0.05; 64.71 and 66.67%) increase in GSH levels, compared to toxic control. The duo-drug combination of RF (4 mg/kg) with FT (14 mg/kg) exhibited significant (p<0.001; 95.34%) increase in GSH levels. The triple drug combination of RF (4 mg/kg) with FT (14 mg/kg) and BC (6 mg/kg) showed significant (p<0.01; 69.13%) escalation in GSH levels. Carbamazepine, standard AED used for comparison also exhibited significant (p< 0.01; 69.34%) increase in GSH levels. Other drugs and their combinations insignificantly increased GSH levels (Fig. 4B).

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d. Impact on Glutathione Peroxidase (GPx) levels (nmol of NADPH oxidized/min/mg of protein) MES (45 mA, 250 V, 0.2 s) significantly (p<0.001; 56.78%) reduced GPx levels compared to normal control (100%). Administration of monotherapy with FT (14 and 22 mg/kg) showed significant (p<0.05; 76.40 and 78.29%) increase in GPx levels, compared to toxic control. The duo-drug combination of RF (4 mg/kg) with FT (14 mg/kg) exhibited significant (p<0.001; 90.74%) increase in GPx levels. The triple drug combination of RF (4 mg/kg) with FT (14 mg/kg) and BC (6 mg/kg) showed significant (p<0.01; 80.52%) increase in GPx levels. Carbamazepine, standard AED used for comparison also exhibited significant (p< 0.01; 81.14%) upsurge in GPx levels. Other drugs and their combinations insignificantly increased GPx levels (Fig. 5A). e. Impact on Glutathione Reductase (GR) levels (nmol of NADPH oxidized/min/mg of protein) MES (45 mA, 250 V, 0.2 s) significantly (p<0.001; 52.36%) reduced GR levels compared to normal control (100%). Administration of monotherapy with FT (14 and 22 mg/kg) showed significant (p<0.05; 68.33 and 69.99%) increase in GR levels, compared to toxic control. The duodrug combination of RF (4 mg/kg) with FT (14 mg/kg) exhibited significant (p<0.001; 94.83%) increase in GR levels. The triple drug combination of RF (4 mg/kg) with FT (14 mg/kg) and BC (6 mg/kg) showed significant (p<0.01; 71.40%) increase in GR levels. Carbamazepine, standard AED used for comparison also exhibited significant (p<0.01; 73.32%) increase in GR levels. Other drugs and their combinations insignificantly increased GR levels (Fig. 5B). 3.4 Impact of Raloxifene (RF), Fluoxetine (FT) and Bromocriptine (BC) in combinatorial therapy on MES induced hippocampal neuronal damage. Hippocampal sections of normal control administered vehicle (2% Tween 80) stained with Haematoxylin and Eosin (H&E) revealed normal morphology, evidenced from noticeable intact neuronal density, null pyknosis and absence of ectopic neurons in all hippocampal subregions. Challenge with MES (45 mA, 250 V, 0.2s) revealed effects of electroshock in the form of severe neuronal loss in all hippocampal regions. The monotherapy with RF (4 and 8 mg/kg) and BC (6 and 10 mg/kg) was foreseen with negligible protection against electroshock induced deterioration

of neurons, as a result extensive loss of neurons and pyknosis was surmountable in regions CA1, CA2 and CA3. While remaining neurons in these regions were also shrunken. Interestingly, 12

