Inhibition of sodium glucose cotransporters following status epilepticus induced by intrahippocampal pilocarpine affects neurodegeneration process in hippocampus

Epilepsy & Behavior 61 (2016) 258–268

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Inhibition of sodium glucose cotransporters following status epilepticus induced by intrahippocampal pilocarpine affects neurodegeneration process in hippocampus Igor S. Melo a, Yngrid M.O. Santos a, Maísa A. Costa a, Amanda L.D. Pacheco a, Nívea K.G.T. Silva a, L. Cardoso-Sousa b, U.P. Pereira c, L.R. Goulart c, Norberto Garcia-Cairasco d, Marcelo Duzzioni a, Daniel L.G. Gitaí a, Cristiane Q. Tilelli e, Robinson Sabino-Silva a,b,⁎, Olagide W. Castro a,⁎ a

Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL), Maceio, AL, Brazil Department of Physiology, Institute of Biomedical Sciences, Federal University of Uberlandia (UFU), Uberlândia, MG, Brazil c Institute of Genetics and Biochemistry, Federal University of Uberlandia, MG, Brazil d Physiology Department, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil e Campus Centro-Oeste Dona Lindu, Federal University of São João del Rei (UFSJ), Divinópolis, MG, Brazil b

a r t i c l e

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Article history: Received 27 January 2016 Revised 22 April 2016 Accepted 23 May 2016 Available online 16 July 2016 Keywords: SGLT1 SGLT2 Phlorizin Hippocampus Neurodegeneration Fluoro-Jade C Pilocarpine Status epilepticus

a b s t r a c t Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, starting from secondary functional disorders due to several insults, including self-sustaining continuous seizures identified as status epilepticus (SE). Although hypoglycemia has been associated with SE, the effect of inhibition of the Na+/glucose cotransporters (SGLTs) on hippocampus during SE is still unknown. Here we evaluated the functional role of SGLT in the pattern of limbic seizures and neurodegeneration process after pilocarpine (PILO)-induced SE. Vehicle (VEH, 1 μL) or phlorizin, a specific SGLT inhibitor (PZN, 1 μL, 50 μg/μL), was administered in the hippocampus of rats 30 min before PILO (VEH + PILO or PZN + PILO, respectively). The limbic seizures were classified using the Racine's scale, and the amount of wet dog shakes (WDS) was quantified before and during SE. Neurodegeneration process was evaluated by Fluoro-Jade C (FJ-C), and FJ-C-positive neurons (FJ-C+) were counted 24 h and 15 days after SE. The PZN-treated rats showed higher (p b 0.05) number of WDS when compared with VEH + PILO. There was no difference in seizure severity between PZN + PILO and VEH + PILO groups. However, the pattern of limbic seizures significantly changed in PZN + PILO. Indeed, the class 5 seizures repeated themselves more times (p b 0.05) than the other classes in the PZN group at 50 min after SE induction. The PZN + PILO animals had a higher (p b 0.05) number of FJ-C+ cells in the dentate gyrus (DG), hilus, and CA3 and CA1 of hippocampus, when compared with VEH + PILO. The PZN + PILO animals had a decreased number (p b 0.05) of FJ-C+ cells in CA1 compared with VEH + PILO 15 days after SE induction. Taken together, our data suggest that SGLT inhibition with PZN increased the severity of limbic seizures during SE and increased neurodegeneration in hippocampus 24 h after SE, suggesting that SGLT1 and SGLT2 could participate in the modulation of earlier stages of epileptogenic processes. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Temporal lobe epilepsy (TLE) is the most common type of epilepsy, and is characterized by a chronic neuronal hyperexcitability and hypersynchrony state that is manifested through recurrent and complex partial seizures, typically with origin in the mesial temporal

⁎ Corresponding authors at: Institute of Biological Sciences and Health, Federal University of Alagoas (UFAL), Av. Lourival de Melo Mota, km 14, Campus A. C. Simões, Cidade Universitária, Maceió, AL CEP 57072-970, Brazil. Tel.: +55 82 3214 1002. E-mail addresses: [email protected] (R. Sabino-Silva), [email protected] (O.W. Castro).

http://dx.doi.org/10.1016/j.yebeh.2016.05.026 1525-5050/© 2016 Elsevier Inc. All rights reserved.

lobe [1,2]. The epileptogenic process is initiated by a cerebral insult, which may be a trauma, infection, stroke, febrile seizures, or status epilepticus (SE) [2]. After the epileptogenic insult, a cascade of cellular events is initiated, and neurobiological, morphological, biochemical, and electrophysiological changes occur. Such events happen during the latency period, ranging from 5–10 years in humans and days– weeks in animal models [1,3]. These changes are neuroplastic events in brain that are supposed to underlie epileptogenesis. Systemic (S) or intrahippocampal (H) administration of pilocarpine (PILO) in rodents leads to continuous or repeated seizures, a pattern called SE, which may last for many hours [4–9]. Afterward, SE results in a variable latent period that precedes a chronic stage that is

