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. 2011 Nov 11;32(2):267–277. doi: 10.1007/s10571-011-9758-2

EGCG Ameliorates the Suppression of Long-Term Potentiation Induced by Ischemia at the Schaffer Collateral-CA1 Synapse in the Rat

Jie Ding 1, Gang Fu 1, Yan Zhao 1, Zhenyong Cheng 1, Yang Chen 1, Bo Zhao 1, Wei He 1, Lian-Jun Guo 1,✉
PMCID: PMC11498421  PMID: 22076575

Abstract

The function of Epigallocatechin gallate (EGCG), a main component of green tea, has been widely investigated, amelioration of synaptic transmission and neuroprotective effects against ischemia-induced brain damage among others. However, the mechanism underlying is still unveiled. We investigated the effects of EGCG on high frequency stimulation-induced long-term potentiation (LTP) in the Schaffer collateral-CA1 synapse with or without cerebral ischemia injury induced by middle cerebral artery occlusion (MCAO) in vivo to examine the possible relations between EGCG and synaptic transmission. Application of EGCG modulated synaptic transmission and produced a dose-dependent improvement of the induction of LTP. However, relative high-dose EGCG can block the induction of LTP at the Schaffer collateral-CA1 synapse in normal rat in vivo. In addition, the effects of EGCG were observed on the infarct volume and neurological deficit in rats subjected to MCAO; furthermore, the cell viability of primary cultured rat hippocampal and cortical neurons suffered from oxygen–glucose deprivation were evaluated with MTT and LDH assay, which showed significant neuroprotective properties in vitro. Surprisingly, the contents of the glutamate (Glu), glycine (Gly), and gamma-aminobutyric acid amino acids were totally disequilibrated before and after cerebral ischemia injury and could be rebalanced to original level by application of EGCG. Our results suggest that EGCG is able to improve the efficiency of synaptic transmission in cerebral ischemia injury with attenuated effect related to the neuroprotection of EGCG through regulating excitatory and inhibitory amino acid balance.

Keywords: (−)-Epigallocatechin gallate, Middle cerebral artery occlusion, Long-term potentiation, Amino acid

Introduction

Recently, it has been generally accepted that ischemia stroke, which develops early neurological deterioration, is a common cause of death in many countries (Macleod et al. 2005; Thanvi et al. 2008). Cerebral ischemia reperfusion injury is a recognized complication of restoring blood flow to ischemic brain tissue (Cheng et al. 2011; Hallenbeck and Dutka 1990), which can incur the release of proteases and the formation of free radicals. This process also accompanies with damage of hippocampus and long-term functional disability of synaptic transmission (Di Filippo et al. 2008) and produces learning and memory deficits (Patil et al. 2006; Sun et al. 2009). Since electrophysiological changes of long-term potentiation (LTP) in function of neurones in the vicinity (Mittmann et al. 1998) and remote from the primary stroke area has been demonstrated that the hippocampus is believed to participate in spatial learning, one can attribute the observed deficits to its dysfunction. Physiological activity-dependent long-term changes in synaptic transmission, as LTP are an attractive model to study cellular mechanisms, are thought to be the substrate of learning and memory.

As we know, green tea, due to its beneficial effects on health, is an extremely popular beverage worldwide. (−)-Epigallocatechin gallate (EGCG) is the major active polyphenol of green tea and primarily responsible for the green tea biological effect (Bae et al. 2002; Yamashita et al. 2000). EGCG has been demonstrated to display potent antioxidant properties (Jung and Ellis 2001; Jung et al. 2001), has been implicated in limiting neuronal loss, and also has been associated with various neurological benefits including neuroprotective effects and cognitive improvement (Kang et al. 2010; Zhang et al. 2008). The beneficial effects may result from EGCG’s antioxidant activity reducing intracellular reactive oxygen species and their detrimental consequences (Higdon and Frei 2003). The beneficial effects may also result from direct interactions with various cellular signaling pathways (Mondaca et al. 2004). Despite its potential neuropharmacological benefits, a connection between EGCG and neuronal activity in learning and memory is still lacking. Previous studies showed that EGCG could inhibit the amplitudes of population spikes (PSs) in the perforant path-CA3 region in rat hippocampus and suppress the LTP induction in this region (Yin et al. 2008). However, little is known about the effect of EGCG on the induction of LTP at the Schaffer collateral-CA1 synapse.

In this study, we investigated the effect of EGCG on high frequency stimulation (HFS)-induced LTP in the Schaffer collateral-CA1 synapse in a rat cerebral ischemia reperfusion model and its potential mechanism of action.