monotherapy with FT (14 and 22 mg/kg) revealed nominal reduction in neuronal densities and mild pyknosis in CA1, CA2 and CA3 regions. However, ectopic neurons were highly surmountable in low dose FT (14 mg/kg) group, which indicates more neuroprotection with high dose FT (22 mg/kg). The duo-drug combination of RF (4 mg/kg) with FT (14 mg/kg), significantly resisted neuronal abatement with electroshock revealed in terms of grossly healthy intact arrangement of neurons, and very few pyknotic neurons in all hippocampal subregions. The combination of BC (6 mg/kg) with each of RF (4 mg/kg) and FT (14 mg/kg) rendered null protection, and that BC (6 mg/kg) reversed neuroprotective effect of FT (14 mg/kg). The triple drug combination of RF (4 mg/kg) with FT (14 mg/kg) and BC (6 mg/kg) although showed neuroprotection evident from intact neuronal morphology with mild pyknosis in all hippocampal regions, the addition of BC (6 mg/kg) to the regimen of duo-drug combination featured to reduce the neuroprotective effects of duo-drug combination. Carbamazepine (20 mg/kg) treatment also protected neurons with mild pyknosis observed in CA1, CA2 and CA3 regions. However, it did not resist scattering of neurons in CA3 region. These results feature that triple drug combination is more neuroprotective than carbamazepine, but less so than duo-drug combination (Fig 6). 4. DISCUSSION Drug combinations are to be rationally selected based on the proof of synergistic efficacy with minimal toxicity (Pottoo et al., 2019b). Nevertheless, experimental and clinical literature to support the notion is rare. The clinical studies have urged synergistic anti-epileptic combination of valproate with lamotrigine, and topiramate with lamotrigine/levetiracetam with encouraging results on seizure control and neurotoxicity, evidenced from animal studies. In contrast, lamotrigine in combination with carbamazepine is recorded with reduced efficacy and escalated toxicity, a pharmacodynamic interaction (Besag et al., 1998). The combinations of oxcarbazepine with lamotrigine /phenytoin are also claimed as pessimistic (Błaszczyk et al., 2018). The failure of combinatorial regimens, seeds the patients with refractory epilepsy and low quality of life (Wickham et al., 2018). Thus, in the pursuit of innovative drug targets and strategies, to restrict inexorable process of epileptogenesis, we evaluated test drugs raloxifene (SERM), fluoxetine (SSRI), bromocriptine (D2 receptor agonist) and their low dose duo and triple combinations against generalized seizures incited with electroshock. Carbamazepine was used as comparator AED. Results of this study undoubtedly specified the potentiating effect of raloxifene on fluoxetine in mitigation of electro-convulsions, given that monotherapy with raloxifene rendered 13

null anti-convulsant effects. This duo-drug combination of raloxifene with fluoxetine exhibited an effect better than carbamazepine, a standard AED. In addition, the triple drug combination of raloxifene with fluoxetine and bromocriptine exhibited significant effect in abrogation of electroshock induced seizures; however less so than duo-drug combination. It seemed out that addition of bromocriptine to the duo-drug combination regimen reduced anti-convulsant potential of the said duo-drug combination. The electroshock-induced seizures were associated with dramatic reduction in hippocampal NPY levels, which were not elevated significantly with test drugs or their combinations, stating other mechanisms in mitigation of seizures. However, the results from measurement of oxidative stress indices and histomorphological findings confirmed results from behavioral findings. The use of estrogen for neuroprotection in epileptic states is overruled due to its pro-seizure effects. This problem was circumvented with the use of SERMs, which act as estrogen agonist and antagonist in a tissue specific manner and have emanated as putative agents for providing neuroprotection avoiding the threat for pro-seizure effects. Bazedoxifene and raloxifene induced neuroprotection independent of caspase-3 and augmented ERα (66 and 46 kDa isoforms) and PPAR-γ protein levels, thereby protected neocortical cells against hypoxic brain injury (Rzemieniec et al., 2018). Pre-treatment of SN4741 cultures with raloxifene, β-estradiol, and α-estradiol prior of exposure to hydrogen peroxide significantly reduced apoptosis caused by oxidative stress (Biewenga et al., 2005). Raloxifene significantly enhanced glutamate uptake and expression of astrocytic glutamate transporters GLT‐1 mRNA and protein levels in rat primary astrocytes. The enhanced expression was said to emulate with activation of ERK, EGFR, and CREB signaling pathways conciliated by estrogen receptors (ERs) ERα, ERβ, and GPR30. Thus, uptake of additional glutamate from the synaptic cleft prevents excitotoxic neuronal death (Karki et al., 2014). Our previous studies and various other studies reported anti-convulsant effects of raloxifene against Pilocarpine and KA induced seizures (Pottoo et al., 2014; Scharfman et al., 2009). Interestingly, the monotherapy with raloxifene (4 and 8 mg/kg) failed to increase threshold towards electroshock convulsions, which indicate that diverse transducer mechanisms are involved in case of seizures elicited via chemicals or electroshock. Fluoxetine (14 and 22 mg/kg) fetched a dose-dependent anti-seizure effect as evidenced from reduction in the ratio of THLE/NO-THLE in mice, showing our results are in accordance with the previous study from Gangopadhyay et al., 2017 who reported that fluoxetine protects against 14

electroconvulsive seizure in albino rats (Gangopadhyay et al., 2017). Borowicz et al., 2012 recited that threshold for PTZ induced clonic convulsions was significantly enhanced with fluoxetine (15 mg/kg), and that fluoxetine (10 mg/kg) also escalated the antiseizure effects of valproic acid (Borowicz et al., 2012). The absence seizures initiation in adult WAG/Rij were significantly reduced after chronic treatment with fluoxetine (30 mg/kg) and duloxetine (10 and 30 mg/kg) (Citraro et al., 2015). The picrotoxin doses required to produce THLE and death in unstressed and swim-stressed mice were enhanced with fluoxetine (Pericić et al., 2005). The density of GABAA receptors in DG, CA1 and CA2 regions of lithium–PILO treated rats was enhanced with fluoxetine, suggestive of anti-seizure effects (Shiha et al., 2015). The plausible mechanism in antiseizure effects of fluoxetine might be due to upregulation of GABA receptors in hippocampus and that serotonin stimulates GABAergic interneurons directly (Chi et al., 2017). The hippocampus is the site of neurogenesis throughout life which is altered by status epilepticus (SE) (Barkas et al., 2012).