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characterized by the occurrence of spontaneous recurrent seizures (SRS). It has been reported that after H-PILO administration, animals presented facial movements, hypersalivation, motor seizures, as well as wet dog shakes (WDS) [10,11]. The central nervous system consumes tenfold more energy than the average of several other peripheral tissues and relies almost exclusively on glucose as energy substrate [12]. Glucose transporter 1 (GLUT1) is the predominant isoform expressed in the blood–brain barrier and glial cells [13], and glucose transporter 3 (GLUT3) is the neuronal isoform [14]. Glucose transporter 3 is the predominant neuronal isoform that fuels ATP generation and thereby the neuronal energy metabolism [15,16]. Sodium/glucose cotransporters (SGLTs) are transporters belonging to the solute carrier family 5 (SLC5A) gene family, which harness the gradient of sodium ions across the plasma membrane to drive glucose and galactose into cells [17–19]. The most studied members are SGLT1 and SGLT2, which are involved in glucose transport in specialized regions of the brain [20,21]. Besides the two Na+ ions plus the one glucose molecule, the SGLT1 protein is also able to transport about 264 H2O molecules across the cell membrane [22,23]. Since the pioneering studies, it has been established that SGLT1 is expressed in many brain areas, including CA1, CA3, and dentate gyrus hippocampal subfields [20,21,24]. Sodium/glucose cotransporter 2 protein has been observed in the hippocampus and cerebellum. The functional assays in rat brain slices suggest that SGLT2 accounts for 20% of the total methyl-4-[F-18] fluoro-4-deoxy-D-glucopyranoside (Me-4FDG), a highly specific SGLT substrate [21]. Both SGLT1 and SGLT2 are specifically inhibited by phlorizin (PZN) [25]; however, the effects of dual SGLT inhibitors in rat brain under epileptic conditions are unknown. High energy expenditure is required for neurons during brain functions [26]. The epileptic focus induced by applying penicillin in the frontal cortex promotes increase in brain uptake of methyl(alpha-D[U-14C]gluco)pyranoside, an isotope-labeled SGLT nonmetabolizable substrate [24]. Sodium/glucose cotransporters generate inward currents in the process of transporting glucose into cells, resulting in depolarization and increased excitability [21]. However, the mechanisms that result in a failure of neurons to compensate during this period of high energetic demand remains unclear [27]. Neurotoxicity may be closely associated with both high and low glucose utilization [28,29]. The effect of experimentally provoked changes in glucose utilization during neurodegeneration in epilepsy is controversial. Intracerebroventricular (i.c.v.) administration of glucose significantly exacerbated the development of neuronal damage [20]. Enhanced cerebral SGLT function has also been associated with development of ischemic neuronal damage [30–32]. The administration of 2-deoxy-D-glucose (2-DG), a glucose analog that is not metabolized by glycolysis, produced dramatic protection against hippocampal damage [33]. Additionally, it was demonstrated that reduced glucose utilization can lead to seizure protection with long-term calorie restriction in epileptic mice [34]. Moreover, infarct area was reduced by i.c.v. administration of PZN following midcerebral artery occlusion [30] and resulted in decreased number of shrunken neurons with pyknotic nuclei in CA3 area of hippocampus, suppressing the development of ischemic neuronal damage [31]. The high demand of glucose during seizure activity can lead to energy failure generated by reduced extracellular glucose, resulting in neurological damage [35]. However, the SGLT role in neurodegeneration during this period of high energetic demand remains also unclear [27]. Therefore, the purpose of this study was to investigate the effects of hippocampal SGLT inhibition in epileptogenesis. We promoted reduction in glucose metabolism restricted to the hippocampus, by inhibition of SGLT using intrahippocampal administration of PZN before pilocarpine-induced SE. We quantified the number of WDS before and during SE induced by H-PILO in the presence of PZN, in order to detect seizure inhibition circuitry activation, and we evaluated severity and evolution of seizures during SE. Finally, we analyzed the pattern of neurodegeneration in hippocampal regions at 24 h and 15 days after SE in