Materials and Methods

Chemicals

EGCG (molecular weight: 458.4, purity > 95%), MTT, and poly-lysine were from Sigma (St. Louis, MO, USA). Modified Dulbecco’s Eagle’s medium/Nutrient Mixture F-12 Ham’s (DMEM/F-12) and B-27 were obtained from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Fetal bovine serum (FBS) was from GIBCO Life Technologies. Sodium hydrosulfite was obtained from Lijing Chemical Factory (Tianjin, China). LDH assay kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). Mobile phase used in HPLC was filtered using a 0.2 μm membrane filter (Eilite Analytical Instruments Co, Dalian, China). All other reagents were of analytical grade and purchased from Shanghai chemical engineering (Shanghai, China).

Animals

Adult male Sprague–Dawley rats weighing 200–250 g were purchased from Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology. All animals were maintained on a 12-h light/dark cycle and had free access to food and water and adapted to these conditions for at least 7 days before experiments. All rats were randomly divided into groups of sham, ischemia, and ischemia treated with EGCG (7.5 and 15 mg/kg). The sham group was treated with saline only without induction of ischemia. The ischemia group was treated with either saline or the drug (EGCG, 7.5 and 15 mg/kg) by intravenous injection (i.v.) after ischemia. All experiments were performed in accordance with the Guidelines of the Care and Use of Laboratory Animals of Tongji Medical College, Huazhong University of Science and Technology. Efforts were made to minimize animal suffering and to reduce the number of subjects used.

Electrophysiological Recordings In Vivo

Extracellular recordings were described in detail previously (He et al. 2008, 2010). Animals were anesthetized with urethane (1.4 g/kg, i.p.), and the body temperature was maintained at 37°C via a constant temperature heating pad. The head was mounted in a stereotaxic frame (SN-3, Narishige, Japan), and both the skin and fascia were retracted to expose the skull under sterile conditions. The tissue was kept moist with gauze moistened with sterile saline throughout the surgical procedures. For i.v. or intracerebroventricular (i.c.v.) administration of drugs, a stainless steel cylindrical cannula (0.7 mm outer diameter) was stereotaxically inserted into the right hemisphere lateral ventricle (0.8 mm posterior to bregma, 1.5 mm lateral to midline, and 3.5 mm ventral to dura). The amplitude of the PS height was measured. Stimulation was achieved with pulses of 150 μs duration delivered at 0.1 Hz (10 s intervals), and evoked field responses were acquired, amplified, monitored, and analyzed with SMUP-PC biology signal processing system (Second Military Medical University, China). For recordings of the amplitude of the PS, the stimulation intensity for the tests and train pulses was set to elicit a response for which the PS amplitude was 50–60% of the maximum response so as to allow increases or decreases of the PS amplitude to be detected. Each recording consisted of an average of 10 consecutive pulses at 10 s interval, and the averaged responses were measured every 5 min throughout the experiment. The LTP of the Schaffer collateral-CA1 synaptic response was induced by HFS (HFS: 4 trains of 50 pulses, 100 Hz, 150 μs duration, and inter-train interval of 10 s) at the test stimulus intensity. After tetanus stimulation, the amplitude of the PS was recorded for at least 120 min. The averaged PS amplitude of five different time points within 30 min before HFS was determined as the baseline value. The percentage of the ratio of absolute PS amplitude to baseline value was used to represent the PS amplitude level. It was defined as a successful induction of LTP if the amplitude of PS change exceeded 20% (Bliss and Collingridge 1993).

MCAO Surgery and Neurological Deficit Evaluation

Rats were anesthetized using 10% chloral hydrate (350 mg/kg, i.p.). The middle cerebral artery was occluded with a 4-0 silicone-coated nylon suture by surgical operation (Li et al. 2009; Longa et al. 1989). Reperfusion was induced after 2 h MCAO by filament withdrawal. Sham-operated animals were subjected to the same surgical procedure, but the suture was not advanced beyond the internal carotid bifurcation. During the surgery, their rectal temperature was monitored and maintained at 37.5 ± 0.5°C. After revival from anesthesia, animals were housed back at room temperature 22 ± 1°C with free access to food and water. Neurological behavior assessment was performed 2 h before MCAO and 3 h, and 24 h after ischemia, and scored on a 6-point scale (Huang et al. 2009a, b; Longa et al. 1989): 0, no neurological deficit; 1, failure to extend left forepaw fully; 2, circling to the left; 3, inability to bear weight on the left; 4, no spontaneous walking with depressed level of consciousness; and 5, death.

