Neuroprotective mechanisms of sildenafil and selenium in PTZ-kindling model: Implications in epilepsy

Author’s Accepted Manuscript Neuroprotective mechanisms of sildenafil and selenium in PTZ-kindling model: implications in epilepsy Karim M. Tawfik, Yasser M. Moustafa, Mona F. El-Azab www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(18)30299-1 https://doi.org/10.1016/j.ejphar.2018.05.035 EJP71817

To appear in: European Journal of Pharmacology Received date: 4 May 2017 Revised date: 19 May 2018 Accepted date: 22 May 2018 Cite this article as: Karim M. Tawfik, Yasser M. Moustafa and Mona F. ElAzab, Neuroprotective mechanisms of sildenafil and selenium in PTZ-kindling model: implications in epilepsy, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.05.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Neuroprotective mechanisms of sildenafil and selenium in PTZ-kindling model: implications in epilepsy

Karim M. Tawfik, Yasser M. Moustafa, Mona F. El-Azab* Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt.

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[email protected] [email protected] [email protected] *Corresponding author: Address for correspondence: Mona F. El-Azab, Professor & Head, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt. Tel. (+2) 0122 140 7777, Fax. (+2) 064 323 0741

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Abstract Epilepsy is one of the furthermost common neurodegenerative diseases affecting above 50 million individuals worldwide. The pathogenesis of epileptic seizures is not satisfactorily explored, and hence more effective anti-convulsive therapies are indispensable. Current study aimed to investigate the mechanisms of the potential neuroprotective effects of sildenafil/selenium on chemically-induced convulsions in mice. Kindling model was induced using pentylenetetrazol (PTZ; 35mg/Kg, 11 doses, intraperitoneally, every other day). PTZ-insulted groups were treated intraperitoneally with sildenafil (20mg/Kg), selenium (0.2mg/Kg) or their combination; 30 min before PTZ administration. PTZ-kindled model showed a significant loss of neuronal cells concurrently with nitrative/oxidative stress and lipid peroxidation. This was associated with enhanced expression of inducible nitric oxide synthase (iNOS), hemeoxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) along with increased activity of thioredoxin reductase (TrxR) in hippocampal tissue. Individual treatment with sildenafil or selenium showed partial neuroprotection, simultaneously with lower hippocampal expression of 4-hydroneonenal (4HNE), nitrotyrosine, iNOS and HO-1, yet without reaching normal levels. Sildenafil, but not selenium, enhanced the expression of VEGF and the endothelial cell marker CD34. The joint treatment with sildenafil and selenium preserved hippocampal neuronal count, improved kindling score, blunted lipid peroxides and nitrotyrosine levels, concomitantly with iNOS inhibition, normalization of TrxR activity and HO-1 expression, and evident neo-angiogenesis. Current study demonstrated the roles of several central signaling cascades in the sildenafil/selenium-evoked neuroprotection represented in, at least in part, amelioration of nitrative/oxidative stress alongside modulation of angiogenesis. Thus, sildenafil combined with selenium could be repurposed as a potential therapeutic regimen for delaying epilepsy progression.

Key words: Angiogenesis; Epilepsy; Kindling; Selenium; Sildenafil; Thioredoxin reductase.

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1. Introduction: Epilepsy is one of the most frequent neurodegenerative diseases affecting millions of people all over the world (Strine et al., 2005). Although the role of nitrative and oxidative stress is evident in epilepsy pathogenesis (Cardenas-Rodriguez et al., 2013), the role of angiogenesis is still elusive. Recent evidence revealed the contribution of excessive production of vascular endothelial growth factor (VEGF), the major mediator of angiogenesis, in epilepsy (Lange et al., 2016). Indeed, the synthesis of VEGF is strongly stimulated by hypoxia, nitric oxide (NO), and reactive oxygen species (Dulak and Jozkowicz, 2003); among other stimuli accompanying epileptic seizures (Striano et al., 2012). Moreover, the direct result of VEGFR-2 activation is the induction of calcium ion influx that enhances the activity of nitric oxide synthase (NOS) leading to enhanced NO release (Stahmann et al., 2010), thereby maintaining the cycle of VEGF/NO production. Nitric oxide (NO) was reported to be a probable neurotransmitter linked to synaptic plasticity (Dinerman et al., 1994) and the regulation of brain excitability (Buisson et al., 1993). The role of NO in epileptogenesis has been examined in several studies with contradictory results where the increased expression and/or activity of iNOS has been reported to play a pivotal role in the pathogenesis of PTZ-induced kindling and cognitive impairments in mice (Abdel-Zaher et al., 2017; Bashkatova et al., 2000; De Sarro et al., 1991; Kaputlu and Uzbay, 1997; Kawamoto et al., 2013; Rehni et al., 2009). Additionally, NO shows a stimulatory effect on VEGF synthesis that involves the activation of hypoxia-inducible factor-1 (HIF-1) (Chen et al., 2015), the key mediator responsible for increased VEGF synthesis under hypoxic conditions (Ruiz de Almodovar et al., 2009) as in epileptic seizures. Besides, NOS is essential for the angiogenic activity of VEGF (Zachary and Gliki, 2001). Sildenafil, mainly used for erectile dysfunction (Pyne et al., 1996), has been reported to directly affect the nervous system in both humans and rodents (Alissa et al., 2003; Corbin et al., 4

