Epilepsy Research (2014) 108, 223—231

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BASIC RESEARCH

Ginseng extract attenuates early MRI changes after status epilepticus and decreases subsequent reduction of hippocampal volume in the rat brain Elena Suleymanova a,b,∗, Mikhail Gulyaev a, Nina Chepurnova a a b

Lomonosov Moscow State University, GSP-1, Leninskie Gory, Moscow 119991, Russia Institute of Higher Nervous Activity and Neurophysiology of RAS, 5A Butlerova St., Moscow 117485, Russia

Received 13 May 2013; received in revised form 15 September 2013; accepted 21 November 2013 Available online 1 December 2013

KEYWORDS Epilepsy; Status epilepticus; MRI; Brain damage; Panax ginseng

Summary Prolonged epileptic seizures during status epilepticus (SE) are known to cause neuronal death and lead to brain damage. Lesions in various brain regions can result in memory and cognitive impairment, thus searching of new neuroprotective drugs is important. We evaluated effects of single and chronic administration of ginseng extract on early and late changes in MRI measurements in the rat brain after lithium-pilocarpine SE. Butanol extract of ginseng root cell culture DAN-25 was administered after termination of SE. MRI study of the rat brain was performed 2, 7, and 30 days after SE. High-resolution T2 -weighed images and T2 -maps were obtained, and total damaged area, hippocampal volume, and T2 relaxation time in several brain structures were calculated. Single administration of ginseng extract attenuated early changes in brain structures found on day 2 after SE. Both single and chronic treatment with ginseng extract decreased brain damage on day 7 after SE. An increase in T2 -relaxation time in the hippocampus on day 2 after SE was less prominent in ginseng-treated rats than in saline-treated rats. 30 days after SE, the reduction of hippocampal volume was found both in saline-treated and ginseng-treated rats; however, it was less pronounced in ginseng-treated rats. We conclude that administration of ginseng extract after SE termination reduced brain damage caused by prolonged seizures. Ginseng extract was effective during early period after SE and had a specific protective effect on the hippocampus. © 2013 Elsevier B.V. All rights reserved.

Introduction ∗ Corresponding author at: Institute of Higher Nervous Activity and Neurophysiology of RAS, Butlerova St. 5A, Moscow 117485, Russia. Tel.: +7 495 3347000; +7 495 334 26 22; fax: +7 499 7430056. E-mail address: [email protected] (E. Suleymanova).

Status epilepticus (SE) is a dangerous neurological condition that is usually defined as seizures lasting for more than 30 min (Chen and Wasterlain, 2006). SE is considered an emergency and requires immediate therapy. SE can lead to