Cerebral Infarct Size Measurement

Cerebral infarct size was assessed with 2,3,5-Triphenyltetrazolium chloride (TTC) staining method. After 2 h MCAO followed by 24 h reperfusion, the animals were anesthetized, and the brains were quickly isolated and sectioned into consecutive 2-mm thick coronal slices using a Vibratome (Campden Instruments, USA). Slices were immediately immersed in 2% TTC medium at 37°C for 30 min. Stained slices were washed with phosphate buffer saline (PBS) for 5 min and fixed in buffered formaldehyde solution for 24 h. Then color image of these slices were captured using a video camera (Olympus, Japan). The infarct size of all brain slices of each animal were analyzed using the Image-Pro plus 5.0 analysis software (Tseng and O’Donnell 2004). Percentage of infarct size was calculated as described (Swanson et al. 1990): [(VC − VL)/VC] × 100%, VC is the volume of control hemisphere and VL the volume of non-infarcted tissue in the lesioned hemisphere.

Cell Culture and Drug Treatment

Primary cultures of hippocampal and cortical neurons were prepared as described previously (Huang et al. 2009a, b). In brief, hippocampus and cortex were dissected from the brains of neonatal Sprague–Dawley rats (day 0–2) and rinsed in ice cold dissection buffer. Brain tissues were treated with 0.125% trypsin in Hanks’ balanced salt solution for 15 min at 37°C and mechanically dissociated using a pipette. Cell suspension was then centrifuged at 1,000 rpm for 10 min for twice, and the cell pellets were resuspended in the DMEM/F-12 with 20% FBS, 100 U/ml penicillin, and 100 mg/l streptomycin. Cells were seeded at a density of 1–5 × 105/ml in 96-well plates pre-coated with poly-l-lysine and kept at 37°C in a 5% CO2 incubator (SHELLAB, Oregon, USA). After 24 h, the culture medium was changed to fresh medium and 5% B-27 supplement (Gibco, USA) and then changed for every 2 days. 10 mg/l arabinosylcytosine was added at 24 h to prevent the growth of non-neuronal cells. All experiments were performed at 6–8 days after primary cells plated.

Oxygen–Glucose Deprivation (OGD) of Primary Cultures of Hippocampal and Cortical Neurons

For OGD model, cells were washed by glucose-free Earle’s solution (mM: NaCl 143, KCl 5.4, CaCl2 1.8, MgSO4 1.0, NaH2PO4 1.0, HEPES 2.4, pH 7.3) for twice. Subsequently, cells were incubated in glucose-free Earle’s solution with sodium hydrosulfite 2 mM at 37°C in the CO2 incubator for 4 h (Huang et al. 2009a, b; Liu et al. 1998). Cells were pre-treated with 25, 50, and 100 μM EGCG for 24 h before OGD and continually treated with EGCG during 4 h OGD insult. After 4 h OGD insult, the cells were used for MTT test, and the culture medium was collected for enzyme assay and amino acid analysis.

MTT Reduction and LDH Release Assays

Cell viability was evaluated by MTT reduction assay. After 4 h OGD injury, culture medium in 96-well plates were incubated with MTT (0.5 g/l, 200 μl per well) at 37°C for 4 h. Then, the blue formazan accumulated in living cells was dissolved in 120 μl DMSO. The formazan was quantified by optical density (OD) measured at 570 nm with 650 nm as the reference filter with a microplate reader (Tecan Sunrise, Switzerland). LDH is released from cells when the cells are injured, so that LDH in the medium is an indicator to the integrity of cell membrane. LDH activity in the medium was measured according to the direction of LDH assay kit as previously described (He et al. 2009; Huang et al. 2009a, b; Koh and Choi 1987). The values of absorbance were read at 440 nm, and the results of the test wells were expressed as percent of the control wells.

Quantification of Amino Acids in Extracellular Fluid Determined by HPLC

After 4 h OGD insult, the extracellular fluid was collected and frozen at −75°C. Concentration of excitability amino acid and inhibitory amino acids were determined by HPLC with fluorescence detection (HITACHI, L2000, Japan) after automatic precolumn derivation with o-phthaldialdehyde as described previously (He et al. 2009). The derivatization of samples and standards was carried out in an autosampler (Prostar, VARIAN, Holland). Derivatives were separated on a Kromasil ODS2 C18 column (Kromasil, AKZONOBEL, Switzerland). The mobile phase gradient elution system consisted of Buffer A: 0.04 M phosphate buffered solution (PBS), pH 6.8, and Buffer B: pure methanol, flow rate 1 ml/min. Data were collected and analyzed using the ANASTAR software. External standard method was used to quantify the concentrations of amino acids according to each peak area. The results of amino acids concentrations of the test samples were expressed as percent of the control samples.