2004). Furthermore, sildenafil has shown an anticonvulsant activity in different animal models (Nieoczym et al., 2010a; Nieoczym et al., 2010b). NO/cGMP pathway, which mediates the vasodilator effect of sildenafil as a result of PDE-5 inhibition, was suggested as a possible mediator in PTZ-induced convulsions (Riazi et al., 2006). Studies have revealed that sildenafil enhances eNOS and iNOS expression, which may participate in the beneficial outcomes of the drug (Das et al., 2005; Salloum et al., 2003). These findings, along with the importance of NO for ischemiainduced angiogenesis (Senthilkumar et al., 2007), incited us to further explore the angiogenic potential of sildenafil therapy in epileptic seizures. Selenium, a trace element important for selenocysteine antioxidant enzymes (Alissa et al., 2003), was demonstrated to improve the cognitive function and prevent brain ischemia and excitotoxic brain damage as well (Schweizer et al., 2004) in addition to its antioxidant and neuroprotective features (Conrad, 2006). An important selenocysteine antioxidant system is the thioredoxin reductase (TrxR) that takes a diverse range of roles in cells through the numerous precise effects of thioredoxin (Trx) (Oien and Moskovitz, 2008). This protein kinase is crucial for multiple signaling pathways that are regulated by the redox status of mammalian cells (Matsuzawa and Ichijo, 2008). In the present study, we used a combination of sildenafil, as a vasodilator with potential angiogenic effect, and selenium, as an antioxidant with probable neuroprotective features, to explore the possible neuroprotective outcomes of sildenafil and/or selenium against neuronal damage induced by PTZ. Kindling mean seizure score along with hippocampal neuronal cell count were assessed as indicators for neuroprotection. To further understand the possible underlying mechanisms by which these drugs could mediate their effects, markers for nitrative/oxidative stress in addition to thioredoxin reductase (TrxR) activity were evaluated. Additionally, modulation of

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angiogenesis was assessed through evaluating vascular endothelial growth factor (VEGF) expression and microvessel density (MVD).

2. Materials and methods: 2.1.

Experimental animals: Experimental procedures were performed according to the “NIH Guide for the Care and

Use of Laboratory Animals” and in line with the guidelines adopted by Research Committee at Suez Canal University. Seventy male Swiss albino mice (weighing 22–26 g) were purchased from Vacsera (Egyptian Organization for Biological Products and Vaccines, Egypt) and housed under standard conditions (25±1°C, 50–60% relative humidity, natural dark/light cycles). After one-week adaptation to laboratory settings, mice were allocated randomly to study groups. Food and water were allowed ad libitum during the study period. 2.2

Chemicals and drugs Sildenafil was a kind gift from El-Nil Pharmaceutical Industrial Co. (Egypt), selenium

was obtained from Gedco Pharmaceutical Industrial Co. (Egypt), and PTZ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Inducible nitric oxide synthase (iNOS, catalog no. bs2072R), 4-hydroxynonenal (4-HNE, catalog no. bs-6313R), nitrotyrosine (catalog no. bs-8551R), hemeoxygenase-1 (HO-1, catalog no. bs-2075R), and cluster of differentiation-34 (CD34, catalog no. bs-8996R) primary antibodies were all purchased from Bioss Inc. (Woburn, MA, USA), while vascular endothelial growth factor (VEGF) primary antibody (catalog no. GTX102643) was purchased from Genetex Inc. (Irvine, CA, USA). Enzyme linked immunosorbent assay (ELISA) kit (catalog no. ab83463) for thioredoxin reductase (TrxR) activity was purchased from Abcam Co. (Cambridge, MA, USA).

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2.3

Pharmacological treatments Seventy mice were divided into 7 main groups, 10 mice each. First group received saline

and served as normal control group. Second and third groups were injected with sildenafil (20 mg/kg, i.p.) (Tahsili-Fahadan et al., 2006) or selenium (0.2 mg/kg, i.p.) (Zafar et al., 2003), respectively, and served as corresponding treatment control groups. Fourth group was insulted with PTZ (35 mg/kg, i.p.) (El-Azab and Moustafa, 2012; Orloff et al., 1949) and served as seizure control group. The last three groups were treated with sildenafil, selenium, or their combination (same previous regimen), 30 min before PTZ administration (Rehni and Singh, 2013). Drug solutions were freshly prepared along the study period. All pharmacological treatments commenced for 11 doses every other day. The experimental paradigm is explained in the provided Schematic Diagram (Fig 1). 2.4

Kindling induction and staging For induction of kindling, PTZ was freshly dissolved in normal saline and a sub-convulsive

dose (35 mg/kg, i.p) was injected every other day, for a total of 11 injections (Inan and Buyukafsar, 2008). After each injection of the sub-convulsive dose of PTZ, mice in different groups were observed for 30 min. PTZ-induced seizures were evaluated blindly by the observer and scored according to Fischer and Kittner scoring system (Fischer and Kittner, 1998) as follow: 1. No evidence of convulsive activity; 2. Ear and facial twitching, head nodding; 3. Myoclonic jerks; 4. Forelimb clonus, full rearing; 5. Generalized-clonic convulsions, rearing, jumping, falling and loss of righting reflex; 6. Tonic-clonic convulsions, tonic hind limb extensions.

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Mean seizure stages were calculated for all groups after each PTZ injection. 2.5

Serum selenium concentration At the end of the experiment, blood samples were collected from mice under slight ether-

anesthesia. Mice were then killed by an overdose of ether. Blood samples were collected from the orbital sinus into sterile tubes without EDTA, allowed for clotting at room temperature, centrifuged at 1968 × g for 20 min. Then, serum samples were collected and kept at -20 ; C for further analysis. Serum selenium levels were determined in the normal control group and selenium control group as well. It was measured by PerkinElmer 5100ZL atomic absorption spectrophotometer, equipped with Zeeman background corrector, a HGA-600 graphite furnace and an AS-60 auto-sampler. Values were expressed as µg/L and the percentage of change in serum selenium level from normal was calculated. 2.6

Histopathological examination For histopathological examinations, brain samples were isolated, cut and fixed

immediately in 10% neutral buffered formalin solution for at least 24 h. Tissues were dehydrated and embedded in paraffin, then were cut at a thickness of 5μm. Sections were stained with hematoxylin and eosin dye. All slides were observed using light microscope (CX21, Olympus Optical Co. LTD, Japan), ten serial fields for each section were captured by a digital microscope camera (Tucsen ISH1000) under High Power Field (HPF) of 40x and intermediate power of 10x (Universal Infinity Optical System, Olympus®). Neuronal cells were counted per constant area in pictures captured at 40x. 2.7

Assay of thioredoxin reductase (TrxR) activity In this assay, TrxR catalyzes the reduction of 5,5’-dithiobis (2-nitrobenzoic) acid

(DTNB) to 5-thio-2-nitrobenzoic acid (TNB2), using NADPH. TNB2 produces a yellow color 8

that can be measured at 412 nm. TrxR specific inhibitor was utilized in a parallel run to determine TrxR specific activity, since other enzymes could also reduce DTNB and thereby interfere with this assay. At the end of the assay, optical density of each well was determined using a microplate reader set to 412 nm, and TrxR activity was determined according to the manufacturer’s protocol. 2.8