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the H-PILO model, using Fluoro-Jade C (FJ-C) histochemistry [6,36], in order to characterize the regional distribution of the neurodegeneration process. 2. Material and methods 2.1. Animals Experiments were conducted in male Wistar rats (Rattus norvegicus [n = 63, 240–340 g, 2–3 months]) from the main breeding stock of the Federal University of Alagoas. They were maintained on a 12 h/12 h light/dark cycle at 21 ± 2 °C, with lights on at 07:00 AM and lights off at 07:00 PM. Animals were individually housed in plastic cages with food and water ad libitum. All experiments were designed to minimize animal suffering and to limit the number of animals used. All experimental procedures were conducted in strict accordance with the guidelines set by Animal Research: Reporting of In Vivo Experiments (ARRIVE) and were approved by the Ethical Committee of the Federal University of Alagoas (permit number: 010/2014). Animals were submitted to surgery/euthanasia procedures under sodium thiopental. The research staff monitored rats at least 2 times per day for signs of illness or impairment by observing the general body condition, respiration rate, signs of dehydration, posture, immobility, social interaction, and response to manipulation. For the animals submitted to SE, monitoring their health was carried out for 2 h/day until the complete postictal recovery (about 2 days after SE; note that the H-PILO model allows rapid recovery and a high rate of survival [6]). During this period, animals were treated with electrolyte and nutrient replacement (i.p. injection of saline 0.9%; and by feeding animals with pasty food). No animals displayed clinical/behavior signal of pain or unexpected distress, and those features (when present) were used as humane endpoint criteria for euthanasia. Animals were euthanized 24 h or 15 days after SE. 2.2. Surgical procedure Animals were anesthetized with sodium thiopental (40 mg/kg, i.p. [Cristália®]) and received 0.1 mL/100 g veterinary pentabiotic (Fort Dodge®, subcutaneous) before the surgery. After fixing on the stereotaxic apparatus, animals received local anesthetic (lidocaine with epinephrine, subcutaneous [Astra®]). A cannula was implanted stereotaxically in the hilus of the dentate gyrus (DG) according to the following coordinates: −6.30 mm anterior–posterior (AP, reference: bregma); 4.50 mm medial–lateral (ML, reference: sagittal sinus); −4.50 mm dorsal–ventral (DV, reference: dura mater) [4,6,37]. The confirmation of the cannula placement into the DG was verified visually at the end of the experiments by hematoxylin and eosin (H&E). Only animals with the cannula exactly into the hilus of DG were included in the analysis. 2.3. Intrahippocampal microinjections Animals received either the drug (PZN, PILO) or its vehicle (VEH), intrahippocampally. The rats were divided into 4 experimental groups: VEH + VEH (n = 12), PZN + VEH (n = 12), VEH + PILO (n = 17), and PZN + PILO (n = 22). Animals were gently immobilized to microinject drugs. Each animal received 1 μL of PZN (50 μg/1 μL) or VEH (saline 0.9%) in the left hilus of the dentate gyrus (DG) 30 min before administration of 1 μL PILO (1.2 mg/μL) to evoke limbic seizures (VEH + PILO or PZN + PILO) or administration of 1 μL VEH (VEH + VEH or VEH + PZN). We used a 5 μL syringe (Hamilton Company, Reno, NV, USA) connected to a microinjection pump (Harvard Apparatus PHD 2000, Holliston, MA, USA) at a speed of 0.5 μL/min. All animals that developed SE were rescued with diazepam (5 mg/kg; i.p.) after 90 min of SE onset.