The

fluoxetine

in

its

capacity

of

powerful

stimulant

for

hippocampal

neurogenesis (Malberg et al., 2000) may counteract hippocampal sclerosis following GTCS. Evidence suggests that Dopamine (D2) receptors seem to exhibit exorbitant capacity to modulate epileptic seizures because D2 receptor agonists (e.g., pergolide and bromocriptine) exhibit anticonvulsant properties while D2 receptor antagonists (e.g antipsychotics) stimulate seizures in patients with no preceding history of the epilepsy (Starr, 1996). Patients with mesial temporal lobe epilepsy (TLE) and hippocampal sclerosis (HS) were reported with reduction in binding of D2/D3-receptor at the pole and in lateral aspects of the epileptogenic temporal lobe (Werhahn et al., 2006). Further Dopamine D2 and D3 receptor agonists are reported to protect differentiated rat cortical oligodendrocytes from oxidative glutamate toxicity and oxygen/glucose deprivation mediated injuries, subsequently protective effect has been reported to be diminished by D2 and D3 antagonists (Rosin et al., 2005). Bromocriptine exhibited neuroprotective efficacy against kainic acid (KA) induced brain damage (Micale et al., 2006) and reduced seizure severity in TLE patients that are primarily affected by pituitary prolactinomas (Deepak et al., 2007) without any severe side effects (Deepak et al., 2007). In our previous studies utilizing PILO induced SE we had reported anti-convulsant properties of bromocriptine (Pottoo et al., 2018, 2016). However, in this study monotherapy with bromocriptine (6 and 10 mg/kg) failed to increase threshold towards electroshock convulsions, which again indicates that pathological condition of brain differs with respect to seizures elicited via chemicals or electroshock. 15

The duo-drug combination of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) displayed potentiation in mitigation of electroshock prompted convulsions, an antiepileptic effect better than carbamazepine was perceived. It is worth recapitulating here that the combination of bromocriptine (6 mg/kg) with each of raloxifene (4 mg/kg) and fluoxetine (14 mg/kg) was unsuccessful to exhibit protective effect. In other words, bromocriptine (6 mg/kg) reversed anticonvulsant effect of fluoxetine (14 mg/kg). Further, on combining bromocriptine (6 mg/kg) with the duo-drug combination regimen of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) the protective effect of the duo-drug combination was reduced, which indicates that dopaminergic neurotransmission confronts serotonergic neurotransmission against electroshock induced seizures. The expression of inflammatory genes is upregulated with oxidative stress via activation of redoxresponsive transcription factors. AP-1 (Activator protein-1) and NF-kB (nuclear factor kB) activation confers to the regulation of diverse inflammatory genes by cellular oxidative stress and/or intracellular glutathione levels (Shin et al., 2011). Glutathione reduced (GSH) is an endogenous intracellular nonprotein thiol antioxidant (Gaucher et al., 2018; Lu, 2013). The modification of levels of which have been shown to regulate seizure susceptibility and neuronal survival (Liu et al., 2012). Glutathione peroxidase prevents oxidative stress induced damage by reducing hydrogen peroxide and alkyl hydroperoxides at the cost of glutathione reduced and form H2O and O2 from H2O2, like Catalase (Cardenas-Rodriguez et al., 2013). The conversion of oxidized glutathione (GSSG) to glutathione (GSH) is catalyzed by Glutathione reductase that is NADPH-dependent (flavoprotein found in cytoplasm) (Untucht-Grau et al., 1981). GR plays an important role in maintaining the required amount of cellular GSH in reduced state. The ratio of GSH/GSSG should be high which is crucial for defense against oxidative stress. The MES (45 mA, 250 V, 0.2 s) induced THLE, culminated in significant escalation in hippocampal MDA levels (a marker of lipid peroxidation) compared to levels in normal mice, which lies in observance with precursory reports (Devi et al., 2012). Monotherapy with Fluoxetine (14 and 22 mg/kg) was effective in restricting increase in MDA levels, while monotherapy with other test drugs raloxifene and bromocriptine failed to restrain the change. Duo- drug combination of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) significantly reduced MDA levels. The addition of bromocriptine (6 mg/kg) to regimen of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg)