Immunohistochemical analysis Five-micrometer-thick paraffin hippocampal sections were deparaffinized in xylene then

hydrated in decreasing concentrations of ethyl alcohol (100%, 90%, 80%, and 70%), followed by immersion in phosphate buffer saline (PBS) for 5 to 10 min. After antigen retrieval using TrisEDTA high pH protocol, sections were incubated overnight at 4°C with primary antibodies for 4HNE (1:300), nitrotyrosine or HO-1 (1:250), iNOS, VEGF or CD34 (1:200). Negative controls included sections incubated with PBS without primary antibodies. Immunohistochemical reaction for each antibody was detected using EnVision™ FLEX, High pH, (Link) detection system then counterstained using Mayer’s hematoxylin. Positive immunoreactions (brown) for all antibodies were captured in ten serial fields for each section (400X). The expression of iNOS, 4-HNE, nitrotyrosine, HO-1, VEGF, and CD34 was quantified densitometrically using ImageJ MacBiophotonics software package (National Institutes of Health, USA). 2.9

Statistical analysis Statistical analysis was done using PASW software (Version 18.0). All data were verified

to be normally distributed and were expressed as the mean ± S.E.M. Overall statistical significance was tested by one-way ANOVA followed by Bonferroni's post-hoc test for multiple comparisons. A confidence limit of P≤ 0.05 was considered statistically significant. For Kindling-induced seizure stage, two-way ANOVA followed by Duncan’s post hoc test for

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multiple range comparisons were used. Correlation studies between kindling dose and mean seizure stage were performed using Spearman’s test.

3. Results 3.1 Sildenafil and selenium co-administration decreased Kindling-induced seizure stage Repetitive injections with PTZ at a sub-convulsive dose, every other day for a total of 11 doses, induced chemical kindling with mean seizure score equals 4 on the last day of PTZ injection. Current data demonstrated that individual treatment with selenium 30 min before PTZ injections showed a tendency to decrease the kindling mean seizure score compared to PTZinsulted mice, yet non-significantly (Fig. 2A). On the other hand, the treatment with sildenafil either alone or combined with selenium significantly (P≤0.05) reduced mean seizure score to reach 3.1 and 1.8, respectively, on the last PTZ dose compared to untreated PTZ-insulted group (Fig. 2A). Moreover, significant changes were observed between differential and combined treatments in PTZ-insulted groups. No seizures were detected in normal mice treated with either sildenafil or selenium. Two-way ANOVA followed by Duncan’s post hoc test using corrected model revealed that the effect of kindling doses (from dose 1 to dose 11) in different treatment groups on mean seizure score was highly significant with group df=6, dose df=10, group X doses df=60, and F ratios= 427, 51.2, and 8.8; respectively (Fig. 2B). The significant difference in mean seizure stage in PTZ groups either untreated or selenium-treated started from 3rd and 4th doses, respectively. On the other hand, the treatment with sildenafil, either individually or combined with selenium, delayed the seizures threshold where the significant difference in mean seizure stage didn’t start till 8th and 9th doses, respectively. 10

Regression trendlines were performed to assess the relationship between kindling doses and mean seizure stage. Positive correlations were observed in all PTZ-insulted groups with least progression in mean seizure stage observed in the group treated with combined regimen (Fig. 2C). Spearman’s test and regression analysis showed strong correlations between different treatment regimens and kindling stages; PTZ (R2=0.9932***), PTZ+Sildenafil (R2=0.9823***) PTZ+Selenium (R2=0.9759***), PTZ+Sildenafil+Selenium (R2=0.9858***). From Fig. 2C, it can be concluded that individual treatment with sildenafil or selenium has decreased Kindlinginduced seizure stage where the highest value for the seizure stage in either treatments was ~ 3 (vs. seizure stage ~ 4 in PTZ control group), with relatively smaller value of the regression line slope (0.2905 and 0.3143, respectively). On the other hand, the combined treatment of PTZinsulted mice with sildenafil and selenium kept the Kindling-induced seizure stage below 2 with a much smaller value for the regression line slope (0.1727). 3.2 Selenium intraperitoneal injections enhanced serum selenium level in normal mice To confirm the absorption of selenium supplementation from the peritoneal cavity, serum selenium levels were determined in the normal control group and the selenium control group as well. Serum selenium level in the normal control mice was found to be 5.11 µg/L. Supplementation of normal mice with selenium showed 16.6% increase above normal serum level to reach 5.96 µg/L. (Fig. 3). 3.3 Sildenafil and selenium co-administration restored normal hippocampal neuronal architecture and preserved hippocampal neuronal cell count of kindled mice Photomicrographs of H&E-stained sections of hippocampus tissues from normal groups, either untreated or treated with sildenafil or selenium, showed normal arrangement of neurons in all hippocampus regions with no loss of neurons or any microglial proliferation (Fig. 4A&B).

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Notably, treatment of normal mice with sildenafil showed focal vascularity increments in the form of scattered small size capillaries. Insult with PTZ demonstrated abnormal arrangement of neurons in hippocampus regions especially PM region (thick black arrow) with widely spaced neurons (Fig. 4A&B). Moreover, moderate to marked degeneration and loss of neurons (green arrow head), microglial cell proliferation, focal apoptotic bodies (thin yellow arrow) associated with moderate edema (black asterisk) were observed in PTZ-insulted group (Fig. 4A&B). Treatment of PTZ-insulted groups with either sildenafil or selenium showed a marked improvement in the hippocampus architecture compared to untreated PTZ group. This was evident in restored arrangement of neurons in hippocampus regions especially PM region with minimal focal degeneration of very few neurons and negligible microglial cell proliferation. Notably, combined treatment of PTZ-insulted group with sildenafil and selenium showed restored arrangement of neurons in hippocampus regions especially PM region with no evident degeneration, yet minimal focal edema was observed (Fig. 4A&B). In line with histopathological changes, neuronal cell count of PTZ-insulted group demonstrated ;