Statistical Analysis

Data were presented as mean ± SD. Statistical significance between the multiple groups, except the neurological score, were determined using one-way analysis of variance (ANOVA) followed by the Fisher LSD test (Least-significant difference). Neurological deficit scores were analyzed by Kruskal–Wallis test followed by the Dunn test (multiple comparisons). Differences at the P < 0.05 level were considered as statistically significant.

Results

Effects of EGCG on Normal SD Rats at the Schaffer collateral-CA1 Synapse In Vivo

In the first series of experiments, we determined whether EGCG regulates the induction of LTP at the Schaffer collateral-CA1 synapse of normal SD rats in vivo. As shown in Fig. 1, application of HFS resulted in a marked increase in the amplitude of PS at Schaffer collateral fiber-CA1 synapses in normal saline-treated rats during 120 min recording period (PS amplitude was 168.20 ± 3.54% of baseline values at 10 min, 171.71 ± 5.71% of baseline values at 60 min, 159.64 ± 3.78% of baseline values at 120 min after HFS application, respectively, P < 0.01 compared with baseline values, n = 5). I.c.v. administration of 12.5 and 25 μg EGCG significantly enhanced the induction of HFS-induced LTP. As we can investigate in Fig. 1b, at 10 min after HFS stimulation, the PS amplitude of administration of 12.5 and 25 μg EGCG was increased to 193.44 ± 4.47% (n = 5) and 201.28 ± 4.46% (n = 5) of baseline values, respectively (P < 0.05 compared with normal saline group). At 60 min after HFS stimulation, the PS amplitude was 197.04 ± 2.98% (n = 5) and 200.85 ± 6.45% (n = 5) of baseline, values, respectively (P < 0.05 compared with normal saline group). At 120 min after HFS stimulation, the PS amplitude was 189.75 ± 3.59% (n = 5) and 190.92 ± 5.48% (n = 5) of baseline values, respectively (P < 0.05 compared with normal saline group). These results suggest that the amelioration of EGCG was maintained without declining throughout 120 min recording period at the Schaffer collateral-CA1 synapse in normal rat hippocampus in vivo. However, when we administrated higher amount of EGCG, an opposite effect was observed. As shown in Fig. 1b, c, i.c.v. administration of 50 and 100 μg EGCG significantly inhibited the induction of HFS-induced LTP and this inhibitory effect was also maintained without declining throughout the recording period. (PS amplitude was 140.70 ± 0.96% (n = 5) and 134.46 ± 1.91% (n = 5) of baseline values at 10 min, 132.42 ± 2.71% (n = 5) and 132.09 ± 3.03% (n = 5) of baseline values at 60 min, 131.12 ± 2.60% (n = 5) and 126.19 ± 1.35% (n = 5) of baseline values at 120 min after HFS application, respectively, P < 0.05 compared with normal saline group) These results indicate that higher amount EGCG can block the induction of LTP at the Schaffer collateral-CA1 synapse in normal rat hippocampus in vivo. Moreover, these results also imply that the property consumption of tea is beneficial to health, but not excessive.

Fig. 1.

Fig. 1

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In vivo effects of EGCG on LTP of hippocampal CA1 region in normal rats at the Schaffer collateral-CA1 synapse. a Representative PS recorded before (left) and after (right) application of HFS. b Normal saline and EGCG (12.5, 25, 50, and 100 μg n = 5) was i.c.v. administered. The linear graph illustrates the relative PS amplitude alteration at different time points within the 120 min after HFS. Application of low-dose EGCG (12.5 and 25 μg) significantly facilitate LTP and without decline throughout 120 min recording period. However, relative high-dose EGCG will block the induction of LTP at the Schaffer collateral-CA1 synapse in normal rat. c The evoked synaptic responses were summarized by calculating the average of PS amplitude 5–120 min after HFS. * P < 0.05 compared with normal saline group. All data are expressed as means ± SD

Effects of EGCG on the Ischemia-Induced Reduction of LTP at the Schaffer Collateral-CA1 Synapse In Vivo

