Neurorestorative effects of epigallocatechin-3-Gallate on cognitive function in a chronic cerebral hypoperfusion rat model

Abstract

Purpose: This study investigated whether (–)-epigallocatechin-3-gallate (EGCG) can enhance cognition by a neurorestorative effect in a rat model of bilateral common carotid artery occlusion (BCCAO).

Methods: Forty-eight male, 8-week-old Sprague-Dawley rats were randomly allocated to four groups 6 weeks after BCCAO or sham operation: EGCG-single intravenous injection (25 mg/kg/day; SIV group), EGCG-multiple intraperitoneal injection (50 mg/kg/day for 5 days; MIP group), untreated BCCAO group (untreated group), and sham-operated group (sham group).

Results: Escape latency was significantly shorter in the SIV and MIP groups than in the untreated group. SIV and MIP groups were significantly different from the untreated group in the activity of superoxide dismutase and the content of malondialdehyde (p < 0.05). Protein expression level of brain-derived neurotrophic factor was not significantly different between groups (p > 0.05), while protein expression of vascular endothelial growth factor was significantly lower in the SIV group than in the untreated group (p < 0.05). Protein expression of N-methyl-D-aspartate receptor subunits NR1 and NR2B was significantly higher in the MIP group than in the untreated group (p < 0.05).

Conclusions: EGCG administration at 6 weeks after BCCAO is neurorestorative via an anti-oxidant effect and synaptogenesis, except for angiogenesis.

Keywords

(–)-Epigallocatechin-3-Gallate neurorestoration cognition vascular dementia rat

Introduction

Vascular dementia is the second largest cause of dementia. With progressive aging of populations, vascular dementia will become the commonest form of dementia. The conventional concept of vascular dementia is that of multi-infarct dementia, arising from chronic cerebral ischemia and various vascular changes in the brain. Reduction in cerebral blood flow and abnormal energy metabolism that occur in chronic cerebral ischemia can induce the accumulation of nitric oxide (NO) and reactive oxygen species (ROS), which lead to neuronal injury in selective and vulnerable regions of the brain, especially the hippocampus and cerebral cortex, accompanied by cognitive decline and some motor disorders (Xiong et al., 2006). Chronic cerebral hypoperfusion is a pathological condition that contributes to establishment of neurodegenerative diseases. In a rat model, the permanent bilateral occlusion of both common carotid arteries causes an abrupt and chronic reduction of whole cerebral blood flow, which affects cortical area and hippocampus. Bilateral common carotid artery occlusion (BCCAO) in rats is widely accepted as an adequate experimental model to investigate the neural impairment and cognitive decline in vascular dementia (Farkas, Luiten, & Bari, 2007; Jiwa, Garrard, & Hainsworth, 2010).

Nok cha is the most common form of Korean green tea. It is derived from dried leaves of the Camellia Sinensis plant. This tea contains many beneficial compounds, especially flavanoids. Flavanoids are low molecular weight compounds that comprise several different classes based on variations of same basic structure. On class is the flavan-3-ols, also referred to as the catechins, which consists of (–)-epicatechin (EC), (–)-epicatechin-3-gallate (ECG), (–)-epigallocatechin (EGC), (+)-gallocatechin (GC), and (–)-epigallocatechin-3-gallate (EGCG) (Cooper, Morre, & Morre, 2005a). EGCG has a neuroprotective effect caused by its’ antioxidant action and free radical scavenging activity (Baluchnejadmojarad & Roghani, 2011; Choi, Kim, Lee, Kim, & Kim, 2004; Hong et al., 2000; Lee, Bae, & Lee, 2004; Mandel et al., 2005; Xu et al., 2010). EGCG can also inhibit angiogenesis and proliferation of cancer cells, indicating potential value as a therapeutic compound for various forms of cancer (Cooper, Morre, & Morre, 2005b; Jung & Ellis, 2001).