52% neuronal loss in hippocampus regions. Neurons were preserved to some

extent when PTZ-insulted mice were treated with either sildenafil or selenium to show 32.5% and 25.5% neuronal cell loss, respectively. Joint treatment with sildenafil and selenium showed the maximal neuronal cell preservation where only 8.5% neuronal loss was evident after PTZ insult, with no notable change in sildenafil and selenium control groups (Fig 4C). 3.4 Sildenafil and selenium co-administration reduced TrxR activity in hippocampal tissue of kindled mice PTZ-insulted group showed a significant (P≤0.05, df=69 and F ratio=13.2) increase in TrxR activity in hippocampal tissue compared to normal control group (Fig. 5). Individual treatment of PTZ-insulted groups with either sildenafil or selenium maintained an activity of 12

TrxR that was comparable to untreated PTZ group and significantly (P≤0.05) higher than normal group. The joint treatment with sildenafil and selenium significantly (P≤0.05) reduced TrxR activity compared to all other PTZ groups; either untreated or individually treated with sildenafil or selenium, that was not significantly different from normal group (Fig. 5). 3.5 Sildenafil and selenium co-administration blunted hippocampal 4-HNE expression in kindled mice A significant (P≤0.05) increase in 4-HNE expression was observed in PTZ-insulted group in comparison to normal control group (df=69 and F ratio=11.42). Individual treatment of PTZ groups with either sildenafil or selenium showed a significant (P≤0.05) decrease in 4-HNE expression compared to untreated PTZ group, yet without normalization. On the other hand, joint treatment of PTZ-insulted group with sildenafil and selenium significantly (P≤0.05) decreased 4HNE expression in comparison to other PTZ groups; either untreated or individually treated with sildenafil or selenium, to reach normal expression level (Fig. 6A left panel & B). 3.6 Sildenafil and selenium co-administration normalized hippocampal nitrotyrosine expression in kindled mice Regarding the effect on nitrotyrosine expression in hippocampus, data came parallel to 4-HNE expression. Results showed that nitrotyrosine expression was significantly (P≤0.05) elevated in PTZ-insulted group compared to normal control group (df=69 and F ratio=20.65). Diseased groups treated with either sildenafil or selenium showed a significant (P≤0.05) decrease in nitrotyrosine expression compared to PTZ group, however, it remained higher than normal control group. On the other hand, treatment of PTZ-insulted group with the combined regimen of sildenafil and selenium successfully reduced nitrotyrosine expression to reach the normal value (Fig. 6A right panel & C).

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3.7 Sildenafil and selenium co-administration normalized hippocampal iNOS expression in kindled mice After 22 days of insulting with PTZ by kindling method, a significant (P≤0.05) increase in iNOS expression in hippocampus tissue was observed compared to normal control group (df=69 and F ratio=16.35) (Fig. 7A right panel & 7B). Individual treatment of kindled groups with either sildenafil or selenium showed a significant (P≤0.05) reduction in iNOS expression compared to PTZ control group, yet normal levels were not achieved. On the other hand, full normalization of iNOS expression was attained by the combined treatment with sildenafil and selenium. It is worth mentioning that normal mice treated with sildenafil exhibited a significantly lower expression of hippocampal iNOS compared to normal control group and jointly-treated PTZ group as well. 3.8 Sildenafil and selenium co-administration blunted hippocampal HO-1 expression in kindled mice The PTZ-kindled control group showed a significant (P≤0.05, df=69, F ratio=14.95) increase in hippocampal HO-1 expression compared to normal control group. Interestingly, treatment of normal mice with sildenafil showed a slight, yet significant (P≤ 0.05), increase in HO-1 expression. Similarly, the treatment of PTZ-insulted mice with sildenafil maintained this slightly elevated HO-1 expression compared to normal group, however, it was significantly (P≤ 0.05) lower than PTZ control group. On the other hand, the treatment of PTZ-insulted groups with selenium, either alone or in combination with sildenafil, significantly (P≤ 0.05) reduced HO-1 expression to reach the normal level (Fig. 7A left panel & 7C).

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3.9 Sildenafil maintained the PTZ-induced VEGF and enhanced its hippocampal expression in normal mice Vascular Endothelial growth factor (VEGF), the key mediator for angiogenesis, was evaluated in the current kindled model. PTZ-insulted control group revealed a significant (P≤0.05, df=69, F ratio=6.28) increase in VEGF expression in hippocampus tissue compared to normal control (Fig. 8A right panel & 8B). The treatment of kindled groups with sildenafil, either individually or jointly with selenium, maintained the PTZ-induced high expression of VEGF with almost 2-fold increment compared to normal level. Interestingly, the treatment of normal mice with sildenafil significantly enhanced VEGF expression to reach a value that was comparable to PTZ control group. On the other hand, the treatment of PTZ-insulted mice with selenium showed a tendency to reduce VEGF expression compared to PTZ-control group, yet with no significance (Fig. 8A right panel & 8B). 3.10

Sildenafil stimulated hippocampal angiogenesis in both normal and kindled mice To evaluate the influence of studied drugs on angiogenesis in the present kindling

model, the marker for endothelial cells, CD34, was evaluated in brain tissue. PTZ control group showed a tendency for increased microvessel density (MVD), yet was insignificant compared to normal control expression. On the other hand, PTZ-insulted groups treated with sildenafil, either alone or in combination with selenium, exhibited an elevated CD34 expression. Remarkably, normal mice treated with sildenafil exhibited a significant (P≤0.05, df=69, F ratio=3.6) increase in MVD compared to normal control group. In contrast, neither PTZ nor selenium could induce MVD significantly, either individually or in combination (Fig.8A left panel & 8C).