To determine whether EGCG can modulate the cerebral ischemia injury induced suppression of LTP at the Schaffer collateral-CA1 synapse in vivo, we use the middle cerebral artery occlusion (MCAO) as an ischemic model, which is usually used to reproduce the pattern of ischemia brain damage (Ginsberg and Busto 1989; Li et al. 2009). As shown in Fig. 2, the induction rate of LTP in the ischemia group (71.38%) was much lower than that in sham-operated group (100%). EGCG (7.5 mg/kg) has no significant effect compared with ischemia group. Relative high-dose (15 mg/kg) of EGCG treatment (120 min) can significantly raise LTP from 71.38% in ischemic group to 87.58% (P < 0.05). More interestingly, this increase maintained in a certain level within the recording time. At 10, 60, and 120 min after HFS stimulation, the PS amplitude of administration of 15 mg/kg EGCG was increased to 155.64 ± 6.07, 140.05 ± 3.20, and 144.05 ± 4.32% (n = 5) of baseline values, respectively, P < 0.05, as shown in Fig. 2b. These results suggest that LTP was inhibited following MCAO and EGCG treatment (15 mg/kg) can restore the suppressed LTP. Furthermore, the improvement effect of EGCG was maintained without declining throughout 120 min recording period (see Fig. 2b).

Fig. 2.

Fig. 2

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In vivo effects of EGCG on LTP of hippocampal CA1 region in rats at the Schaffer collateral-CA1 synapse with cerebral ischemia injury after MCAO. a Representative PS recorded before (left) and after (right) application of HFS. b Sham, ischemia, and EGCG (7.5 and 15 mg/kg n = 5) was i.v. administered. The linear graph illustrates the relative PS amplitude alteration at different time points within the 120 min after HFS. Application of EGCG significantly attenuated cerebral ischemia injury induced suppression of HFS-induced LTP at the Schaffer collateral-CA1 synapse and the ability of EGCG to modulate the induction of LTP was dose-dependent. c The evoked synaptic responses were summarized by calculating the average of PS amplitude 5–120 min after HFS. Sham group includes rats without MCAO, ischemia group was NS-treated rats with MCAO, and the EGCG-treated groups were administrated with EGCG (7.5 and 15 mg/kg i.v.) before and after MCAO, respectively. \ P < 0.001 compared with sham group; ## P < 0.01 compared with ischemia group. All data are expressed as means ± SD

Effects of EGCG on Cerebral Infarct Size and Neurological Deficit Score In Vivo

To further investigate the effect of EGCG on improving the decline of learning and memory induced by cerebral ischemia, representative consecutive 2-mm thick coronal slices stained with 2% TTC from one sample of sham, ischemia, and EGCG-treated (7.5 and 15 mg/kg) groups respectively were presented in Fig. 3a. In EGCG-treated group, the rats were administered with EGCG (7.5 and 15 mg/kg, i.v.) 30 min before and immediately after MCAO, whereas the ischemia group was given the same volume of saline. 7.5 mg/kg EGCG treatment had no significant effect on improving cerebral infarct size and neurological deficit score after 2 h MCAO and 24 h reperfusion, however, 15 mg/kg EGCG treatment could significantly decrease cerebral infarct size (Fig. 3b) and improve neurological deficit (Fig. 3c), compared with ischemia group (P < 0.05).

Fig. 3.

Fig. 3

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In vivo effects of EGCG on cerebral infarct size and neurological score after 2 h MCAO and 24 h reperfusion (n = 5). a Representative brain sections stained with 2% TTC from sham, ischemia, EGCG-treated (7.5 and 15 mg/kg) groups, respectively. b Quantitative analysis of cerebral infarct size from ischemia, EGCG-treated 7.5 mg/kg, P > 0.05 compared with ischemia groups and EGCG-treated 15 mg/kg groups, # P < 0.05 compared with ischemia groups. c Neurological deficit score was measured at 3 and 24 h after MCAO, 24 h EGCG-treated 15 mg/kg group, # P < 0.05 compared with ischemia groups. Sham group includes rats without MCAO, ischemia group was NS-treated rats with MCAO, and the EGCG-treated groups were administrated with EGCG (7.5 and 15 mg/kg i.v.) before and after MCAO, respectively. All data are expressed as means ± SD

Effects of EGCG on OGD-Induced Hippocampal and Cortical Neurons Injury

As shown above, EGCG can attenuate cerebral ischemia injury induced suppression of LTP at the Schaffer collateral-CA1 synapse in vivo and decrease cerebral infarct size, further improving neurological deficit.