Many attempts to treat cerebrovascular disease have focused on neurorestorative therapies rather than acute neuroprotection (Chen et al., 2005; Hong et al., 2000; Mandel et al., 2008; H. Sun et al., 2010; Zhang & Chopp, 2009). Neurorestorative events include angiogenesis and synaptic plasticity, as well as neurogenesis. Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are two important neurotrophic factors that have multiple effects on sustaining and evoking elements of brain plasticity (Jin et al., 2002; Y. Sun et al., 2003). BDNF regulates neuronal survival, cell migration, and synaptic function. VEGF is an angiogenic agent, which promotes neurogenesis and stem-progenitor cell migration.

N-methyl-D-aspartate (NMDA) receptors, which play a key role in the synaptic plasticity, are composed of NR1, NR2 (A, B, C, and D) and NR3 (A and B) subunits (Goebel & Poosch, 1999). NR1 and NR2B subunits have been implicated as important contributors to synaptic responses in the hippocampus (Kirson & Yaari, 1996; Kohr et al., 2003).

In a BCCAO rat model, gradual recovery of regional cerebral blood flow (CBF) in the hippocampus has been demonstrated to begin 1 week after operation, but recovery remains significantly lower than the control values at 4 weeks post-operatively (about 66% of the control level). At 8 weeks after BCCAO, hippocampus CBF is indistinguishable from the initial pre-operative value (Otori et al., 2003).

The neurorestorative effect of EGCG in chronic neuronal degenerative disease has not been reported. This study investigated whether EGCG can enhance cognition by a neurorestorative effect including neurogenesis, angiogenesis, and synaptogenesis in a rat model of BCCAO with cognitive dysfunction.

2 Materials and methods

2.1 Animal models and EGCG administration

All animal experimental protocols were carried out in accordance with the guidelines of the Chonnam National University Animal Care and Committee. Forty-eight male Sprague-Dawley rats, 8-weeks-of-age, weighing 250∼280 g (Samtako, Osan, Korea) were maintained under controlled temperature (23.0±1.0°C) and controlled light conditions (light, 07:00∼19:00 hours). Food and water were supplied ad libitum. Twelve rats underwent sham operation (sham group). Thirty-six rats were randomly allocated to three groups 6 weeks after BCCAO: EGCG-single intravenous injection (25 mg/kg/day; SIV group, n = 12), EGCG-multiple intraperitoneal injection (50 mg/kg/day for 5 days; MIP group, n = 12), and untreated BCCAO group (untreated group, n = 12). The polyphenol EGCG was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in physiological saline for injection. This experimental study had an 8-week schedule (Fig. 1) after which all rats were sacrificed and prepared for analyses.

2.2 BCCAO model

The common carotid arteries of 36 rats were bilaterally and permanently occluded by ligation as previously described (Payan, Levine, & Strebel, 1965). The operation was performed under inhalation anesthesia with 2% isoflurane in 70% nitrous oxide and 30% oxygen mixture using a face mask. A ventral midline incision was made to expose the bilateral common carotid arteries, which were carefully separated from carotid sheath and vagus nerve. The bilateral common carotid arteries were double ligated with 4–0 type surgical non-absorbable silk and were not ligated in sham operation group. During the operation, body temperature was maintained at 37.5±0.5°C, then rats were placed on a homeothermic tapetum (Harvard Apparatus, South Natick, MA, USA) until they recovered from anesthesia. After the operation, all rats were injected intramuscularly with gentamicin sulfate (5 mg/kg) for 3 days and housed under usual controlled conditions for 6 weeks.

2.3 Behavioral test

Cognitive function, especially spatial learning and memory, were assessed by Morris water maze (MWM) training and test based on the previously described method (Morris, Garrud, Rawlins, & O’Keefe, 1982). The water maze pool consisted of a circular water tank (diameter 184 cm, depth 60 cm) with water temperature maintained at 20∼25°C, and a circular hidden platform (diameter 10 cm, height 38 cm). The pool was divided into four equal quadrants (zones) and one was allocated as the target quadrant. The circular hidden platform was placed in the center of the target quadrant. Visual cues were placed at the perimeter of each quadrant. The water level in the pool was adjusted so that the platform was submerged 1 cm below the surface of the water.