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4. Discussion: Maintaining the balance between inhibitory and excitatory processes in the CNS is crucial for the brain to function normally thus preventing neuronal disorders such as epilepsy. It has been reported previously that an elevated level of NO in the CNS had modulated neuronal glutamate release in a chemically-induced neurotoxicity model (Bogdanov and Wurtman, 1997). Nitric oxide synthase (NOS) mediates enzymatic synthesis of NO from L-arginine amino acid resulting in guanylyl cyclase (GC) activation and consequently increased concentration of cGMP. Intracellular level of cGMP, as a vital secondary messenger in the brain, is regulated by GC isoforms and phosphodiesterases (PDEs) as well (Esplugues, 2002). The elevation in cGMP level might boost glutamate release centrally (Prast and Philippu, 2001). Taken together, NO acts not only as a signaling molecule in the CNS by itself, but also as a modulator of inhibitory and excitatory neurotransmission through NO/cGMP pathway (Esplugues, 2002). Sildenafil inhibits PDE-5 and consequently reduces cGMP degradation, leading to smooth muscles relaxation. Sildenafil can cross “blood brain barrier” and hence affect PDE-5 in the brain resulting in CNS effects (Uthayathas et al., 2007). There is a discrepancy regarding the effects of sildenafil on seizures’ activities. A proconvulsant effect of sildenafil was firstly reported in humans (Gilad et al., 2002) and later in experimental models (Montaser-Kouhsari et al., 2011; Nieoczym et al., 2010b). Other studies demonstrated the influence of PDE-5 inhibitors on seizures in different epilepsy models (Kruuse et al., 2002; Nieoczym et al., 2010b; Riazi et al., 2006), relying on the contribution of NO/cGMP pathway in the neurotransmission processes. On the other hand, some studies showed that sildenafil has elevated electroconvulsions’ threshold in mice with reduction of seizures intervals and after discharges in amygdala kindling model (Nieoczym et al., 2010b). The present study demonstrates for the first time that sildenafil significantly increased seizures threshold in PTZ-kindled mice which is 16

contradictory to the previous observation reported by Montaser-Kouhsari et al. (2011), who used an acute PTZ model. This discrepancy in sildenafil effect could be attributed to the use of different PTZ models and different treatment regimens as well in both studies. The previously reported activation of NO/cGMP pathway by sildenafil (Nieoczym et al., 2012) could explain the currently observed anticonvulsant activity. Clinical and experimental evidence has revealed that nitrative and oxidative stress contributes to the pathogenesis of epileptic seizures (de Freitas et al., 2010; Dupont and Vercueil, 2015). Moreover, antiepileptic drugs disrupt oxidant/antioxidant homoeostasis by exerting extra burden of oxidative stress and thereby worsening the antioxidant status in the brain, an effect that may hinder their antiepileptic effects (Shin et al., 2011). Previous studies investigated whether protracted seizure activities have led to amplified production of reactive oxygen species and if oxidative damage has participated in seizure-induced brain injury (Ashrafi et al., 2007; Shin et al., 2011). Elevation of hippocampal nitrotyrosine with subsequent lipid peroxidation that were observed in the current PTZ-kindled model lend an additional evidence to the involvement of nitrative/oxidative stress in seizures-mediated degeneration of hippocampal neurons. Nitrotyrosine residues are widely considered as biomarkers for the contribution of reactive nitrogen species in a diversity of pathophysiological conditions including neurodegenerative diseases. The mitochondrial respiratory chain is sensitive to both NO and peroxynitrite; the product of NO reaction with superoxide anion (Carreras et al., 2004). Excessive NO produced during seizures inhibits mitochondrial oxidative phosphorylation (Poderoso et al., 1996), leading to dysfunction of mitochondrial resistance and consequently apoptotic neuronal death (Carreras et al., 2004). Additionally, neuronal cell membrane comprises elevated levels of polyunsaturated fatty acids (Benatti et al., 2004). Thus, brain cells 17

are enormously vulnerable to peroxidative injury (Shin et al., 2011), reflecting the critical importance of prooxidants/antioxidants homeostasis in epileptogenesis. The specific lipid peroxidation aldehydic product, 4-HNE, is recognized as a significant marker and mediator of cellular dysfunction and degeneration in a plethora of diseases (Bruce-Keller et al., 1998). Elevated expression of 4-HNE in hippocampal neurons that was evident in the current study is supported by the previous observation by Tang and his co-authors, who proved that 4-HNE played an essential role in neuronal degeneration post-stroke (Tang et al., 2007). This was associated with excessive nitrotyrosine expression in hippocampus, which confirms the involvement of nitrative/oxidative stress and subsequent lipid peroxidation in the neuronal loss that was observed in the current PTZ-kindling model. The PTZ-induced neurodegeneration was alleviated in this model by sildenafil or selenium treatment, yet only combined regimen achieved full protection, demonstrating efficient prevention of nitrative/oxidative stress against the detrimental effects of PTZ. Low level of reactive oxygen species is crucial for the cell to function normally, however, extensive elevation in reactive oxygen species represents an intrinsic risk of exacerbating neurodegeneration (Ashrafi et al., 2007) as seen in epilepsy. Evidence proposes redox regulation as a connection between selenium and neuronal disorders. Indeed, low levels of selenium in the human body were found to be linked with anxiety, depression, cognitive impairment, and aggression (Rayman, 2002). Current study proofed the ability of exogenously administered selenium in combination with sildenafil to attenuate seizures generation and the associated nitrative/oxidative burden, thus inhibiting consequent lipid peroxidation and neuronal injury. Regarding the antioxidant activity in the brain, thioredoxin reductase (TrxR) and all Trxrelying system, among other selenium-containing selenoproteins, have been assumed to be protective against neurodegeneration through the regulation of oxidation/reduction mechanisms 18

centrally (Arner, 2009; Steinbrenner and Sies, 2009). In addition to its importance in the reduction of hydroperoxides, TrxR controls redox state of Trx, thus conferring protection against reactive oxygen species at both cellular and mitochondrial levels (Rubartelli et al., 1992). To the best of our knowledge, this study is the first to evaluate the role of TrxR in PTZ-induced neurodegeneration and its modulation by sildenafil and/or selenium. Current data revealed that only the combined regimen of sildenafil and selenium could successfully reduce PTZ-induced over-activity of TrxR, indicating the resumption of antioxidant defense and potential neuroprotection that was evident in the form of preserved neuronal cell count and minimal percentage of neuronal loss among all PTZ-insulted groups. It was previously demonstrated that the expression of iNOS, an inducible enzymatic mediator of NO synthesis (Taylor et al., 1997), has been markedly induced throughout epileptic events in the CNS (Murashima et al., 2000). The currently used PTZ kindling model showed consistent findings. Furthermore, reduction in iNOS expression that was evident after individual treatment with either sildenafil or selenium came in harmony with other studies (Liu et al., 2014; Morsy et al., 2014). Additionally, current investigations showed an elevated expression of HO-1, a stress-inducible enzyme that is stimulated by reactive oxygen species and NO, in PTZinsulted group that was lessened in all treated groups. Present results came in parallel with previous studies, which proved that both iNOS and HO-1 could be induced by various stresses in the brain, including seizures (Murashima et al., 2000; Parfenova et al., 2005; Parfenova et al., 2012). The normalization of HO-1 and iNOS that was observed in the current study after treatment with sildenafil and selenium could be attributed to the effectively decreased oxidative and nitrative stress in hippocampal neurons. Interestingly, NO is also recognized as an upstream mediator of VEGF synthesis (Dulak et al., 2004). Angiogenesis occurring during pathological conditions, like epilepsy, is driven by 19

