Morphofunctional Changes in Field CA1 of the Rat ... - Springer Link

ISSN 0022-0930, Journal of Evolutionary Biochemistry and Physiology, 2014, Vol. 50, No. 6, pp. 531—538. © Pleiades Publishing, Ltd., 2014. Original Russian Text © D.S. Vasil’ev, N.L. Tumanova, I.A. Zhuravin, K.Kh. Kim, N.Ya. Lukomskaya, L.G. Magazanik, A.V. Zaitsev, 2014, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2014, Vol. 50, No. 6, pp. 463—469.

MORPHOLOGICAL BASICS FOR EVOLUTION OF FUNCTIONS

Morphofunctional Changes in Field CA1 of the Rat Hippocampus after Pentylenetetrazole and Lithium-Pilocarpine Induced Seizures D. S. Vasil’eva, N. L. Tumanovaa, I. A. Zhuravina, K. Kh. Kima, N. Ya. Lukomskayaa, L. G. Magazanika, b, and A. V. Zaitseva a Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences,

St. Petersburg, Russia b Faculty of Medicine, St. Petersburg State University, St. Petersburg, Russia

E-mail: [email protected] Received May 18, 2014

Abstract—Animal models of seizures and epilepsy are very diverse and instrumental for elucidating the mechanisms that underlie convulsive states and epileptogenesis. A single injection of pentylenetetrazole (PTZ) induces seizures, however, does not raise the risk of further development of epilepsy. Pilocarpine, immediately after injection, evokes epileptical state and, following a latent period, results in the development of spontaneous seizures, i.e. the drug triggers epileptogenesis. Assuming that in the PTZ model morphofunctional changes are mainly transient, while changes in the lithium-pilocarpine (PC) model may indicate the brain epileptization, we set ourselves the task of comparing morphological and functional characteristics of the hippocampal field CA1 in control and two experimental animal groups in 24 h after injection of the convulsants. We revealed the changes specific to the PC model and indicating neurodegeneration: a decrease in the cell spacing density, a diminution in the number of the viable NeuN-expressing neurons, an increased activity of the proapoptotic protease caspase-3. A characteristic feature of the PTZ model was the appearance of hyperchromic neurons with normal viability. In both models, the expression of the excitatory amino acid carrier EAAT1 increased by about 40% as compared to control. These morphofucntional correlates of reversible changes in the nervous tissue caused by seizures, as well as the early disorders leading to long-term brain epileptization can be used as indicators allowing assessment of a therapeutic potential of novel anticonvulsive drugs. DOI: 10.1134/S0022093014060088 Key words: epilepsy models, hyperchromic neurons, caspase-3, carriers of excitatory amino acids, NeuN.

INTRODUCTION Animal models of seizures and epilepsy play a crucial role in unraveling the mechanisms of convulsive states and epileptogenesis. These models are also instrumental for searching and pre-clin-

ical trials of new anticonvulsant drugs [1]. A lot of models have been developed to date which employ both genetically altered animals with spontaneous seizures, or seizures induced by sound or light stimulation, and normal animals in which seizures are induced by electric shock or chemiconvulsants