To become accustomed to the water maze, the rats performed two trials a day for 3 consecutive days before BCCAO. The animals were placed into the water maze facing the maze wall at random entry points, which were equally distributed around the perimeter of the maze. After finding the platform, the rats were allowed to remain there for 10 sec until the next trial. If a rat could not find the hidden platform within 120 sec, the animal was guided to the platform and allowed to remain for 15 sec before being removed from the pool, dried, and placed back in its holding bin for 5 min. A second trial was then conducted. The water maze trials were analyzed using an Ethovision Color-Pro automated tracking system (Nodulus, Wageningen, The Netherlands).

MWM training was performed at 7 weeks after BCCAO using the same protocol as the pre-training. Each rat received two trials a day for 5 consecutive days.

The spatial learning performance of rat was evaluated by escape latency of the MWM the next day after the fifth day of MWM training. Escape latency, which has been generally used to assess performance of learning and memory of the rats, was measured as the time to reach the platform and was recorded as the average value of two trials by researchers not directly involved in the procedures of animal models and EGCG administration.

2.4 Superoxide dismutase (SOD) and malondialdehyde (MDA) analyses

The oxidant-antioxidant status of the rat brains subjected to chronic cerebral hypoperfusion was assessed by determining level of lipid peroxidation and activity of SOD. Lipid peroxidation was determined by measuring the level of MDA, which is considered to be an important parameter of oxidative damage determination. The activity of SOD and the level of MDA play critical roles in anti-oxidation in tissues and organs, and indicate the level of anti-oxidation. Hippocampal tissues were collected, weighted, and homogenized in 9 volumes of ice-cold 0.9% NaCl. After centrifugation at 13,000 rpm for 10 min at 4.0°C, the supernatant was used to determine the activity of SOD and the level of MDA. SOD activity was measured as previously described (Y. Sun, Oberley, & Li, 1988) according to the spectrophotometrical procedure provided with the assay kits (Cayman Chemical Company, Ann Arbor, USA). The principle of the method is based on the inhibition of Nitro Blue Tetrazolium (NBT) reduction by the xanthine-xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant after 1 ml of ethanol-chloroform mixture (5:3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the amount of enzyme causing 50% inhibition in the NBT reduction rate. Tissue MDA levels were measured as previously described (Draper & Hadley, 1990) based on the reaction of MDA with thiobarbituric acid (TBA) at 95.0°C according to the spectrophotometrical procedure provided with the assay kits (OxisResarch, Portland, USA). In the TBA test reaction, MDA and TBA react to form a pink pigment with an absorption maximum at 540 nm. The reaction was performed at pH 2∼3 at 95.0°C for 15 min. The sample was mixed with 2.5 volumes of 10% trichloroacetic acid to precipitate the protein. The precipitate was pelleted by centrifugation and supernatant was reacted with 0.67% TBA in a boiling water-bath for 60 min. After cooling, the absorbance was read at 540 nm. The values obtained were compared with a series of standard solutions.

2.5 Histopathologic studies

The same day of biochemical analysis, rats from the four groups were deeply anaesthetized with 2.5% pentotal sodium, and the heart of each rat was exposed. Each rat was transcardially perfused with 100 ml saline followed by phosphate buffer (0.1 mol/l, pH 7.4) containing 400 ml of 4% paraformaldehyde. The brain of each rat was removed and postfixed overnight in the same fixative solution at 4.0°C. After paraffin embedding, coronal and serial sections of the hippocampus (2 μm thick) were made. The sections selected at the same layers from each rat brain were stained with hematoxylin and eosin (H&E) to check neuron density and distribution. Light microscopy examination (Nikon, Tokyo, Japan) was carried out at a magnification of 200 and the counts of cells were determined per square mm iusing a standardized ocular grid. The numbers of normal pyramidal cells in the CA1, CA2, CA3, and CA4 hippocampal regions were counted. The criteria used to distinguish normal from abnormal cells were as follows. Cells with regular-shaped soma with clear cytoplasm and intact nuclear membranes were determined as normal. Cells with irregular shape, with or without chromatin compaction or a nuclear membrane, DNA marginalization and darkened cytoplasm, or blackened cytoplasm with extensive vacuolarization, were considered to be abnormal cells. Enumeration was done by two persons blinded to the details of theexperiment.