16

although significantly reduced MDA levels, but less so than duo-drug combination. The effect of carbamazepine (20 mg/kg) treatment was similar to triple drug combination. Challenge with MES (45 mA, 250 V, 0.2 s) significantly reduced hippocampal glutathione reduced (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) levels, compared to levels in normal hippocampus, which is in conformity with the previous report (Bhosle, 2013; Reddy et al., 2018). Monotherapy with Fluoxetine (14 and 22 mg/kg), significantly increased levels of glutathione reduced, glutathione peroxidase and glutathione reductase, compared to levels in toxic control. Similarly, duo- drug combination of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) significantly increased their levels. The addition of bromocriptine (6 mg/kg) to regimen of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) although significantly increased glutathione reduced, glutathione peroxidase and glutathione reductase levels, but less so than duodrug combination. Again, the effect of carbamazepine (20 mg/kg) treatment was similar to triple drug combination. NPY given

into the lateral ventricle is a powerful inhibitor of motor as well as

electroencephalographic (EEG) seizures prompted by KA (Woldbye et al., 1997). It is recited that gene therapy with NPY reduces chronic spontaneous seizures in a rat model of TLE (Noè et al., 2008). NPY reduces synaptic excitation interceded with glutamate release (Hollopeter et al., 1998). NPY mRNA was found reduced in (PTZ) induced seizures in zebrafish. The fish treated with Gabapentin (GBP), Diazepam (DZP), exhibited escalation in the mRNA expression of NPY gene (Kundap et al., 2017). NPY dramatically reduces stimulation-induced EPSPs in DG and CA1 (up to 30 and 55%, respectively) via Y2 receptors in human hippocampal slices in vitro (Ledri et al., 2015). Thus NPY acts an endogenous anti-convulsant. THLE significantly decreased (p<0.001) the NPY levels in comparison to

levels in normal mice. All drug(s) and their

combinations exhibited insignificant increase in NPY levels compared to levels in toxic mice. It might not be the possible mechanism in abrogation of MES induced THLE with test drugs/ or their combinations. In patients with epilepsy (PWE), generalized epileptic seizures are reported with hippocampal neuronal debt (Dam, 1980). Challenge with MES (45 mA, 250 V, 0.2 s), showed marked effects of THLE in the form of widespread neuronal loss in CA1, CA2, CA3 and DG regions, indicative of extensive neuronal pyknosis which is in agreement with previous study (Zarubenko et al., 17

2005). The electroshock induced neuronal apoptosis and derangement was effectively counteracted with fluoxetine (14 and 22 mg/kg) and more so with duo drug combination of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) as evident from normal layer organization, minimal reduction in healthy neurons, mild pyknosis and absence of shrinkage of neurons in treatment with said duo-drug combination. Again, the addition of bromocriptine (6 mg/kg) to said duo-drug combination reduced its neuroprotective potential. Carbamazepine (20 mg/kg) revealed neuroprotective effects but the effect was less pronounced in comparison to triple drug combination. The best neuroprotective effect was substantiated with duo-drug combination.

5. CONCLUSIONS The possible failure with current AEDs and their combinations can be attributed to their meager neuroprotective potential. The present study recites the potentiating effect of raloxifene on fluoxetine (at low doses) as optimum strategy against electrically induced convulsions and neurodegeneration. The study emphasizes that protection of serotonergic neurons by raloxifene, facilitates GABAergic signaling through serotonergic pathway, which has profound effect against the deteriorating process of epileptogenesis, further the two drug synergize in preventing glutamate release and/or increase glutamate uptake thereby intercepting excitotoxicity.

6. CONFLICT OF INTEREST The authors declare no conflict of interest.

7. ACKNOWLEDGMENTS The authors would like to acknowledge Unicure (India) Pvt. Ltd., Noida, for carrying out the pharmaceutical analysis of APIs.

Faheem Hyder Pottoo: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. Nahida Tabassum: Conceptualization, Methodology, Data curation, writing, writing – reviewing

18

and editing. Md. Noushad Javed: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. Shah Nigar: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. Shrestha Sharma: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. Md. Abul Barkat: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. Harshita: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. Md. Sabir Alam: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. Mohammad Azam Ansari: Conceptualization, Methodology, Data curation, writing, writing – reviewing and editing. George E. Barreto: Conceptualization, writing, writing – reviewing and editing, Supervision. Ghulam Md Ashraf: Conceptualization, writing, writing – reviewing and editing, Supervision.