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224 the development of temporal lobe epilepsy (TLE) later in life (Fujiwara et al., 1979; Loscher and Brandt, 2010). SE can be considered as an insult that induces a variety of pathologic changes including neurodegeneration, axonal sprouting, inflammation, and gliosis. These processes take place during the seizure-free latent period after SE that can last for several years and are believed to underlie epileptogenesis (Loscher and Brandt, 2010; Pitkanen and Lukasiuk, 2009). Pilocarpine and lithium-pilocarpine models are wellestablished experimental models of SE and subsequent TLE. Time course of pathologic processes in this model is similar to human acquired TLE and includes initial insult (SE), seizure-free latent period, and chronic period (Andre et al., 2007; Turski et al., 1989). Prolonged seizures during SE are known to induce neuron death in several subfields of the hippocampus, amygdala, thalamus, cerebellum, piriform, and entorhinal cortex both in humans (DeGiorgio et al., 1992; Fujikawa et al., 2000a) and experimental SE models (Honchar et al., 1983; Treiman, 1990). Neuroprotection is one of promising strategies in prevention or modification of pathologic consequences of SE. Though prevention of neurodegeneration in the hippocampus seems not to be sufficient for prevention of epileptogenesis (Brandt et al., 2006), neuroprotection may be important for attenuation of behavior impairment, learning, and memory deficits, associated with post-SE brain damage, and also reducing of resistance to antiepileptic drugs (AEDs) (Loscher and Brandt, 2010). Thus, searching for new neuroprotective drugs is important. Ginseng is one of the most famous herbs that have been used in Asia for more than 2000 years. Many studies have shown numerous effects of ginseng and its components on brain including neuroprotective and anti-inflammatory effects Glycosides of ginseng called ginsenosides or ginseng saponins are responsible of most pharmacological effects of ginseng (Attele et al., 1999). Neuroprotective activity of whole ginseng extracts and single ginsenosides has been found in vivo and in vitro. Ginseng extract and its components prevented or attenuated neuron death induced by 3-nitropropionic acid (Lian et al., 2005a), kainic acid (KA) (Shin et al., 2009), and glutamate (Li et al., 2010). Ginsenosides Rb1, Re, Rb3, Rg3 decreased brain damage in cerebral ischemia in rodents (Chen et al., 2008; Tian et al., 2005; Ye et al., 2011; Zhu et al., 2012). Besides neuroprotective effect, ginsenosides were shown to have anticonvulsive properties: administration of ginsenosides attenuated seizures induced by KA and pentylenetetrazol (Lee et al., 2002; Lian et al., 2005b), and prevented pentylenetetrazol-induced kindling (Gupta et al., 2001). Pre-treatment with ginsenosides reduced neuron death in hippocampus of KA-treated rats (Lee et al., 2002; Shin et al., 2009). Thus components of ginseng have both anticonvulsant and neuroprotective effect. However, protective effects of ginseng against seizure-induced neuron damage were found either in vitro or in pre-treated animals. To our knowledge, an effect of administration of ginseng after an initial insult, such as SE, on subsequent pathologic changes in brain structures was not investigated. In this study we investigated effects of administration of Panax ginseng root cell culture extract after termination of seizures on brain damage induced by lithium-pilocarpine SE. We used structural and quantitative MRI for assessment of ginseng effects

E. Suleymanova et al. on changes in the rat brain after SE. MRI is a powerful non-invasive tool for assessment of brain damage that allows investigating the temporal evolution of pathological changes in the brain. There is strong evidence that MRI findings can be used for accurate quantifying brain lesions in vivo in brain ischemia, traumatic brain injury, and SE models (Allegrini and Sauer, 1992; Kharatishvili et al., 2009; Niessen et al., 2005; Roch et al., 2002). An increase in T2 -relaxation time is observed in damaged brain tissues after an insult such as ischemia or SE and can indicate irreversible neuron damage (Choy et al., 2010; Ebisu et al., 1996; Fabene et al., 2003; Hoehn-Berlage et al., 1995; Scott et al., 2002). For an objective and detailed analysis of changes in brain tissue after SE, we used quantitative MRI in addition to structural MRI.

Materials and methods Animals Adult male wistar rats (n = 100) weighting 300—350 g at the beginning of experiments were used in this study. All animal experiments were carried out according to EU Directive for animal experiments and approved by the local bioethical committee.

Lithium-pilocarpine model of SE Seizures were induced by administration of 40 mg/kg pilocarpine hydrochloride (Acros Organics, USA). In order to potentiate SE, 127 mg/kg lithium chloride (Acros Organics, USA) was injected 24 h prior the administration of pilocarpine. Seizures were observed and scored using Racine scale (Racine, 1972) for 2 h after the onset of generalized seizures. After that SE was terminated by administration of 0.6 ml/kg paraldehyde (Acros Organics, USA). All drugs were freshly dissolved in 0.9% saline and administered intraperitoneally. Control rats received equivalent volume of saline instead of pilocarpine.