2.6 Western blot analysis

The hippocampal tissue was homogenized with 500 μl PRO-PREP^TM Protein Extraction Solution (iNtRON BIOTECHNOLOGY, Seongnam city, Kyeonggi-do, Republic of Korea). After centrifugation at 12,000 rpm for 30 min at 4.0°C, the supernatant was collected. For detection of BDNF and VEGF proteins, equal amounts of protein (20 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). NR1 and NR2B protein (20 μg) in each sample was separated by 8% SDS-PAGE. The resolved proteins were electrotransferred to a polyvinylidene difluoride membrane at 300 mA for 1 h. Membranes were stained with Ponceau Red to ensure equal protein loading. Immunodetection of the protein of interest was carried out by first blocking the membrane in 5% skim milk in 20 mM Tris-buffered saline containing 0.1% Tween-20 (TBST, 80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, 0.1% Tween-20, pH 7.5) for 1 h at room temperature. Each membrane was washed with TBS-T four times within 20 min and incubated in primary antibody against BDNF (rabbit polyclonal antibody, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit VEGF (1:500; Novus Biologicals, San Diego, CA, USA), anti-NR1 (1:1,000; Novus Biologicals), or anti-NR2B (1:1,000; Novus Biologicals) overnight at 4.0°C in TBS-T with 2% skim milk. Excess antibody was removed by TBS-T three times, 5 min each. The membranes were incubated with goat anti-rabbit IgG-HRP (1:5,000; Santa Cruz Biotechnology) secondary antibody and TBS-T with 3% skim milk in blocking solution for 1 h. The membranes were washed again three times with TBS-T within 30 min and immunoreactive protein bands were visualized using Enhanced Chemiluminescence plus (Immobilon Western Chemiluminescence; Millipore Corporation, Billerica, Massachusetts, USA) and LAS-3000 Image Reader (Fuji Photo Film, Tokyo, Japan). Densitometrical analyses for specific protein signals were carried out using Image J 1.37 software (National Institutes of Health, Bethesda, MD, USA) with the aid of a computer. The density of the signal for specific protein was normalized by dividing by the density of the β-actin immunoreactive signal in the same sample. Values are expressed as the relative amount of protein expression (%), with the mean density in the sham group being 100%.

2.7 Statistical analysis

Statistical analyses were performed using the Kruskal-Wallis test with Dunn-Bonferroni post-hoc method. All statistical analyses were performed using IBM SPSS Statistics ver. 21.0 (IBM Inc., Armonk, NY, USA). The results are expressed as mean±standard deviation. Statistical significance was accepted at the conventional p < 0.05 level.

3 Results

3.1 Behavioral test

Learning and memory ability were examined in the MWM test. As shown in Figs. 2 and 3, escape latency was significantly shorter in the SIV and MIP groups (65.5±48.8 and 48.8±42.5 sec, respectively; p < 0.05) than in the untreated group (172.5±90.8 sec). Rats in the sham group spent 50.8±41.9 sec finding the platform.

3.2 SOD activity and MDA level

The activity of SOD was significantly higher in the SIV, MIP, and sham groups than in the untreated group (all p < 0.05). MDA is a product of lipid peroxidation that is useful to evaluate lipid oxidative damage. Increasing concentrations of MDA indicate increasing oxidative damage. In the present study, the content of MDA was significantly lower in the SIV, MIP, and sham groups than in the untreated group (all p < 0.05; Fig. 4).

3.3 Histopathological findings

BCCAO caused histological appearance of neuronal loss in CA1 of the hippocampal area. A few eosinophilic neurons and phyknotic neurons were noted at the hippocampus in the untreated group (Fig. 5). The quantitative number of hippocampal neurons was less in the untreated group than the in sham group at CA1, CA2, and CA4 hippocampal areas (all p < 0.05). Hippocampal neurons in the SIV and MIP groups were significantly more numerous than that in the untreated group in the CA1 and CA2 region (all p < 0.05; Fig. 6).