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Figure Legends

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Fig. 1. Effect of RF (4 and 8 mg/kg), FT (14 and 22 mg/kg), BC (6 and 10 mg/kg) and their combinations against MES induced THLE in mice. Fischer’s exact test was used to compare qualitative variables (drug treated vs toxic control) to determine the Ratio of THLE/NO THLE. ###

p<0.001, ## p<0.01, #p<0.05 (compared with toxic control group).

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Fig. 2. Effect of RF (4 and 8 mg/kg), FT (14 and 22 mg/kg), BC (6 and 10 mg/kg) and their combinations on hippocampal NPY levels post MES induced THLE in mice. Data is represented as Mean ± SEM of 6 mice per group. Analyzed by one-way ANOVA followed by Tukey Kramer’s multiple comparison test. ###

***

p<0.001, **p<0.01, *p<0.05 (compared with normal control group).

p<0.001, ##p< 0.01, #p<0.05 (compared with toxic control group). 27

Fig. 3. Effect of RF (4 and 8 mg/kg), FT (14 and 22 mg/kg), BC (6 and 10 mg/kg) and their combinations on hippocampal protein levels post MES induced THLE in mice. Data is represented as Mean ± SEM of 6 mice per group. Analyzed by one-way ANOVA followed by Tukey Kramer’s

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multiple comparison test. ###

***

p< 0.001, **p<0.01, *p<0.05 (compared with normal control group).

p<0.001, ##p<0.01, #p<0.05 (compared with toxic control group).

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Fig. 4. Effect of RF (4 and 8 mg/kg), FT (14 and 22 mg/kg), BC (6 and 10 mg/kg) and their combinations on hippocampal: (A) Lipid Peroxidation and (B) Glutathione reduced (GSH) levels post MES induced THLE in mice. Data is represented as Mean ± SEM of 6 mice per group. Analyzed by one-way ANOVA followed by Tukey Kramer’s multiple comparison test. ***

p<0.001, **p< 0.01, *p<0.05 (compared with normal control group). ###p<0.001, ##p<0.01, #p<0.05

(compared with toxic control group).

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Fig. 5: Effect of RF (4 and 8 mg/kg), FT (14 and 22 mg/kg), BC (6 and 10 mg/kg) and their combinations on hippocampal: (A) Glutathione peroxidase (GPx) and (B) Glutathione reductase (GR) levels post MES induced THLE in mice. Data is represented as Mean ± SEM of 6 mice per group. Analyzed by one-way ANOVA followed by Tukey Kramer’s multiple comparison test. ***

p<0.001, **p< 0.01, *p<0.05 (compared with normal control group). ###p<0.001, ##p<0.01, #p<0.05

(compared with toxic control group).

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Fig. 6. Representative photomicrographs depicting H/E stained hippocampal sections of swiss albino mice comparing MES-induced neuronal damage in different groups. Hippocampal transverse section of mice observed 24 hrs post MES (45 mA, 250 V, 0.2 sec) revealed absolute loss of pyramidal neurons in CA1, CA2, CA3 and DG regions (Group II). Monotherapy with raloxifene (4 and 8 mg/kg) and bromocriptine (6 and 10 mg/kg) failed to restrain neuronal abatement (GroupsIV-V and VIII-IX). However, treatment with fluoxetine (14 and 22 mg/kg) revealed neuroprotective effects as evidenced from reduced the number of pyknotic neurons in CA1, CA2 and CA3 regions. While scattered arrangement of neurons was not averted with low dose fluoxetine (Groups VI-VII). The duo-drug combination of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) resisted electroshock induced neuronal damage as evident from intact neuronal structure with very few pyknotic nuclei in CA1 and CA3 regions (Group-X). The combinations of bromocriptine (6 mg/kg) with each of raloxifene (4 mg/kg) and fluoxetine (14 mg/kg) revealed null neuroprotective effects. The bromocriptine (6 mg/kg) seems to inverse neuroprotective effects of fluoxetine (14 mg/kg) (Groups XI-XII). Triple drug combination of raloxifene (4 mg/kg) with fluoxetine (14 mg/kg) and bromocriptine (6 mg/kg) although prevented the neuronal abatement, but less so than duo-drug combination (Group-XIII). The neuroprotective effect was also contrived with standard AED, Carbamazepine with nominal loss of healthy neurons, few pyknotic neurons in CA1, CA2 and CA3 regions, while scattering of healthy neurons in CA3 region wasn’t averted (Group-III).

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GA

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