Experimental groups Animals were divided into the following experimental groups: 1. 18 mg/kg ginseng extract treated rats (n = 16). Single injection was made 30 min after SE termination; 2. 180 mg/kg ginseng extract treated rats (n = 8). Single injection was made 30 min after SE termination; 3. VPA-treated rats (n = 12). Single injection was made 30 min after SE termination; 4. saline treated rats, single injection was made 30 min after SE termination (n = 24); 5. rats receiving chronic treatment with ginseng extract during 4 weeks after SE (n = 11); 6. rats receiving chronic treatment with saline during 4 weeks after SE (n = 11); 7. control rats (no SE) (n = 18). Freeze-dried butanol extract of Panax ginseng C.A. Meyer root cell culture DAN-25 was used in the study. This strain

MRI changes after status epilepticus in the rat brain

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Fig. 1 (A) An MR image with ROIs selected by manual thresholding (on the right), ROIs are marked with arrows, and an original image (on the left); (B) ROIs in the rat brain selected for evaluation of T2 -relaxation time. 1—hippocampus; 2—entorhinal cortex; 3—parietal cortex; 4—piriform cortex; 5—amygdala; 6—thalamus.

is characterized by glycoside profile typical for wild ginseng root (Konstantinova et al., 1995) The preparation was provided by BIOKHIMMASH (Moscow, Russia). The concentration of ginsenosides Rb1 , Rb2 , Rc, Rg1 , Re, Rd, Rf was estimated as 50 mg/1 g of dry extract. Freshly dissolved extract was administered intraperitoneally (18 mg/kg or 180 mg/kg). During chronic treatment, bolus injections were administered with 24-h intervals. Valproic acid (VPA) is a conventional AED with neuroprotective properties (Brandt et al., 2006). 150 mg/kg VPA (Convulex injectable 100 mg/ml, Gerot Pharmazeutika, Austria) was administered intraperitoneally 30 min after termination of seizures.

MRI procedure MRI study of the rat brain was performed using Bruker BioSpec 70/30 USR system with 7 T magnetic field (Brucker, Germany). Monitoring of changes in rat brains was performed 7 days prior SE and 2, 7, and 30 days after SE. Animals were anaesthetized with 350 mg/kg (i.p.) chloral hydrate (Sigma) and placed in a cradle equipped with stereotaxic holder and heating system to maintain normal body temperature. The receiving coil was positioned on a rat’s head for detection of MR signal. The cradle was placed in the center of the MR scanner. High-resolution T2 -weighed axial images were acquired using RARE sequence with the following parameters: 6000 ms repetition time (TR), RARE factor 8, 13.7 ms echo time (TE), matrix size 152 × 152, 26 × 26 mm field of view (FOV), 0.5 mm slice thickness, and 20 slices. Duration of acquisition was 3 min 15 s.

Multi-sliced T2 -weighed axial images with 16, 32, 50, 66, 82, 100, 116, 132, 150, 166, 182, 200, 216, 232, 250, and 266 ms TE (T2 -maps) were made to calculate T2 -relaxation time. Image acquisition time was 11 min.

Image analysis High-resolution images were analyzed using ImageJ program (Schneider et al., 2012). To evaluate brain damage after prolonged seizures, total area of regions of interest (ROIs) with signal hyperintensity was calculated. Images were segmented by manual thresholding and brighter pixels were assigned to ROIs; threshold values for ROIs were determined with the help of an image histogram. Examples of thresholded images are shown on Fig. 1A. 12 continuous slices from AP −7.3 mm to AP +4 mm from bregma (Paxinos and Watson, 1998) were analyzed. Results from these slices were combined to compute the total area with signal hyperintensity. To evaluate hippocampal volumes, ROIs on 6 consecutive slices were outlined using standard rat brain atlas (Paxinos and Watson, 1998). The total hippocampal volume was computed by combining the results of these slices. Each hemisphere was analyzed separately. The analysis of T2 -maps was carried out using ParaVision Image Sequence Analysis software (Brucker, Germany). T2 -relaxation time was evaluated in several ROIs including the hippocampus, parietal cortex, entorhinal cortex, piriform cortex, amygdala, and thalamus (Fig. 1B). ROIs were drawn manually on high-resolution images and then cloned to corresponding slices on T2 -maps.