numerous inflammatory mediators (Rigau et al., 2007) that modulate the synthesis of growth factors (Zhang et al., 2003). Moreover, an early report has demonstrated that an ischemiainduced NO production had facilitated subsequent angiogenic responses (Murohara et al., 1998). Current PTZ kindling model confirmed the previous observations. It has been established that VEGF synthesis in many cell types, including vascular smooth muscle cells (Dulak and Jozkowicz, 2003), is stimulated by NO. The enhancing effect of NO on VEGF production comprises the stimulation of hypoxia-inducible factor-1 (HIF-1) (Kimura and Esumi, 2003), the main transcription factor responsible for increasing VEGF production under reduced oxygen tension (Semenza, 2001). Moreover, HO-1 was revealed to be implicated in angiogenesis as the attenuation in HO activity significantly inhibited angiogenesis (Bussolati et al., 2004). The role of sildenafil in angiogenesis was evident in the current study as VEGF expression and consequently MVD were significantly enhanced in hippocampus and surrounding brain tissue of normal group, an effect that was maintained in PTZ-insulted groups treated with sildenafil. This may indicate that the role of sildenafil in angiogenesis is independent from iNOS and HO-1. Current data are in part consistent with previous reports that demonstrated an inhibitory effect of sildenafil on ischemia-induced NO production (Senthilkumar et al., 2007), while enhancing VEGF expression and capillary-like tube formation (Zhang et al., 2003). Previous clinical reports have proposed that seizures complicating cerebral hypoxia are connected to increased risk of epilepsy (So et al., 1996; Thundiyil et al., 2011). In addition, experimentally hypoxia-induced seizures further increased seizure susceptibility (Ho et al., 2015; Kubova and Mares, 2007) and accelerated neuronal death in hippocampal tissue (Dzhala et al., 2000). Seizures are associated with hypoxic responses that may exacerbate brain injury (Patel et al., 2008). Hypoxia is the most consistently confirmed trigger of VEGF expression. Upregulation of VEGF after cerebral ischemia in hippocampus has been previously reported 20

(Marti et al., 2000). Cerebral ischemia and VEGF upregulation were observed in association with seizures (Croll et al., 2004). The upregulation of VEGF and subsequent increase in MVD could result from increased neuronal activity (Abraham et al., 2004). Croll and Wigand (2001) have reported an increased expression of VEGF in the hippocampal neurons in a cerebral ischemia model (Croll and Wiegand, 2001). Current data in PTZ-kindled mice came in line with previous studies. On the other hand, VEGF has been reported to be neuroprotective in the context of cerebral ischemia (Jin et al., 2000). Agents inducing VEGF level or expression in brain tissue, as achieved in the present study by sildenafil, may represent promising neuroprotective intervention for neuro-degenerative diseases involving hypoxia in its pathogenesis like epilepsy. Current study suggests that using sildenafil alone in the presence of increased activity of iNOS shall cause NO accumulation that, under conditions of excess reactive oxygen species generation as evident in epileptic seizures, will be converted into peroxynitrite leading to further oxidative/nitrative stress with consequent lipid per-oxidation and neuronal damage (Fig. 9). The concomitant use of antioxidants, like selenium, may allow for any accumulated NO to favor physiological direction towards vasodilatation and increased blood supply, and hence can allow for therapeutic angiogenesis leading to more efficient neuroprotection.

Conclusion: Pathophysiology of epilepsy, as a multifactorial disease, involves excessive oxidative and nitrative stress along with inappropriate pathological angiogenesis. In addition, antiepileptic drugs are not satisfactory and usually hamper antioxidants’ homeostasis centrally. Improving the microenvironment in the brain using agents like selenium, that will help to restore antioxidant defense, may grant for certain degree of neuroprotection. This also can allow for angiogenesis modulatory agents, such as sildenafil, to encourage the formation of new healthy and functional 21

blood vessels that in turn will afford improved blood supply and more efficient nutrients and antioxidants delivery for neurons, and consequently enhanced neuroprotection. Sildenafil/selenium combined treatment may represent an attractive approach for neuroprotection in neurodegenerative diseases. Further studies are needed to assess the beneficial outcomes for such a regimen in conjunction with anti-epileptic drugs.

Acknowledgements The authors thank Dr. Mohamed Kamal Abdel-Monim El-Kherbetawy, Assistant Lecturer of Pathology, Faculty of Medicine, Suez Canal University, for his valuable assistance with the evaluation of histopathology photomicrographs. Sildenafil was a kind gift from El-Nil Pharmaceutical Industrial Co. (Egypt). Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of Interest Statement The authors do not have any financial, personal or other relationships with other people or organizations that might create a conflict of interest about the manuscript.