We used cultured neurons suffering from OGD, which can mimic oxygen and glucose reduction as ischemic model for pathophysiological investigation in vitro. In cultured hippocampal and cortical neurons, marked morphological changes were visualized after 4 h OGD and pre-treatment with EGCG reduced these pathological changes (Fig. 4a). The viability of hippocampal and cortical neurons were estimated by the MTT assays. The viability of hippocampal and cortical neurons were significantly decreased after 4 h OGD insult, with the OD values of cells lower than the normal cultured neurons (P < 0.01). Low-dose EGCG (25 and 50 μM) pre-treatment markedly increased the cell viability in a concentration-dependent manner. Surprisingly, high-dose EGCG (100 μM) had no statistical effect compared with OGD group (Fig. 4b). Moreover, pre-treatment with low-dose EGCG (25 and 50 μM) can also concentration-dependent prevent OGD-induced LDH efflux. However, high-dose EGCG (100 μM) could also attenuate LDH leakage but had less effect compared with low-dose EGCG (Fig. 4c). These results indicated the cell viability of primary cultured rat hippocampal and cortical neurons depressed by exposed to 4 h OGD were improved by pre-treated EGCG at appropriate concentrations, but not excessive, which showed significant neuroprotective properties of EGCG.

Fig. 4.

Fig. 4

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In vitro effects of EGCG on cells morphologic changes, MTT, and LDH activity induced by 4 h OGD in hippocampal neurons and cortical neurons of rats (n = 5). a Marked morphological changes were visualized in 4 h OGD group, and EGCG 25 μM reduced the pathological changes. Magnification is ×250 and ×500, respectively. b MTT assay showing the effect of a 24 h treatment with EGCG on mitochondrial enzymatic dehydrogenase activity of rat hippocampal neurons (left) and cortical neurons (right). Compared with control group, OD value was decreased in OGD group, whereas EGCG 25 and 50 μM reversed this tendency in a concentration-dependent manner. c Effects of EGCG on LDH activity of rat hippocampal neurons (left) and cortical neurons (right). Drugs were pre-incubated at different concentrations for 24 h. Data were expressed as a control percentage. Readings were performed at 570 nm with 630 nm as the reference filter and 440 nm. ** P < 0.01 when compared to control group; # P < 0.05 and ## P < 0.01 when compared to OGD group. All data are expressed as means ± SD

Effects of EGCG on Extracellular Amino Acid Level of Hippocampal and Cortical Neurons

In consideration of the function of EGCG may be attributed to an imbalance between excitatory and inhibitory neurotransmission (Qu et al. 1998), the extracellular fluid from primary cultured rat hippocampal and cortical neurons as samples were collected for the following investigation.

In order to observe the effects of EGCG on the excitatory and inhibitory amino acids changes in vitro, glutamate (Glu), glycine (Gly), and gamma-aminobutyric acid (GABA) amino acids were detected by high performance liquid chromatography (HPLC) from the extracellular fluid in normal, OGD, and EGCG-treated groups(25, 50, and 100 μM). Compared with normal group, the content of Glu in 4 h OGD group of hippocampal and cortical neurons were enhanced as expected nearly 5- and 2.5-fold, respectively (P < 0.05). Interestingly, pre-treatment of EGCG at 25 and 50 μM decreased Glu levels to 122.56 ± 46.58% (n = 5) and 114.83 ± 31.83% (n = 5), respectively, P < 0.05, compared with OGD group on hippocampal neurons and 116.39 ± 46.34% (n = 5) and 97.47 ± 37.03% (n = 5), respectively, P < 0.05, compared with OGD group on cortical neurons. However, high-dose EGCG (100 μM) had no significant effect compared with OGD group (P > 0.05) (see Fig. 4a).

Further investigation on inhibitory amino acid, the contents of Gly and GABA in 4 h OGD group of hippocampal and cortical neurons were all depressed compared with the normal cultures, as shown in Fig. 4b, c. Surprisingly, pre-treatment of EGCG at appropriate concentration can reverse this tendency and rescue Gly and GABA levels to the similar level of normal group. Whereas, the relative high-dose EGCG (100 μM) had less effect than low-dose EGCG (25 and 50 μM) either on rescuing Gly or GABA.

These results demonstrated that the concentration of the Glu, Gly, and GABA amino acids were totally disequilibrated before and after cerebral ischemia injury and could be rescued by application of EGCG at appropriate concentration.

Discussion

This study demonstrates that EGCG at appropriate concentration can not only facilitate LTP on normal rats, but also ameliorate depression of synaptic transmission efficiency induced by MCAO in vivo. Further investigation in vitro suggests that EGCG can decline the infarct volume, elevate neurological deficit, and enhance the cell viability of primary cultured rat hippocampal and cortical neurons exposed to 4 h OGD, which shows significant neuroprotective properties. Surprisingly, the concentration of excitatory amino acid (EAA) (Glu) and inhibitory amino acids (Gly and GABA) were totally disequilibrated before and after cerebral ischemia injury, however, application of appropriate EGCG can restore amino acid level to similar normal condition. Taken together, these results suggest EGCG has the ability to facilitate the efficiency of synaptic transmission with or without cerebral ischemia injury. Furthermore, these neuroprotection maybe related to EGCG′ function of modulating excitatory and inhibitory amino acid transmitters balance.