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Fig. 2 High-resolution T2 -weighed images of saline-treated (A) and 18 mg/kg ginseng-treated (B) rat brain before SE (pre SE), on 2, 7, and 30 days after SE. Images at the top row correspond to slices on approximately AP 3.6 mm from bregma; images at the bottom row correspond to slices approximately on AP 5.3 mm from bregma (Paxinos and Watson, 1998) Hip—hippocampus; Pir—piriform cortex; Ent—entorhinal cortex. Areas with signal hyperintensity (bright areas) predominantly in the hippocampus were observed on day 2 after SE; then it became less prominent on day 7. On day 30, a reduction in the hippocampal volume was observed. Brain damage is less prominent in a ginseng-treated rat than in a saline-treated rat on day 2 after SE. Areas with signal hyperintensity in the hippocampus, pirifirm cortex, entorhinal cortex, and cerebral ventricles are marked with arrows.

Analysis of images was carried out by an investigator blinded to the grouping of brains.

Statistical analysis Changes in the total area with signal hyperintensity were analyzed using Friedman test and Wilcoxon test; intergroup differences were evaluated using Kruskal—Wallis test and Mann—Witney U-test with correction for multiple comparisons. Differences in hippocampal volumes and T2 -relaxation times were analyzed with ANOVA for repeated measurements and Newman—Keuls post-hoc analysis.

Results No animals were found to have pre-existing brain abnormalities; there were no significant differences in baseline areas with signal hyperintensity, hippocampal volumes, and T2 values in studied ROIs. Administration of pilocarpine induced generalized convulsive SE with clonic, tonic, and tonic-clonic seizures. The analysis of severity of seizures showed that there were no intergroup differences in seizure scores.

Single administration of ginseng extract attenuated early MRI changes in rat brain after SE MRI study of rat brains on day 2 after SE showed the presence of signal hyperintensity in the hippocampus, entorhinal cortex, piriform cortex, amygdala, and in some rats in the neocortex. We calculated the total area of these regions. On day 7 areas with signal hyperintensity decreased and included mostly the hippocampus and cerebral ventricles. On day 30 an increased signal was received mostly from cerebral ventricles (Fig. 2A). Control rats that did not receive pilocarpine and did not have seizures showed no significant changes in areas with signal hyperintensity throughout the experiment. In these animals an increased signal was received mostly from cerebral ventricles. 18 mg/kg ginseng-treated rats had less pronounced increase in MRI signal in hippocampus than saline-treated rats on day 2 after SE (Fig. 2B). Most animals that received 180 mg/kg ginseng extract had areas with hyperintense signal in neocortex on day 2 and day 7 after SE. Only 3 rats survived by the end of the study, so this group was excluded from further statistical analysis due to insufficient number of animals. Data on 180 mg/kg ginseng-treated rats are not shown.

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Fig. 3 Total areas with hyperintense MR signal found on brain scans in control rats (no SE) and saline-, 18 mg/kg (gin SE), and VPA-treated (VPA SE) rats before SE (baseline), on 2, 7, and 30 days after SE. Areas with hyperintense signal were smaller in 18 mg/kg ginseng-treated rats than in saline-treated rats on day 2 and 7 after SE (*p < 0.05, Kruskal—Wallis test). # p < 0.05; ## p < 0.005 (Wilcoxon test, in comparison with baseline). Data shown as mean ±sem.

Total areas with signal hyperintensity in rat brains before SE and on day 2, day 7, and day 30 after SE are shown on Fig. 3. The analysis of temporal dynamics of such areas in rats treated with saline, ginseng extract, and VPA showed the increase in the total area with signal hyperintensity on day 2 after SE in comparison with pre-SE values. On day 7 such increase was found in saline and VPA-treated rats but not in 18 mg/kg ginseng-treated rats (Friedman test, posthoc Wilcoxon test, p < 0.005, p < 0.05). The analysis of intergroup differences between salinetreated, ginseng-treated, and VPA-treated rats showed that total brain areas with hyperintense signal were smaller in 18 mg/kg ginseng-treated rats than in saline-treated rats on day 2 and day 7 after SE (Kruskal—Wallis test, p < 0.05). VPA-treated rats also had smaller damaged areas in brain in comparison with saline-treated rats, however this difference was not statistically significant. There were no significant intergroup differences in areas with hyperintense signal on day 30 after SE.