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2.4. Behavioral analysis Behavioral activity was recorded by means of a video camera (Full HD Digital Camcorder Sony DCR-PJ6) for a period of 90 min after the beginning of SE, enough time to further detect neurodegeneration [6]. Behavioral analysis was done according to Racine's scale (1972) [38], and the following stages were observed: (0) immobility, (1) facial movements, (2) head nodding, (3) forelimb clonus, (4) rearing, (5) rearing and falling, and (6) multiple class 5 seizures. This analysis was performed during SE, in order to study the neurophysiological changes in temporal lobe epilepsy that account for the behavioral changes. The 90-minute observation time was split into 18 windows of 5 min, and the most severe seizure that happened with greater frequency was used to represent the window. The number of WDS was quantified before and during SE. To examine the severity of seizures, the representative scales of each window were summed, and the result was divided by 18, the total amount of windows. 2.5. Tissue sampling Animals were injected with an overdose of sodium thiopental at 24 h or 15 days after SE induction and were transcardially perfused with 0.1 M phosphate-buffered saline with pH 7.4 (PBS) and 4% paraformaldehyde solution in PBS. Afterward, the brains were removed, cryoprotected with sucrose 20%, frozen at − 20 °C for 3 h, and stored at − 80 °C. Sections were then cut (30-μm thickness) using a cryostat (Leica CM 1850) at a temperature ranging from − 18 to − 22 °C and were processed for FJ-C staining [6,36]. 2.6. FJ-C staining procedure Sections were subjected to successive washes of 100% ethanol for 3 min, 70% ethanol for 1 min, and distilled water for 1 min. Afterward, slides were transferred to a solution of 0.06% potassium permanganate for 15 min on a rotating platform. Slides were rinsed three times for 1 min in distilled water and were then transferred to the FJ staining solution (0.0001%) for 30 min. After, slides were again rinsed in distilled water three times for 1 min. Finally, slides were coverslipped using fluoromount (EMS). The sections were examined and images captured using a fluorescence microscope (Nikon DS RI1). 2.7. Cell counting Fluoro-Jade C positive cells were counted by using the ImageJ software (Wayne Rasband; Research Services Branch, National Institute of Mental Health, Bethesda, MD, USA). For the quantification of neurodegenerating cells, we sampled in three different coordinates of the hippocampus: CA1, CA3, and hilus of dentate gyrus, (AP: − 2.56 mm, AP: − 3.30 mm, and AP: − 6.30 mm [37]), as shown by Castro et al. [6]. These regions were selected because of the high sensitivity to the neurodegenerative process. All cells were counted on the contralateral hippocampus because animals that received microinjection of PILO developed an obvious lesion and scar around the microinjection site [6]. 2.8. SGLT1 and SGLT2 structure assembly and PZN interaction The SGLT1 and SGLT2 proteins of rodent (Mus musculus, access number in Gene Bank: AF208031 and NP_573517) were submitted online in I-TASSER platform to generate high-quality predictions of 3D protein structure of both Na +/glucose cotransporters [39]. After that, in silico analyses of docking were performed to predict the interaction of PZN with SGLT1 and SGLT2 proteins. The 3D protein structures were submitted with PZN structure (access number Zinc: 3875408) to in silico interaction using Patchdock tool (http://bioinfo3d.cs.tau.ac.il/PatchDock/) with analysis of protein with small ligands [40,41]. To generate

visualization of drug interactions of PZN-protein we used the PyMOL 1.3 software. 2.9. Statistical analysis All values are presented as mean ± SEM. To analyze WDS patterns and the FJ-C+ cells, we performed the unpaired t test. In addition, to determine the evolution of seizures, we compared the classes of Racine's scale by one-way analysis of variance (ANOVA), followed by Student– Newman–Keuls posttest (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA). The significance level was 5% (described as p b 0.05). 3. Results 3.1. SGLT1 and SGLT2 structure assembly and PZN interaction Fig. 1 shows examples of successful modeling on SGLT1 and SGLT2 proteins by the I-TASSER server. Fig. 1A and B shows the interaction of PZN (red) with SGLT1 (green) and SGLT2 (green), respectively. In Fig. 1C, D, E and F, the representation of extracellular loops (blue), transmembrane loops (green), and cytoplasmic loops (orange) of both SGLT1 and SGLT2 proteins permits the identification of PZN (red) binding. We observed a PZN binding domain in a transmembrane segment of both SGLT1 and SGLT2 proteins. 3.2. Development of SE rate In VEH + PILO group, 18 of 25 (72%) animals that received microinjections of PILO developed SE, and one died because of severity of seizures; in PZN + PILO group, 23 of 30 (77%) animals developed SE, and one died also because of severity of seizures. Animals that received VEH microinjections instead of PILO (VEH + VEH or PZN + VEH) did not develop SE, so behavioral analysis was not performed. 3.3. Evaluation of latency time and WDS Immediately after microinjection of PILO, animals had not limbic seizures for a short interval. Regarding this latency to seizure onset (Fig. 2A), there was no significant difference between VEH + PILO group (30.6 ± 2.9 min) and PZN + PILO (26.4 ± 3.6 min). Wet dog shakes were considered present when the animals developed intense shakes of head and body. The PZN + PILO animals (113.7 ± 14.5) presented a higher (p b 0.05) amount of WDS when compared with VEH + PILO (73.5 ± 9.4) as shown in Fig. 2B. 3.4. Classification and temporal pattern of limbic seizures In VEH + PILO group, among the 17 surviving animals that developed SE, 12% presented classes 2–3, 12% classes 3–4, 47% classes 4–5, and 29% classes 5–6, while in PZN + PILO, among the 22 surviving animals that developed SE, 14% exhibited classes 2–3, 4% classes 3–4, 55% classes 4–5, and 27% classes 5–6. We analyzed the pattern of limbic seizures in both groups, PZN + PILO and VEH + PILO, for 90 min after SE induction (Fig. 3A and B). In VEH + PILO, at 10 min after the beginning of SE, the amount of classes 2 and 4 seizures was higher than that of classes 3 and 5 (p b 0.05). However, at 20 and 30 min of SE, the amount of class 2 seizures decreased, and class 4 was higher than all the other classes (p b 0.05). In contrast, no difference was observed in times 50, 60, and 70 min of SE among all classes observed (Fig. 3A). Similarly, in PZN + PILO, after 10 min of SE, classes 2 and 4 seizures were observed in higher amount than the classes 3 and 5 (p b 0.05). Subsequently, after 30 min of SE, the number of class 2 seizures decreased, and class 4 remained higher (p b 0.05) than classes 2 and 3