Teas, the most commonly consumed beverage, represent a large family of plants containing high amounts of polyphenols that may confer health benefits. The present electrophysiological experiments on LTP show appropriate concentration of EGCG can facilitate LTP, however, high concentration EGCG blocks the induction of LTP at the Schaffer collateral-CA1 synapse in normal rat, as shown in Fig. 1. These results indicate the moderate consumption of tea is beneficial to health in our daily life, but not excessive.

The mechanisms of plasticity at the Schaffer collateral-CA1 region synapses of hippocampus have been extensively investigated (Bliss and Collingridge 1993). It is believed that the induction of LTP at this pathway involves the synaptic activation of NMDA type of glutamate receptors, and the consequent increase in postsynaptic intracellular calcium concentration to lead to LTP (Mondaca et al. 2004). Ischemia could inhibit the synaptic transmission by preventing the production and storage of ATP and neurotransmitter (Lyubkin et al. 1997). Furthermore, under cerebral ischemia injury, malfunction of brain Glu system has been suggested to be associated with cognitive impairment of several neurodegenerative diseases and influence learning and memory ability (Lipton et al. 1994). These insults contribute to excessive synaptic-glutamate accumulation, triggering a series of intracellular biochemical changes and finally inducing neuron death. Therefore, neurotoxicity of EAA appears to contribute to the pathogenesis of the death of neurons caused by ischemia (Wang et al. 2006). It is also reported that the way to limit excitotoxicity can be obtained by increasing GABA-mediated inhibition and some inhibitory amino acids in the central nervous system (CNS), such as Gly, has displayed neuroprotection in rat cortical neurons under hypoxia (Zhao et al. 2005).

In our research, animal models of focal cerebral ischemia, for which MCAO is usually used, reproduced the pattern of ischemia brain damage observed in many human ischemic stroke patients (Ginsberg and Busto 1989; Li et al. 2009). Our present experiments in rats with ischemia following 2 h MCAO and reperfusion 24 h showed that EGCG, administrated by i.v. at 7.5 and 15 mg/kg, can dose-dependent modulate the Schaffer collateral-CA1 synapse in the rat with ischemia. The induction rate and the average amplitude of LTP in the ischemia group were lower than those in sham group. Administration of EGCG abolished the inhibition of LTP in rats with ischemia (Fig. 2), indicating that EGCG could facilitate the synaptic transmission. These electrophysiology data are in line with previous reports that EGCG can alleviate the hippocampal LTP deficiency of the Ts65Dn mouse (Xie et al. 2008). One possible mechanism by which EGCG may ameliorate synaptic transmission could involve an interaction with Ca2+ influx and serves as a negative feedback molecule to NMDA function under ischemic conditions (Yu et al. 2004). Another possible mechanism underlying this amelioration of synaptic transmission induced by ischemia may result from the neuroprotective effect attribute to suppression of the release of EAA neurotransmitter and excitotoxicity of neuron.

In consideration of the amelioration on the impaired LTP induced by cerebral ischemia is due to EGCG′ neuroprotective effect. Therefore, cerebral infarct size and neurological deficit score after 2 h MCAO and 24 h reperfusion were examined to investigate if EGCG have neuroprotection. Representative consecutive coronal slices stained with 2% TTC from one sample of sham, ischemia, and EGCG-treated (7.5 and 15 mg/kg) groups show that 15 mg/kg EGCG treatment can significantly decrease cerebral infarct size and improve neurological cerebral infarct size from ischemia, which suggests a neuroprotective effect of EGCG (Fig. 3). Furthermore, cell viability of primary cultured rat hippocampal and cortical neurons suffered from OGD, which can mimic oxygen and glucose reduction as ischemic model for pathophysiological investigation, were used to investigate the neuroprotective effect of EGCG. The cell viability of primary cultured rat hippocampal and cortical neurons exposed to 4 h OGD and administration of EGCG at appropriate concentrations were evaluated by MTT and LDH assay. Pre-treatment with EGCG (25 and 50 μM) showed protective effects against the OGD damage in both cortical and hippocampal cultures in a concentration-dependent manner (Fig. 4). These data demonstrate EGCG can rescue neuronal injury in vitro, which is consistent with our results before (Fig. 3). Surprisingly, relative high-dose EGCG (100 μM) have no or less effect compared with low-dose (25 and 50 μM). Together with the results in Fig. 2, high-dose EGCG is not the best choice for treatment, even shows negative effect sometimes.