Hippocampal volumes after SE Changes in hippocampal volumes were similar in all rats after SE. Hippocampal volumes increased in saline-treated, ginseng-treated, and VPA-treated rats in comparison with control rats on day 2 after SE. Hippocampal volumes were increased by 24, 21, and 31% in comparison with pre SE values in saline-, 18 mg/kg, and VPA-treated rats respectively. On day 7 hippocampal volumes reversed to normal and did not differ from control rats. A significant decrease in hippocampal volumes was found in saline, 18 mg/kg ginseng-, and VPA-treated rats on day 30 after SE. The analysis of changes in hippocampal volumes on day 30 in comparison with pre SE showed that in saline-, 18 mg/kg, and VPAtreated rats the hippocampus decreased by 18, 9, and 21% respectively. Hippocampal volumes of control rats (no SE) did not change on days 2 and 7 and slightly increased by the end of the experiment (Fig. 4).

T2 -relaxation time in rat brain structures after SE Evaluation of T2 -relaxation time was performed in several ROIs including the hippocampus, entorhinal cortex, piriform

Fig. 4 Change in hippocampal volumes of control rats (no SE), saline-treated (saline SE), 18 mg/kg ginseng-treated (gin 18 SE), and VPA-treated (VPA SE) rats on day 2, 7, and 30 after lithiumpilocarpine SE. A significant increase in hippocampal volumes was found on day 2 by 24, 21, and 31% in comparison with pre SE values in saline-, 18 mg/kg, and VPA-treated rats respectively. On day 30 hippocampal volumes decreased by 18% and 21% in saline- and VPA-treated rats respectively, but only by 9% in 18 mg/kg ginseng-treated rats, *p < 0.05.

cortex, amygdala, thalamus, and parietal cortex. A significant increase in T2 values was found in the hippocampus, entorhinal cortex, piriform cortex, amygdala, thalamus on day 2 after SE; on day 7 and 30 slight increase in T2 was found in the hippocampus of saline-treated rats. Though all rats demonstrated a highly significant increase of T2 -relaxation time in studied ROIs, except the parietal cortex, in comparison with its values in control rats and pre-SE values, the increase in T2 in the hippocampus was significantly less prominent in 18 mg/kg ginseng-treated rats than in saline-treated rats. There were no differences in T2 values in the other studied structures between rats that received ginseng extract or saline (Fig. 5). Thus administration of 18 mg/kg ginseng extract after SE reduced the development of the early increase of T2 -relaxation time

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Fig. 5 T2 -relaxation time in the hippocampus, entorhinal cortex, piriform cortex, amygdala, thalamus, and parietal cortex before SE (baseline), on day 2, 7, and 30 after SE in saline-treated (saline SE), 18 mg/kg ginseng treated (gin 18 SE), and control rats (no SE). Data are shown as mean ± standard error, *p < 0.05.

specifically in the hippocampus, but not in the other studied limbic structures. Interestingly T2 was increased in the parietal cortex of 180 mg/kg ginseng-treated rats on day 2 and day 7 after SE (data not shown). There was no such increase in the parietal cortex of other rats after SE.

Chronic administration of ginseng extract attenuates early brain damage after SE and does not prevent the reduction of the hippocampal volume MRI study of brains of rats receiving either 18 mg/kg of ginseng extract or the equivalent volume of saline daily for 4 weeks starting from the day of SE showed that 7 days

after SE there was hyperintense signal predominantly in the hippocampus, cerebral ventricles, and neocortex. Areas with increased MR signal measured in rat brains 7 days after SE were smaller in ginseng-treated rats than in saline-treated rats. Areas with hyperintense signal in brains of saline-treated rats were significantly larger than in control rats, while such regions in brains of ginseng-treated rats did not differ from controls (Fig. 6). 30 days after SE regions with hyperintense signal mostly included dilated cerebral ventricles. There were no significant intergroup differences; however, these areas tended to be larger in rats receiving saline than in control rats and rats receiving ginseng extract. 30 days after SE, there were rats with the reduced hippocampal volume both in groups receiving saline and ginseng extract in comparison with their hippocampal volumes on

MRI changes after status epilepticus in the rat brain

Fig. 6 The total area with signal hyperintensity in rats after SE. Rats were daily administered either 18 mg/kg ginseng extract (gin SE) or saline (saline SE) during 4 weeks after SE, *p < 0.05, Mann—Witney, in comparison with control rats (no SE).

day 7 and control rats. Reduction of the hippocampus was highly significant in rats receiving saline (p = 0.001); while it was statistically insignificant in rats receiving ginseng extract (p = 0.13). The analysis of individual changes in the hippocampus of rats after SE showed that the hippocampal volume was reduced in all saline-treated rats by more than 10%, but such reduction was found only in 3 of 7 (43%) ginseng-treated rats.