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Fig. 1. SGLT1 and SGLT2 Structure assembly and PZN interaction (A–B) the interaction of PZN (red) with SGLT1 (green) and SGLT2 (green), respectively. (C–F) The representation of extracellular loops (blue), transmembrane loops (green) and cytoplasmic loops (orange) of both SGLT1 and SGLT2 proteins and the identification of PZN (red) binding.

Fig. 2. Latency (minutes) to SE (A) and number of WDS before and during the SE (B). Unpaired t test, *p b 0.05.

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Fig. 3. Evolution of SE in VEH + PILO (A) and PZN + PILO (B). Number at all classes of Racine's scale for window (10 min) in VEH + PILO and PZN + PILO. ANOVA, post-hoc of Student– Newman–Keuls, p b 0.05. (A–B) # compared with classes 3 and 5, * compared with classes 2, 3 and 5, and & compared with classes 2, 3 and 4.

(Fig. 2B). Furthermore, class 5 seizures occurred more often (p b 0.05) than the other classes in the PZN group at 50, 60, and 70 min after SE induction (Fig. 3B). Qualitatively, analyzing Fig. 3, it is possible to observe that the VEH + PILO group presented a peak on seizure severity at 60 min of SE, while there is a longer time of higher seizure severity 40–60 min after PILO injection in the PZN + PILO group. Nonetheless, at 90 min post PILO injection, the proportion of seizures grades 2, 3, 4 and 5 is similar in both groups. 3.5. Neurodegeneration: FJ-C+ neurons in dentate gyrus, hilus, and CA3 and CA1 regions of the hippocampus after 24 h and 15 days of SE As expected, there were no FJ-C + cells in VEH + VEH in dentate gyrus, hilus, and CA3 and CA1 regions of hippocampus (Figs. 4A, 5A, 6A, and 7A). The PZN administration in the left hilus of the dentate gyrus (PZN + VEH) also did not promote presence of FJ-C+ cells in all areas analyzed (Figs. 4B, 5B, 6B and 7B). After 24 h of SE, all hippocampal areas evaluated presented higher number of FJ-C+ cells in the PZN + PILO brains, when compared with VEH + PILO ones. There were higher numbers of FJ-C + cells in DG (1205.0 ± 116.9), hilus (277.3 ± 23.2), CA3 region (338.0 ± 36.8), and CA1 area (400.2 ± 60.5) of PZN + PILO than that in DG (693.3 ± 109.0), hilus (141.5 ± 45.1), CA3 region (170.2 ± 61.1), and CA1 area (221.0 ± 52.7) of VEH + PILO group. The charts and the corresponding pictures for DG and hilus are shown in Fig. 4, for CA3 in Fig. 5, and for CA1 in Fig. 6. On the other hand, 15 days after SE, PZN + PILO animals had a decreased number (p b 0.05) of FJ-C+ in CA1 (245.3 ± 45.9) compared with the same region of VEH + PILO (425.8 ± 47.5) group (Fig. 7E). However, no significant difference was found in DG and hilus of hippocampus (Supplementary data 1) and in the CA3 region of the hippocampus (Supplementary data 2) when both groups were compared. 3.6. Temporal profile of neurodegeneration in dentate gyrus, hilus, and CA3 and CA1 regions of the hippocampus after 24 h and 15 days of SE Regarding the kinetics of neurodegeneration, in VEH + PILO group, the number of FJ-C + significantly decreased in dentate gyrus after 15 days of insult (360.2 ± 56.7) when compared with 24 h after SE (693.3 ± 109.0). However, in the CA1 region, there was an increase in FJ-C+ neurons after 15 days (425.8 ± 47.5) compared with the initial time (221.0 ± 52.7). No significant difference was found in the hilus (24 h, 141.5 ± 45.1; 15 d, 83.8 ± 17.3) and CA3 (24 h, 170.2 ± 61.1; 15 d, 235.5 ± 40.1) regions when the different times were compared (Fig. 8A). In PZN + PILO, 15 days after SE, there was a decrease in FJ-C+ cells in the dentate gyrus (262.3 ± 38.9) and hilus (120.3 ± 24.9) compared with 24-h postinsult (dentate gyrus, 1205.0 ± 116.9; hilus, 277.3 ± 23.2). On the other hand, CA3 (24 h, 338.0 ± 36.8; 15 d, 358.8 ±