It was reported previously that high content accumulation of excitatory neurotransmitter specifically referred to as excitotoxicity is involved in ischemic neuronal damage and degenerative disorders in CNS. Increasing evidences also indicate that inhibitory neurotransmitters may antagonize such neuronal excitotoxicity (Schwartz-Bloom and Sah 2001). Therefore, we assume the neuroprotection of EGCG may be related to the regulation of excitatory and inhibitory amino acid transmitters balance, further avoiding excitotoxicity. Further investigations by HPLC on the contents of Glu, Gly, and GABA in the extracellular fluid from primary cultured rat hippocampal and cortical neurons were detected. The results of HPLC analysis showed that the extracellular Glu level of hippocampal and cortical neurons in 4 h OGD group were enhanced nearly 5- and 2.5-fold, respectively compared with the normal cultured neurons. Pre-treatment with EGCG at 25 and 50 μM could significantly reverse this tendency (Fig. 4). Surprisingly, the contents of Gly and GABA in 4 h OGD group were significantly depressed in both hippocampal and cortical cultures compared with the normal cultured neurons, with pre-administration of EGCG 25, 50, and 100 μM could obviously increase these inhibitory amino acids level (Fig. 5). These results collectively suggest OGD-induced ischemic neuronal damage, which was involved in EAA (Glu) accumulation specifically referred to as excitotoxicity and depressed the level of inhibitory amino acid (Gly and GABA). However, EGCG exerts a significant neuroprotective effect on cerebral ischemia damage. The mechanism is likely to be associated with down and up regulation of excitatory and inhibitory amino acid release and then achieve a new balance. The inhibition of Glu level of EGCG may contribute to its antioxidant effect and Ca2+-antagonistic effect (Bae et al. 2002; Lee et al. 2004), which can sustain the membrane intactness. Further investigations on molecular mechanisms are underway in our laboratory.

Fig. 5.

Fig. 5

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In vitro effects of EGCG on extracellular level of amino acids (Glu, Gly, and GABA) from extracellular fluid of primary cultured rat hippocampal and cortical neurons. Glutamate (Glu), glycine (Gly), and gamma-aminobutyric acid (GABA) amino acids from the extracellular fluid in normal, OGD, and EGCG-treated Groups (25, 50, and 100 μM) were detected by HPLC to observe the effects of EGCG on the excitatory and inhibitory amino acids changes in vitro. The bar chart had depicted the effect of EGCG on both hippocampal neurons (left) and cortical neurons (right) suffered from oxygen–glucose deprivation. a Compared with control group, Glu level in extracellular fluid was markedly increased (* P < 0.05, compared to control group), whereas pre-treated with EGCG 25 and 50 μM for 24 h reversed this tendency (# P < 0.05, compared to OGD group). However, relative high-dose EGCG 100 μM have no statistical significance compared with OGD group. b The contents of Gly in 4 h OGD group of hippocampal and cortical neurons were depressed compared with the normal cultures (* P < 0.05). However, pre-treatment of EGCG at appropriate concentration can reverse this tendency and rescue Gly level to the similar level of normal group. Whereas, the relative high-dose EGCG (100 μM) had less effect than low-dose EGCG (25 and 50 μM). c Compared with control group, GABA level in extracellular fluid was markedly decreased in 4 h OGD group of hippocampal and cortical neurons (* P < 0.05), whereas pre-treated with EGCG 25 and 50 μM for 24 h reversed this tendency (# P < 0.05). High-dose EGCG (100 μM), compared with the OGD group, which can also increased GABA levels (# P < 0.05), but the effect was not as strong as relative low-dose EGCG (25 and 50 μM). Drugs were pre-incubated at different concentrations for 24 h. Data (n = 5) were expressed as a control percentage. *P < 0.05 when compared to control group; # P < 0.05 when compared to OGD group. All data are expressed as means ± SD

In conclusion, our results demonstrated that cerebral ischemia injury induced suppression of LTP at the Schaffer collateral-CA1 region synapses of rat hippocampus and EGCG can ameliorate the efficiency of synaptic transmission partly due to the neuroprotective properties through regulating excitatory and inhibitory amino acid transmitters’ balance.

Acknowledgments

This study was supported by the National Foundation of Nature and Science of China (No. 81173038) and by the Foundation of Central Authorities of an Institution of Higher Learning of Scientific Research Special Fund (No. 2011TS073).

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