Discussion Our main finding is that administration of a relatively low dose (18 mg/kg) of ginseng extract after SE decreased severity of early transient changes found in the rat brain on day 2 after SE. Administration of 18 mg/kg ginseng extract reduced early changes in brain structures found on highresolution T2 -weighed images and partially prevented an increase in T2 -relaxation time in the hippocampus in comparison with saline-treated rats. At the same time, an increase in T2 -relaxation time was found in the parietal cortex of 180 mg/kg ginseng-treated rats. The higher dose of 180 mg/kg of ginseng extract also caused an increase in death rate that could be due to toxic effects of high dose of crude ginseng extract. The progressive brain edema could be the cause of the increased mortality rate in this group of rats. Despite widespread use of ginseng and large number of studies of ginseng effects, there are few studies of ginseng toxicity. In humans ginseng abuse syndrome has been described: long-term use of products containing ginseng induced hypertension, nervousness, euphoria, insomnia, diarrhea, skin eruptions, and edema (Siegel, 1979). Chronic treatment with root extract has been reported to have no toxic effects in animals in several studies (Bittles et al., 1979; Chan et al., 2011; Hess et al., 1983). LD50 of ginseng root extract in rodents is high and ranges from 1000 to 3000 mg/kg (Brekhman and Dardymov, 1969; Takagi et al., 1972); so 180 mg/kg dose was not expected to be toxic. However, pre-treatment with high doses of ginseng root extract (600, 450 and 150 mg/kg) was also found to increase the mortality rate of rats after pilocarpine-induced SE (Lian et al., 2005b). Apparently rats after SE are more susceptible to ginseng toxicity.

229 We also found that administration of 18 mg/kg ginseng extract attenuated the increase in T2 -relaxation time in the hippocampus, but not in the piriform and entorhinal cortex, thalamus, and amygdala on day 2 after SE. Thus active components of ginseng extract had an effect specifically on the hippocampus but not on the other studied limbic structures. The transient increase in signal on T2 -weighed images and T2 -relaxation time in the hippocampus and associated structures on day 2 after SE resolved, at least partially, by day 7 after SE. These data on MRI changes in the rat brain after prolonged seizures are in agreement with findings of other authors (Andre et al., 2007; Nairismagi et al., 2004; Roch et al., 2002). The reverse of MRI changes was found by day 7 in saline-, ginseng-, and VPA-treated rats, however some residual increase in the MR signal was found in the hippocampus. Both single and chronic administration of ginseng extract attenuated changes in the hippocampus found on day 7 in comparison with saline- and VPA-treated rats; and chronic daily treatment caused the significant decrease in total brain damage in comparison with salinetreated rats. These results indicate that administration of ginseng extract during acute and latent period after SE could lead to faster reverse of early pathologic changes in rat brain. SE is believed to be a trigger initiating a cascade of processes including neurodegeneration, inflammation, and an increase in blood-brain barrier permeability (Pitkanen and Lukasiuk, 2009). These processes can result in the development of brain edema and an increase of brain tissue water content that cause the increase in T2 -relaxation time and the appearance of hyperintense signal in limbic structures. Our findings suggest that active components of ginseng extract are capable of reducing the development of these processes particularly in the hippocampus during the initial days after SE. A significant reduction in the hippocampal volume took place 30 days after SE which is consistent with other studies (Andre et al., 2007; Chan et al., 2011; Nairismagi et al., 2004; Roch et al., 2002). Pilocarpine and lithium-pilocarpine SE is known to induce massive neuron death in limbic structures including hippocampus and parahippocampal region, amygdala, and thalamus (Fujikawa et al., 2000b). Hippocampal volumes correlate well with neuron count in CA1 and CA3 subfields of the hippocampus (Choy et al., 2010; Fabene et al., 2003). Therefore the reduction of the hippocampal volume can be an evidence of neuron loss in the hippocampus. Both single and chronic administration of ginseng extract did not completely prevent the reduction of hippocampal volumes in rats 30 days after SE; however, hippocampal reduction was less prominent in the group of ginseng-treated rats than in saline-treated rats. Since chronic treatment with ginseng extract during 30 days after SE did not improve the outcome of SE in comparison with single treatment 30 min after SE, and did not prevent further development of brain lesions, we assume that ginseng extract components can be effective during early period after SE. There is evidence that early MRI changes in brain after SE such as increase in T2 -relaxation time correlates with subsequent hippocampal reduction and hippocampal neuron loss (Choy et al., 2010). Therefore the attenuation of hippocampal reduction in single and chronically treated with ginseng extract rats can be attributed to the effect of