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Figures Legends

Fig. 1. Schematic diagram of the study paradigm. Fig. 2. Differential and combined effects of sildenafil (20 mg/kg, i.p.) and/or selenium (0.2 mg/kg, i.p.) on Kindling-induced seizure stage. Kindling was induced in mice by repeated injections of pentylenetetrazol (PTZ; 35 mg/kg, i.p.). All pharmacological treatments commenced for 11 doses every other day. Mice were observed for 30 min after PTZ injections and scored using Fischer and Kittner seizure stage rating scale. (A) Data presented as seizure stage means ± S.E.M.; #P≤0.05 vs PTZ control group, (n=10), •P≤0.05 vs combined treatment PTZ group (i.e. sildenafil and selenium). (B) Data presented as seizure stage means ± S.E.M. Data were analyzed using Two-way ANOVA followed by Duncan post hoc analysis. *P≤0.05 vs initial dose. (C) Regression trendline were performed to assess the relationship between increasing kindling doses (1-11) and mean seizure stage (0-5). Fig. 3. Differential and combined effects of sildenafil (20 mg/kg, i.p.) and/or selenium (0.2 mg/kg, i.p.) on serum selenium level. The bar chart is showing the percentage change in serum selenium level based on a single measurement performed on a pooled sample in normal mice. Selenium treatment commenced for 11 doses every other day. Serum selenium level was measured in μg/L by graphite-furnace atomic absorption spectrometry. The percentage change from normal (%) was calculated relative to normal control group. Fig. 4. Differential and combined effects of sildenafil (20 mg/kg, i.p.) and/or selenium (0.2 mg/kg, i.p.) on histopathological changes and neuronal cell count. Photomicrographs represent H&E-stained hippocampal sections from different treatment groups captured at 100x (A) and 400x (B). Thick black arrow: abnormal arrangement of neurons; Green arrow head: degeneration and loss of neurons; Thin yellow arrow: focal apoptotic bodies; Black asterisk: moderate edema. Bar charts represent neuronal cell count (C) in normal mice and PTZ-kindled mice as well. *P≤0.05 vs normal group, #P≤0.05 vs PTZ control group, •P≤0.05 vs combined treatment PTZ group (i.e. sildenafil and selenium), ◦P≤0.05 vs corresponding treatment normal control group (i.e. sildenafil or selenium), (n=10). The explanation and doses are identical to that in Fig. 2.

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Fig. 5. Differential and combined effects of sildenafil (20 mg/kg, i.p.) and/or selenium (0.2 mg/kg, i.p.) on TrxR activity in hippocampus. TrxR activity was assessed colorimetrically and expressed in nmol/min/ml. *P≤0.05 vs normal group, #P≤0.05 vs PTZ control group, •P≤0.05 vs combined treatment PTZ group (i.e. sildenafil and selenium), (n=10). The explanation and doses are identical to that in Fig. 2. Fig. 6. Differential and combined effects of sildenafil (20 mg/kg, i.p.) and/or selenium (0.2 mg/kg, i.p.) on 4-HNE and nitrotyrosine expressions in hippocampus. Photomicrographs are representative of 4-HNE and nitrotyrosine assessed immunohistochemically (A). Bar graphs (B & C) represent optical density of positive reactions (brown stain) of both antibodies determined using ImageJ program. *P≤0.05 vs normal group, #P≤0.05 vs PTZ control group, •P≤0.05 vs combined treatment PTZ group (i.e. sildenafil and selenium), ◦P≤0.05 vs corresponding treatment normal control group (i.e. sildenafil or selenium), (n=10). The explanation and doses are identical to that in Fig. 2. Fig. 7. Differential and combined effects of sildenafil (20 mg/kg, i.p.) and/or selenium (0.2 mg/kg, i.p.) on iNOS and HO-1 expressions in hippocampus. Photomicrographs are representative of iNOS and HO-1 assessed immunohistochemically (A). Bar graphs (B & C) represent optical density of positive reactions (brown stain) of both antibodies determined using ImageJ program. *P≤0.05 vs normal group, #P≤0.05 vs PTZ control group, •P≤0.05 vs combined treatment PTZ group (i.e. sildenafil and selenium), ◦P≤0.05 vs corresponding treatment normal control group (i.e. sildenafil or selenium), (n=10). The explanation and doses are identical to that in Fig. 2. Fig. 8. Differential and combined effects of sildenafil (20 mg/kg, i.p.) and/or selenium (0.2 mg/kg, i.p.) on VEGF and CD34 expressions in hippocampus. Photomicrographs are representative of VEGF and CD34 assessed immunohistochemically (A). Bar graphs (B & C) represent optical density of positive reactions (brown stain) of both antibodies determined using ImageJ program. *P≤0.05 vs normal group, #P≤0.05 vs PTZ control group, •P≤0.05 vs combined treatment PTZ group (i.e. sildenafil and selenium), ◦P≤0.05 vs corresponding treatment normal control group (i.e. sildenafil or selenium), (n=10). The explanation and doses are identical to that in Fig. 2.

30

Fig. 9. Schematic representation demonstrating the possible mechanisms by which sildenafil and selenium may exert their neuroprotective effects against epileptic-induced seizures.

Graphical abstract Seizures induced in PTZ-kindling model are associated with pathological changes in the brain specifically in the hippocampus region. These changes are accompanied by excessive production of ROS and activation of iNOS leading to accumulation of NO centrally, thereby generating a status of oxidative and nitrative stress with subsequent lipid peroxidation and neurodegeneration. Additionally, the seizures-induced hypoxia will trigger VEFG-mediated pathological angiogenesis along with increased HO-1 expression that together are still insufficient to protect hippocampal neurons against PTZ insult, thus allowing for sustained seizures and leading to exacerbated neuronal loss. Sildenafil treatment of PTZ-kindled mice activates NO/cGMP pathway as a result of PDE-5 inhibition. Accumulated NO, in the presence of a powerful antioxidant as selenium-the important element for TrxR activity-will further activate VEGF at the expenses of nitrative stress and lipid peroxidation. Moreover, VEGF activation by sildenafil will promote iNOS expression and stimulate NO/cGMP pathway that, in the presence of antioxidants like selenium, will preferentially mediate the loop of angiogenesis. The newly formed blood vessels shall keep providing nutrients and antioxidants

to

hippocampal

neurons,

thereby

retarding

seizures-induced

neurodegeneration and mediating neuroprotection. 4-HNE: 4-Hydroxynonenal; cGMP: cyclic guanosine monophosphate; GMP: guanosine monophosphate; GTP: guanosine triphosphate; HIF-1: hypoxia inducible factor-1; HO-1: hemeoxygenase-1; iNOS: inducible nitric oxide synthase; NADPH: Nicotinamide adenine dinucleotide phosphate; NO: nitric oxide; ONOO-: peroxynitrite; PDE-5: phosphodiesterase-5; PTZ: pentylenetetrazol; ROS: reactive oxygen species; sGS: soluble guanylyl cyclase; Trx: thioredoxin; TrxP: thioredoxin peroxidase; TrxR: thioredoxin reductase; VEGF: vascular endothelial growth factor.