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[2–5]. A part of chemiconvulsants, e.g. an inhibitor of GABAergic synaptic transmission pentylenetetrazole, after a single administration induce a bout of seizures which, as a rule, results in some delayed consequences not accompanied by seizure relapse [6, 7]. Some other chemiconvulsants, such as a muscarinic receptor agonist pilocarpine, immediately after injection induce epileptical state in experimental animals, while later, after a latent period lasting several days or weeks, lead to the development of spontaneous seizures in these animals [8]. Since the long-term behavioral consequences resulting from application of the pentylenetetrazole (PTZ) and litium-pilocarpine (PC) models are different, the specific morphological changes can be expected. However, the data on the probable morphological differences induced by the application of the PTZ and PC models are quite fragmentary. It is necessary to distinguish between the reversible changes in the nervous tissue caused by a convulsive state, and the early disorders that lead to long-term brain epileptization and can be used as early biomarkers of epileptogenesis [9]. If we knew the specific biomarkers, we could use them as indicators to assess the therapeutic potential of new anticonvulsants, particularly, to predict their ability to prevent epileptogenesis. We hypothesized that morphological changes induced in the PTZ model can be mainly transient, whereas changes in the PC model should rather indicate rearrangements leading to brain epileptization. The goal of the study was to compare the morphological and functional characteristics of the control and two experimental animal groups. Nervous tissue of the hippocampal field CA1 was examined at the light microscopic level using Nissl staining; besides, we evaluated the viability of the neurons in this area, the expression level of the excitatory amino acid transporter EAAT1, and the activity of caspase-3. MATERIALS AND METHODS Pentylenetetrazole (PTZ) and lithium-pilocarpine (PC) models of seizures in rats. 36 male Wistar rats aged 6 weeks were used in the study. All experiments were performed in compliance with the Protocol of handling the laboratory animals

adopted at the Sechenov Institute of Evolutionary Physiology and Biochemistry (St. Petersburg, Russia) and based on the European Communities Council Directive #86 / 609 for the Care of Laboratory Animals. PTZ seizures were induced by intraperitoneal (i. p.) injection of pentylenetetrazole (Sigma, USA, 70 mg/kg). In the PC model, a day before pilocarpine injection (Sigma, 30 mg/kg, i.p.), the animals were injected with LiCl (Sigma, USA, 127 mg/kg, i.p.) and an hour before pilocarpine injection—with methylscopolamine (Sigma, 1 mg/kg, i.p.) to prevent excessive stimulation of peripheral muscarinic receptors. Control animals were injected with physiological saline. Volume of the injected solutions was 0.2 ml/100 g of the rat mass. Video recording of convulsive reactions of each animal was performed, and the intensity of the convulsions was measured in points on a modified Racine’s scale [10, 11]. The intensity of seizures in all the experimental animals was no lower than 3–5 points, and the first-day lethality was 29% in the PTZ model and 33% in the PC model. Light microscopy of the hippocampal tissue. 24 h after injection of chemiconvulsants in the experimental groups, or saline in the control group, all survived rats were anesthetized by diethyl ester and decapitated. The brains were rapidly removed and fixed in 10 % formalin in 0.1 M phosphate buffer (pH 7.4) for a week, and then the brains were immersed in the 20 % sucrose. 20 μm-thick sections were prepared on a cryostat Leica CM 1510S and Nissl stained. The field CA1 of the rat dorsal hippocampus (Fig. 1a) was examined in the microscope AF7000 (Leica, Germany), the images were digitized using a camera DFC495 (Leica, Germany). The neuronal spacing density in the pyramidal layer was estimated by counting their number in the middle of the 1000 μm-long segment of the pyramidal layer. The number of normal and hyperchromic neurons was determined using Videotest Master-Morfologiya software (VideoTest, Russia). Neurons were considered hyperchromic if their absorbance exceeded the absorbance of other neurons more than 2 times. Immunohistochemistry. Neuronal marker Fox3 (NeuN) expressed only in living and normally functioning neurons [12] was detected in the pyramidal layer of the rat hippocampal area CA1 using indirect immunohistochemical technique. Prima-

JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 50 No. 6 2014

MORPHOFUNCTIONAL CHANGES IN FIELD CA1

ry antibodies to NeuN (ab104224, Abcam, dilution 1:1000) and FITC-conjugated secondary antibodies (ab97022, Abcam, 1:200) were used. The background staining of nuclei was achieved with the fluorochrome Hoechst 33342 (Invitrogen, USA) and allowed estimation of the total number of cells. The immunofluorescent study was carried out using a microscope Leica DMR equiped with the confocal scanner Leica TCS SL (Leica Microsystems, Germany). Cells differing in brightness from the background more than 3 times were considered immunopositive. For each section, the ratio of NeuN-positive cells to the total number of Hoechst 33342-positive cell nuclei was calculated. Immunoblotting. Homogenate of the dorsal hippocampus was centrifuged (2500 g, 5 min, +4°C), the supernatant was collected for investigation and stored at –80°C. Cocktail of protease inhibitors (Roche cat# 11 836 170 001) was used for preservation of the material. Liver tissue homogenate was used as a negative control. Protein was determined in the sample according to Bradford [13]. The proteins were denaturized for 5 min at 95°C and placed into the wells with SDS-polyacrylamide gel (8%) at the rate of 25 g protein per well. Electrophoresis was carried out for 1–1.5 h at 120–150 V, and then the proteins were transferred onto PVDF membrane (0.5–1 h at 100 V). The membranes were blocked in 5% dry milk in 0.1% Tween 20/phosphate buffer for one hour at +4 °C. Monoclonal antibodies to the excitatory amino acid transporter protein EAAT1 (Abcam ab416, 1:10000) and rabbit anti-actin antibody (Sigma, А5060, 1:10000) were used. Incubation with the primary antibodies was performed for 20 h at +4°C. Visualization was carried out using HRP-conjugated secondary antibodies (Abcam, ab6721-1, 1:5000). To visualize the stripes the Optiblot ECL Ultra Detect Kit (1.2pg-2ng) (Abcam, ab133409) was used according to the protocol of the manufacturer. For each sample a ratio of EAAT1 stripe absorbance to that of actin was calculated. Caspase-3 activity. Hippocampal tissue was homogenized in 50 mM Tris-HCl buffer (pH 7.4) with addition of 1% (v/v) Triton X100. The homogenate was centrifuged (2500 g for 5 min at +4°C) and the supernatant was collected for determination of the caspase-3 activity. The samples

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were normalized to the protein concentration at the rate of 100 μg of protein per 50 μl of sample. Caspase-3 activity was determined colorimetrically using the Caspase 3 Assay Kit (ab39401, Abcam) according to the protocol recommended by the manufacturer, based on comparing the sample optical density with a chromogenic substrate (200 μM DEVD-p-NA) before incubation and after one hour of incubation at 37°C. Statistics. Comparison of the mean values was performed using Student t-test for unrelated samples or by using the nonparametric Mann– Whitney test; differences were considered significant when p ≤ 0.05. All the data are presented as mean ± SEM. RESULTS Light microscopic study of the field CA1 in the rat dorsal hippocampus. Investigation of the Nisslstained hippocampal field CA1 in rats of the control group (n = 7), after PTZ (n = 12) and PC (n = 8) seizures revealed significant morphological differences between the groups. In the pyramidal layer of the hippocampus, in 24 h after PTZ-induced seizures, a lot of elongated shrunken hyperchromic neurons were found characterized by a loss of turgor (29.9 ± 3.5% of the total number of cells, Figs. 1d, 1e) as compared with controls (3.1 ± 1.2%, Figs. 1b, 1c). However, cell spacing density, calculated as a number of cells within the 1000 μm-long segment of the field CA1, did not change after PTZ seizures (Fig. 2a). After PC seizures, the number of hyperchromic neurons in the rat hippocampus did not differ from the control (2.6 ± 0.5%, Fig. 2c), but the neuronal spacing density in the pyramidal layer decreased as compared with control by 21% (Fig. 2a). In the pyramidal layer, the sparse neurons characterized by a higher turgor and a lysis of the Nissl bodies in the cytoplasm were observed in the state of chromatolysis (Figs. 1e, 1f). These neurons were regularly surrounded by a large number of glial cells. Neurons in the state of chromatolysis were almost absent in the rats of the control group and after PTZ seizures. Viability of neurons. Viability of neurons was estimated using two parameters. Firstly, the expression of the protein NeuN (Fox3) was estimated in