77.9) and CA1 (24 h, 400.2 ± 60.5; 15 d, 245.3 ± 45.9) subfields did not show any considerable change (Fig. 8B). 4. Discussion The decline in cytosolic ATP and glucose-6-phosphate in neurons may drive neuronal glucose consumption during functional activation [12]. We showed that decrease of SGLT1 activity in hippocampus by PZN increases FJ-C + neurodegeneration in the dentate gyrus, hilus, and CA3 and CA1 regions of the hippocampus 24 h after SE, associated with increases in the severity of limbic seizures. Epileptic seizures are related to a set of behavioral, electrophysiological, cellular, and molecular changes [3,42], including changes in neuronal energy metabolism [43]. Previously, it has been shown that glucose uptake is increased in an epileptic focus induced by penicillin injection, suggesting a protective role via SGLT in metabolic stress conditions [20, 21,24]. The increased expression of SGLT1 and SGLT2 proteins in plasma membrane of neurons may play a protective function when the energy supply is reduced (such as during ischemia and hypoglycemia) or when the energy consumption is increased during epilepsy [20,21]. Thus, SGLTs could be essential for the survival of neurons that are under low glucose concentrations or anoxia [20]. Possibly, SGLT inhibition shown in this study is able to compromise the neuronal survival, since less glucose enters in the cell, and consequently, less ATP is generated. Adenosine triphosphate, provided by mitochondrial oxidative phosphorylation, is the source of energy to exert various cell functions, including the operation of sodium–potassium and chloride pumps and maintaining the resting membrane potential [42,44]. Therefore, the commitment of glucose supply to neurons significantly affects brain function as well as the development of and susceptibility to seizures [45], possibly increasing neuronal susceptibility to activation of cell death molecular cascades. We analyzed the behavioral patterns of epileptic and PZN-treated epileptic rats, in order to observe whether hippocampal SGLT inhibition would cause any alteration in motor seizures during SE. It is known that severity of seizures increases with time [5], but few studies provide explanations for this behavioral pattern during the SE. After H-PILO administration, animals displayed predominantly classes 2 and 4 seizures and progressed to a pattern of increased seizure severity up to the 90-min observation interval. The increased severity of the seizures was not confirmed statistically, though. This pattern of SE development can be explained by the fact that initially the seizures are focal, centralized in a few brain areas, which accounts for behavior expressions such as facial automatism and head and neck myoclonus. However, as SE progresses, other areas are recruited, which is seen as the development of generalized motor seizures. At this point, the mild seizure expressions, such as automatism and head and neck myoclonus, are incremented and intermingled with forelimb myoclonus, rearing, and frequent fallings. According to Lothman and Collins [43], the first classes of Racine's scale [38] occur as restricted epileptic expression

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Fig. 4. Qualitative and quantitative analysis of FJ-C+ neurons in dentate gyrus and hilus of hippocampus after 24 h of SE. Dentate gyrus and hilus in VEH + VEH (A), PZN + VEH (B), VEH + PILO (C) and PZN + PILO (D). Total number of FJ-C+ in dentate gyrus (E) and hilus (F). Arrows represent the dentate gyrus and arrow heads, the hilus. (A–D) Magnification, 100×; scale bar, 100 μm. (C′–D′) Magnification, 200×; scale bar, 50 μm. Unpaired t test, p b 0.05 and p b 0.01. *, ** compared with VEH + PILO.

of the hippocampus and lateral septum activation, then during the occurrence of intermediate classes, other limbic centers would be recruited, and at the final stage, extralimbic regions would also be involved. On the other hand, in the presence of SGLT inhibition, PILO-injected rats presented class 5 seizures significantly more often than the other seizure classes at 50, 60, and 70 min after SE onset, indicating increase of seizure severity in these specific time points when compared with VEH–PILO group. The increased seizure severity detected statistically in the PZN group during the 30-minute observation might be related to the higher incidence of FJ-C + cells in the hippocampus observed 24 h after SE induction (see below).