230 ginseng components on early processes taking place during the initial days after SE. At the same time, single administration of VPA did not significantly attenuate early transient MRI changes and later decrease of hippocampal volumes. VPA protected the hippocampus from SE-induced neurodegeneration if administered at high dose or after chronic treatment (Brandt et al., 2006). In contrast to ginseng extract, single low-dose treatment with VPA could be insufficient for neuroprotection. In this study an extract of ginseng root cell culture containing ginsenosides, oligo, and polysaccharides was used; so the mechanisms of its protective effect remains unknown. Usually most effects of ginseng extract are attributed to ginseng saponins ginsenosides (Attele et al., 1999). Neuroprotective effects of ginsenosides may be carried out via several mechanisms. Ginsenosides, particularly ginsenoside Rg3, were found to diminish NMDA-dependent Ca2+ influx in cultured hippocampal neurons via a competitive interaction with the glycine-binding site of NMDA receptors thus attenuating NMDA-mediated neurotoxicity (Kim et al., 2004). At the same time, excessive glutamate-mediated activation of NMDA receptors during SE is considered to be an important pathway of neuron death induced by prolonged seizures (Deshpande et al., 2008; Fujikawa et al., 2000b). Ginsenosides also inhibited SE-induced Ca2+ influx and subsequent development of spontaneous recurrent epileptiform discharges (SREDs) and further neuron death in cultured neurons (Kim and Rhim, 2004). Thus ginsenosides could partly prevent the development of early pathologic changes in the rat hippocampus by attenuating activation of NMDA receptors and subsequent neuron death. Ginsenosides could also reduce further changes by attenuating SE-induced SREDs. Ginsenoside Rb1 was shown to selectively inhibit the activity of L-type Ca2+ channels in hippocampal neurons (Lin et al., 2012). Inhibitors of these channels have neuroprotective effects that are possibly carried out via their anti-inflammatory properties (Hashioka et al., 2012; Liu et al., 2011). Since Rb1 is one of the main components of DAN-25 Panax ginseng extract, the reduction in early changes observed on MRI after SE could occur due to attenuation of inflammation by Rb1, however further studies are needed to confirm this assumption.

Conclusion We conclude that administration of ginseng extract after SE termination reduced brain damage caused by prolonged seizures. Our data suggest that ginseng extract is effective during early period after SE; its active components have a specific protective effect on hippocampus. Both single and chronic treatment with ginseng extract attenuated hippocampal reduction. Protective effect of ginseng extract can be attributed to ginsenosides, but further studies with purified ginsenosides are necessary to determine the effective components and their mechanisms of action.

Acknowledgement We thank Dr. Lyudmila Vinogradova for her help with editing of this manuscript.

E. Suleymanova et al.

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Ginseng extract attenuates early MRI changes after status epilepticus and decreases subsequent reduction of hippocampal volume in the rat brain.

Prolonged epileptic seizures during status epilepticus (SE) are known to cause neuronal death and lead to brain damage. Lesions in various brain regio...
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