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the hippocampal neurons using immunofluorescent technique, as it has been demonstrated previously that this protein is expressed only in the normally functioning neurons [12, 14]. Secondly, the proapoptotic caspase-3 activity in the hippocampal tissue was measured. We found that the protein NeuN was expressed after PTZ seizures in all the neurons including the hyperchromic cells in the pyramidal layer of the hippocampal field CA1. A total number of NeuNpositive neurons in these animals did not differ from that in the control (Fig. 2b). The caspase-3 activity in the hippocampal tissue did not change after PTZ seizures, but increased by 60% after PC seizures (Fig. 2d). Our results suggest that in the PTZ model no death of neurons occurs in the hippocampal field CA1 in the first day, whereas in the PC model a loss of neurons is significant and appears to be in progress, as the caspase-3 activity in the tissue is increased. It should be emphasized that the hyperchromatosis of neurons is not a sign of their death, as these neurons continue to express the protein NeuN. Level of the excitatory amino acid transporter EAAT1. Seizures can evoke an increase in the intracellular glutamate concentration [15–17], while excessive glutamate in nervous tissue, in its turn, induces an elevation of the excitatory amino acid transporter EAAT1 level [18, 19]; that is why we investigated how the EAAT1 level changes in the hippocampal tissue in the PTZ and PC models. Both models demonstrated an increase in the EAAT1 level by nearly 40% as compared with control (Fig. 2e). DISCUSSION In the present work, we report for the first time a comparative study of a complex of morphofunctional changes in the rat hippocampus in the PTZ and PC seizure models using a unified experi-

Fig. 1. Nervous tissue of field CA1 in rat dorsal hippocampus in control (b, c) and in 24 h after injection of pentylenetetrazole (d, e), or pilopcarpine (f, g). Nissl staining, scale bar 30 μm. (a) Topography of rat hippocampal fields CA1, CA2, CA4; DG—dentate gyrus; the frame marks the area studied. (b) Field CA1 in control rat hippocampus. (c) Neurons in control rat tissue at higher magnification. (d) Hyperchromic neurons (HN) in hippocampal pyramidal layer (arrows). (e) Hyperchromic neurons (HN) at higher magnification. (f) Neuronal chromatolysis (ChN) in hippocampal pyramidal layer. (g) Neuronal chromatolysis (ChN) and glial cells (Gl) at higher magnification.



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Fig. 2. Morphofunctional characteristics of the nervous tissue of the hippocampal field CA1 in control rats (n = 7) and rats after single injection of pentylenetetrazole (PTZ, n = 12), or pilocarpine (PC, n = 8). Significance of differences with control group: * p ≤ 0.05; ** p ≤ 0.01. (a) Neuronal spacing density in hippocampal pyramidal layer. Ordinate: mean number of neurons per 1000 μm-long segement. (b) % of NeuN-positive neurons relative to total number of neurons. (c) % of hyperchromic neurons relative to total number of neurons. (d) Caspase-3 activity in rat hippocampus homogenate. Ordinate: difference between optical densities before incubation and after 1 h-incubation, in arbitrary units. (e) Level of excitatory amino acid transporter EAAT1. Ordinate: ratio of optical density of EAAT1 stripe to that of actin.

mental and data processing methodology. This suggests that the observed differences between the experimental groups are due to the specific features of the molecular-cellular mechanisms of seizure implementation in these models. The PTZ and PC models are often used to study the human temporal lobe epilepsy [20], however they differ significantly in their manifestations and distant consequences. The PTZ model reproduces the

processes occurring in acute seizures to a greater extent [20]. According to the literature data, a single injection of pentylenetetrazole normally does not induce considerable long-term disorders in behavior, memory and learning ability [6] and does not lead to pronounced long-term morphological abnormalities [21]. The PC model is considered as one of the most adequate to reproduce basic characteristics of temporal lobe epilepsy;