We showed in this study that SGLT1 and SGLT2 inhibition by PZN results in increased FJ-C+ neurons in the dentate gyrus (Fig. 4E), hilus (Fig. 4F), and CA3 (Fig. 5E) and CA1 (Fig. 6E) regions, after 24 h of SE. Sodium/glucose cotransporter 1 protein is expressed in the dentate gyrus granule cells and pyramidal cells of CA1 and CA3 subfields [20]. Although, in those studies, authors have not mentioned immunostaining of SGLT1 in hilar interneurons, their photomicrographs indicated that SGLT1 is also present in the hilus of the dentate gyrus. Since SGLT1 is expressed in these four regions, increased number of FJ-C + in PZNtreated rats directly reflect neuronal impairment associated with SGLT inhibition due to SE. Interestingly, the inhibition of SGLT in the hippocampus, followed by intrahippocampal administration of VEH, in other

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Fig. 5. Qualitative and quantitative analysis of FJ-C+ neurons in CA3 region of hippocampus after 24 h of SE. CA3 region in VEH + VEH (A), PZN + VEH (B), VEH + PILO (C) and PZN + PILO (D). Total number of FJ-C+ in CA3 (E). Arrows represent the CA3 region. (A-D) Magnification, 100×; scale bar, 100 μm. (C′–D′) Magnification, 200×; scale bar, 50 μm. Unpaired t test, p b 0.05. * compared with VEH + PILO.

words, SGLT inhibition not associated to seizures, did not promote neuronal death evidenced by FJ-C+ cells. The early vulnerability of hippocampus shown by the increased presence of FJ-C + cells in temporal lobe epilepsy under SGLT inhibition, coupled with the high SGLT1 densities in these neuronal bodies and terminals [21], are strongly indicative of the critical role of SGLT1 in maintaining neuronal survival during SE. Furthermore, after 15 days of SE, when comparing PZN + PILO with VEH + PILO groups, neuronal death was decreased in the CA1 region (Fig. 7E) while the other subareas remained unchanged (Supplementary data). This fact may be explained by (1) poor neuronal survival acutely, which would result in a lower number of surviving neurons to

continue to be impaired at 15 days after SE, (2) elongation of neuronal impairment in part of the population initially detected, without associated neuronal death, (3) neurons that were not FJ-C+ 24 h after SE but became FJ-C+ later, or (4) an earlier presence of spontaneous recurrent seizures in animals subjected to PZN injection, that would induce the increase of the number of impaired cells 15 days after SE. Such possibilities, however, were not evaluated in the current work and need to be further investigated. As soon as highly specific SGLT1 and SGLT2 tracers become available, the investigation of mechanisms underlying SGLT role in epileptogenesis will be improved. Kinetic analysis of neurodegeneration was performed in this study, 24 h and 15 days after SE in groups VEH + PILO and PZN + PILO

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Fig. 6. Qualitative and quantitative analysis of FJ-C+ neuron in CA1 region of hippocampus after 24 h of SE. CA1 region in VEH + VEH (A), PZN + VEH (B), VEH + PILO (C) and PZN + PILO (D). Total number of FJ-C+ in CA1 (E). Arrows represent the CA1 region. (A–D) Magnification, 100×; scale bar, 100 μm. (C′–D′) Magnification, 200×; scale bar, 50 μm. Unpaired t test, p b 0.05. * compared with VEH + PILO.

(Fig. 8), evidencing a region and time dependency of neuronal death provoked by SE evidenced with the FJ-C technique: a decrease in the number of FJ-C + in DG and hilus cells was observed after 15 days of SE when compared with the 24-h interval, while in CA1 and CA3, no changes were present. In agreement with our data, Castro et al. [6] have shown FJ-C + neurons at all time points after SE in the hilus of the dentate gyrus, and in CA1 and CA3, with the number of FJ-C + cells differing over time. Those authors analyzed neuronal loss in hippocampal subfields at 12, 24, and 168 h (7 days) after SE, also using the HPILO model, showing that neuronal loss in the hilus was reduced after 7 days, when compared with 12 h but was not different from the 24-h time point. On the other hand, although neuronal death in CA3 at 24 h

was elevated when compared with 12 h post-SE, there was no difference between 24 h and 7 days. Such data are in agreement with our current findings, since the maintenance in the number of FJ-C+ neurons is observed at 15 days, when compared with the 24-h group, in CA3. In the same study, the authors showed that FJ-C+ neurons increased in later points when compared with acute (12 h and 24 h) time in the CA1 subarea, which was also corroborated by us, since 15 days after SE, the number of FJ-C+ was higher than that at 24 h [46,47]. Additionally, according to Poirier et al. [48], the best time to detect neurodegeneration with Fluoro-Jade histochemistry is 24 h after SE, but these authors also found neurodegeneration after 15 days of the initial insult. Although earlier time points after SE have been suggested as better for FJ

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Fig. 7. Qualitative and quantitative analysis of FJ-C+ neuron in CA1 region of hippocampus after 15 days of SE. CA1 region in VEH + VEH (A), PZN + VEH (B), VEH + PILO (C) and PZN + PILO (D). Total number of FJ-C+ in CA1 (E). Arrows represent the CA1 region. (A–D) Magnification, 100×; scale bar, 100 μm. (C′–D′) Magnification, 200×; scale bar, 50 μm. Unpaired t test, p b 0.05. * compared with VEH + PILO.