31

N.B. Measured parameters are highlighted in red color.

32

Figure

Fig. 1

Day 1 Day 3

Day 5

Day 7

Day 9

Day 11 Day 13 Day 15 Day 17 Day 19 Day 21

Dose 1

Dose 3

Dose 4

Dose 5

Dose 6

Adaptation

7 Days

Dose 2

Dose 7

Dose 8

Dose 9

Dose 10

21 Days (Kindling) Number of days for PTZ insult (35 mg/kg, i.p.)

Dose 11

Termination & Harvesting

Treatment

Day 22

Figure

Fig. 2 A. 4.5

PTZ PTZ+Sildenafil PTZ+Selenium PTZ+Sildenafil+Selenium

4

Mean seizures stage

3.5

#•

3 2.5 2

# 1.5 1 0.5 0 1

2

3

4

5

6

7

8

9

10

11

Number of PTZ injections

B. PTZ

5

PTZ+Sildenafil

PTZ+Selenium

PTZ+Sildenafil+Selenium

4.5

* * 4

*

Mean seizure stage

3.5

* *

*

3

*

*

*

*

*

*

*

*

*

2.5

* 2

* *

*

*

*

* *

* 1.5

1

0.5

0 1

2

3

4

5

6

7

Number of PTZ injections

8

9

10

11

C. 5

5

PTZ

PTZ+Sildenafil 4

4 y = 0.3664x + 0.1382 R² = 0.9932

3 2

2

1

1 0

0

Mean seizure stage stage

y = 0.2905x + 0.13 R² = 0.9823

3

0

1

2

3

4

5

6

7

8

0

9 10 11

1

2

5

5

3

4

5

6

7

8

9

10 11

PTZ+Sildenafil+Selenium

PTZ+Selenium 4

4 y = 0.3143x + 0.1753 R² = 0.9759

3

2

2

1

1

0

0 0

1

2

3

4

5

y = 0.1727x - 0.1273 R² = 0.9858

3

6

7

8

9 10 11

0

1

2

3

Number of PTZ injections

4

5

6

7

8

9 10 11

Figure

Fig. 3

Percentage change from normal (%)

150

116.6

100 100

50

Normal

Selenium

PTZ+Sildenafil+Selenium

PTZ+Selenium

PTZ+Sildenafil

PTZ

Selenium

Sildenafil

Normal

Figure

Fig. 4 A

10μm

B

40μm

C 250

Neuronal Cell Count

200

◦ 150

100

50

0

Normal

Sildenafil

Selenium

PTZ

PTZ +Sildenafil

PTZ +Selenium

PTZ +Sildenafil +Selenium

Figure

Fig. 5

70

TrxR activity (nmol/min/ml)

60

50

#

40

30

20

10

0

Normal

Sildenafil

Selenium

PTZ

PTZ +Sildenafil

PTZ +Selenium

PTZ +Sildenafil +Selenium

PTZ+Sildenafil+Selenium

PTZ+Selenium

PTZ+Sildenafil PTZ Selenium

Sildenafil

Normal

Figure

Fig. 6

A 4-HNE Nitrotyrosine

40μm 40μm

B

220

* * # • º

200

4-HNE expression (O.D)

180 160

* # • º

140

# 120 100 80 60 40 20 0

Normal

Sildenafil

Selenium

PTZ

PTZ+Sildenafil

PTZ +Selenium

PTZ +Sildenafil +Selenium

C

220

*

200

Nitrotyrosine expression (O.D.)

180

* # • º

160

* # • º #

140 120 100 80 60 40 20 0

Normal

Sildenafil

Selenium

PTZ

PTZ +Sildenafil

PTZ +Selenium

PTZ +Sildenafil +Selenium

PTZ+Sildenafil+Selenium

PTZ+Selenium

PTZ+Sildenafil

PTZ

Selenium

Sildenafil

Normal

Figure

Fig. 7 A iNOS HO-1

40μm μm

40μm

B 180 160

iNOS expression (O.D.)

140 120 100 80 60 40 20 0

Saline

Sildenafil

Selenium

PTZ

PTZ+Sildenafil PTZ+Selenium

PTZ +Sildenafil +Selenium

C 200

*

180

* #

160

HO-1 expression (O.D.)

*

# º

140

#

120 100 80 60 40 20 0

Normal

Sildenafil

Selenium

PTZ

PTZ +Sildenafil

PTZ +Selenium

PTZ +Sildenafil +Selenium

PTZ+Sildenafil+Selenium

PTZ+Selenium

PTZ+Sildenafil

PTZ

Selenium

Sildenafil

Normal

Figure

Fig. 8 A VEGF

40μm

CD34

B 180 160

VEGF expression (O.D.)

140 120 100 80 60 40 20 0

Normal

Sildenafil

Normal

Sildenafil

Selenium

PTZ

PTZ +Sildenafil

PTZ +Selenium

PTZ +Sildenafil +Selenium

C 180 160

CD34 expression (O.D.)

140 120 100 80 60 40 20 0

Selenium

PTZ

PTZ +Sildenafil

PTZ +Selenium

PTZ +Sildenafil +Selenium

Figure

Fig. 9 PTZ-Kindling

Initial seizures Hypoxia

iNOS

ROS

HIF-1

NO

O2-

GC

Nitrative stress (Nitrotyrosine)

Reduced uced TTrx r rx

Oxidative stress

VEGF Angiogenesis

Sildenafil

TTrx rx Oxidized

Lipid peroxidation (4-HNE) Neurodegeneration Sustained epileptic seizures

Exacerbated neuronal loss

Selenium

PDE-5

GTP

TrxR

cGMP

ONOO-

TrxP

GMP

HO-1

Graphical Abstract

Graphical abstract

PTZ-Kindling

Initial seizures Hypoxia

iNOS

ROS

HIF-1

NO

O2-

GC

Nitrative stress (Nitrotyrosine)

Reduced uced TTrx r rx

Oxidative stress

VEGF Angiogenesis

Sildenafil

Trx Trx Oxidized

Lipid peroxidation (4-HNE) Neurodegeneration

Sustained epileptic seizures

Exacerbated neuronal loss

Selenium

PDE-5

GTP

TrxR

cGMP

ONOO-

TrxP

GMP

HO-1