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even a single injection of pilocarpine causes serious morphological abnormalities and triggers epileptogenesis [8]. Thus, the early specific morphological changes in the PC model may serve the markers of epileptogenesis [9]. We chose the hippocampal area CA1 for the comparative study because significant morphological disorders have been previously demonstrated in the rat PC model [22]. In this brain area, there occurs a death of both pyramidal cells and some types of interneurons as well as active neurogenesis, changes in arborization of the neuronal dendrites, and pathogenesis of the neuronal networks [8]. Apparently, this leads to the appearance of epileptic foci. Also it should be noted that the hippocampus is one of the key areas of the brain that are involved in seizure implementation both in the PTZ [23] and in PC [8] models. The analysis of morphological and functional changes in the hippocampal CA1 area in convulsive states shows that they can be divided into those specific to either PC or PTZ model and those observed in both models. Thus, we obtained a clear evidence of hippocampal neurodegeneration in the rat PC model, in contrast to the PTZ model and control group. Firstly, as early as in 24 h there occurs a reduction in the spacing density of neurons by about 20%. Secondly, among the remaining neurons nearly 15% do not express the protein NeuN, while the decrease in the expression level of this protein is a reliable indicator of the death of differentiated neurons [14]. Thirdly, the rat hippocampal tissue in the PC model exhibited an increase by nearly 60 % of the caspase-3 activity, a trigger and regulator of cell apoptosis [24]. Thus, our data show that early neurodegenerative changes in the hippocampal CA1 area may be a sinificant prerequisite of brain epileptization. In our experimental conditions, the appearance of a large number of hyperchromic neurons was specific to the PTZ model, in contrast to the control and PC model where these neurons were almost completely absent in the rat hippocampus. The appearance of hyperchromic neurons have been shown previously in the studies of electroconvulsive and bicuculline epilepsy models [25, 26], as well as in the PTZ model [27, 28], although in some cases a single injection of PTZ in convulsive doses did not cause the appearance of hyperchromic neu-

rons [7]. Hyperchromic neurons in the hippocampus have been found in the PC model as well [22]. Currently, hyperchromic neurons with a reduced turgor are considered not as dying cells, but as neurons with temporary impaired vital functions, since they can recover their functions with high degree of probability [29, 30]. Our experimental data are consistent with these ideas, as we have shown the ability of hyperchromic neurons to express the protein NeuN which is an indicative of their normal vitality. We can assume that the appearance of hyperchromic neurons is not a prerequisite of incipient epileptization of the brain. The nature of hyperchromic neurons is still unclear, but presumably their origin may relate to a prolonged membrane depolarization induced, among other reasons, by an excessive activation of glutamate receptors [31]. In our experiments using both models, we could have expected a seizure-induced increase in the glutamate level in the nervous tissue. The literature data indicate that glutamate concentration actually rises in this area during seizures [15, 16, 32], being typically higher in seizures induced by the blockade of GABAergic transmission, i.e. in disinhibition [15]. It has been shown that pentylenetetrazole is an antagonist of GABAA receptors (IC50 = 0.6 mmol/l) [33], hence it is likely that the differences in the glutamate concentration and its dynamics in the PC and PTZ models are causative for the appearance of hyperchromic neurons. Our data on the increased expression of the excitatory amino acid transporter EAAT1 argue indirectly in favor of the increased concentration of glutamate in the hippocampus in both models. EAAT1 level in glial cells have been shown to rise in compensatory response to the excess of glutamate in the extracellular medium [18, 19, 34]. CONCLUSION Our comparative morphofunctional study of the rat hippocampal field CA1 in the PC and PTZ seizure models revealed the following correlates of reversible seizure-induced changes in the nervous tissue: the appearance of hyperchromic neurons and the elevated level of the excitatory amino acid transporter EAAT1. Furthermore, we obtained the evidence of neurodegeneration in



the PC model, specifically, a decreased cell spacing density, a decreased NeuN expression and an increased caspase-3 activity; altogether, they may lead to long-term epileptization of the brain. These features can be used as indicators (biomarkers) for the evaluation of therapeutic potential of new anticonvulsive drugs.

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ACKNOWLEDGMENTS The study is supported by the grants nos. 12-0401080, 13-04-00453, 13-04-00388, 13-04-00224 from the Russian Foundation for Basic Researches and by the Programs of the Presidium of the Russian Academy of Sciences no. 5 and 7.

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