evaluation of neuronal impairment, neuronal death has been detected up to 3 months after SE induced by PILO-lithium [49]. Furthermore, our results showed for the first time, to our knowledge, that WDS number was increased following intrahippocampal administration of PZN in SE induced by H-PILO microinjection. This motor pattern has been studied in different models of epilepsy in rats, such as kindling [50–53], kainic acid, and PILO [11,43,54]. Wet dog shakes have been approached in the literature as limbic seizure-associated behavior, with focal epilepsy induced by kindling stimulation or local injections of kainic acid or PILO. Wet dog shakes gradually disappear during generalization, which has been suggested as an indicator of progression toward

the generalized limbic seizures [53]. However, it is noteworthy that WDS expression and seizures do not depend on a path in common, as they can be propagated through distinct pathways [54,55]. In the H-PILO model, Rodrigues et al. [11] have shown that the presence of WDS can be associated with the potential activation of endogenous anticonvulsant systems. They report that animals that developed SE showed a large number of WDS initially which declined sharply after SE onset. In our current study, hippocampal PZN, an SGLT inhibitor, increased the number of WDS, which we speculate may be explained as a compensatory mechanism due to increased seizure severity [11].

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Fig. 8. Analysis of neurodegeneration kinetics in VEH + PILO (A) and PZN + PILO (B). Kinetics of FJ-C+ neurodegeneration after 24 h and 15 days in dentate gyrus (DG), hilus, CA3 and CA1, respectively. Unpaired t test, p b 0.05. (A) In DG and CA1, * 24 h versus 15 d. (B) In DG and hilus, *** 24 h versus 15 d.

Finally, the glucose availability for the proper maintenance of neuronal function during high-energy demand events such as seizures must be considered. Epilepsy is frequently observed in patients with diabetes mellitus [56]. The present results can be considered an alert to the potential risk of neurological damage using SGLT inhibitors to improve glycemic control, especially in patients with comorbid diabetes and epilepsy. The urgent need for new antidiabetic agents brought dual SGLT2 and SGLT1 inhibitors, such as canagliflozin and LX4211 to clinical use, although important aspects of their collateral effects were still unknown [57]. 5. Conclusion In summary, we showed for the first time that inhibition of SGLT in hippocampus increases the severity of limbic seizures and WDS during SE induced by H-PILO. Furthermore, inhibition of hippocampal SGLT function also increases FJ-C + neurodegeneration in dentate gyrus, hilus, and CA3 and CA1 regions of hippocampus 24 h after SE. However, we found that this inhibition promotes decreased FJ-C+ neurodegeneration in the CA1 region 15 days after SE. These data support an important role for SGLT during the development of SE, possibly ensuring the survival of neural cells during earlier stages of the epileptogenic processes. We believe that this study will contribute to the understanding of the role of SGLT in epilepsy and seizures, as well as the complexity of behavioral expression of TLE and its association with hippocampal cell death. Finally, our data open perspectives for the study of novel therapeutic strategies to reduce acute neuronal death caused by prolonged seizures, by preventing the reduction of hippocampal SGLT function during ictal events. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yebeh.2016.05.026. Conflicts of interest There is no conflict of interest. Acknowledgements This study was supported by FAPEAL (Grant number: (60030 000709/ 2013), CNPq (Grant number: 484911/2012-0), CAPES (Grant number: 02417/09-0) and FAPESP (Grant number: 2007/50261-4). Melo IS is a CNPq-Brazil MSc fellow. DG and NGC are CNPq Research Fellows. References [1] Sharma AK, Reams RY, Jordan WH, Miller Ma, Thacker HL, Snyder PW. Mesial temporal lobe epilepsy: pathogenesis, induced rodent models and lesions. Toxicol Pathol 2007;35:984–99. http://dx.doi.org/10.1080/01926230701748305. [2] Van Liefferinge J, Massie A, Portelli J, Di Giovanni G, Smolders I. Are vesicular neurotransmitter transporters potential treatment targets for temporal lobe epilepsy? Front Cell Neurosci 2013;7:139. http://dx.doi.org/10.3389/fncel.2013.00139. [3] O'Dell CM, Das A, Wallace G, Ray SK, Banik NL. Understanding the basic mechanisms underlying seizures in mesial temporal lobe epilepsy and possible therapeutic targets: a review. J Neurosci Res 2012;90:913–24. http://dx.doi.org/10.1002/jnr.22829.

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