3.4 Western blot analysis for BDNF, VEGF, NR1, and NR2B

Protein expression of BDNF did not differ significantly between groups (p > 0.05; Fig. 7). The protein expression of VEGF was significantly lower in the SIV group than in the untreated and sham groups (p < 0.05; Fig. 8). The protein expression of NR1 and NR2B was significantly higher in the MIP group than in the untreated and sham groups (p < 0.05; Figs. 9 and 10).

4 Discussion

Vascular dementia represents dementia due to problems in supply of blood to the brain and is thought to be an irreversible form of dementia. Clinically, the aim of vascular dementia management is the prevention of further cerebrovascular lesions. Many studies have sought to discover find the drug and/or material for the treatment of vascular dementia. No neurorestorative agent has been found. Over the past few years, most attempts to treat cerebrovascular disease have focused on neuroprotection, although more recently the focus has shifted to neurorestorativetherapies, which are likely to be far more involved than acute neuroprotection (Mandel et al., 2008; Mandel et al., 2005).

BCCAO models have been most effective in evaluating the relationship between chronic/moderate cerebral hypoperfusion and cognition. (Ginsberg & Busto, 1989) Rats with chronic cerebral hypoperfusion suffer from progressive spatial memory impairment, selective CA1 neuronal damage, reactive astrocytosis in the hippocampus, increased membrane phospholipid synthesis in the hippocampal region, reduction of cerebral pre-terminal noradrenergic varicosities, decreased immunoreactivity of microtubule associated protein-2, and significantreductions of hippocampal-parietal cortex blood flow (de la Torre & Fortin, 1991; de la Torre et al.,1992).

EGCG has two main effects. A neuroprotective effect is caused by antioxidant action and free radical scavenging activity (Baluchnejadmojarad & Roghani, 2011; Choi et al., 2004; Hong et al., 2000; Lee et al., 2004; Mandel et al., 2005; Xu et al., 2010). Cancer-prevention and treatment effect is due mainly to anti-angiogenesis action and apoptosis activity in cancer cells (Cooper et al., 2005b; Jung & Ellis, 2001; Jung et al., 2001).

The aim of this study was to investigate the neurorestorative capability of EGCG including angiogenesis, neurogenesis, synaptic plasticity, as well as anti-oxidative effects, when EGCG was administrated 6 weeks after BCCAO operation. The regional cerebral blood flow (CBF) in the hippocampus has begun to recover at 1 week after BCCAO, but remain significantly lower than the control values 4 weeks after BCCAO in rats (about 66% of the control level). At 8 weeks, either only a slight reduction or virtually no reduction has been reported (Otori et al., 2003).

This behavioral test in this study revealed that EGCG has a neurorestorative effect on cognitive function in a BCCAO rat model. Biochemical examinations showed that EGCG has antioxidant activity. SOD is an enzyme with antioxidative effect. MDA is highly reactive and responsible for cytotoxic effects and neuronal death. The contents of MDA indicate the extent of brain damage due to free radicals in cerebral ischemia (Shang, Miao, Cheng, & Qi, 2006). Presently, SOD increased and MDA decreased in EGCG-treated groups compared with the untreated group.

BDNF and VEGF are two important neurotrophic factors that have multiple effects on sustaining and evoking elements of brain plasticity. BDNF regulates neuronal survival, cell migration, and synapticfunction (Aguado et al., 2003; Gorski, Balogh, Wehner, & Jones, 2003; Yamada, Mizuno, & Nabeshima, 2002). Likewise, VEGF is only one of several angiogenic agents that promotes neurogenesis and stem-progenitor cell migration (Jin et al., 2002; Y. Sun et al., 2003). BDNF mRNA expression in hippocampus induced by BCCAO in the rat transiently increases because of homeostatic signs of cerebral circulation failure after BCCAO (Schmidt-Kastner et al., 2001). But, generalized stress is reportedly associated with a down-regulation of BDNF mRNA in granule cells of the dentate gyrus (Smith, Makino, Kvetnansky, & Post, 1995). Another study observed that VEGF mRNA levels were significantly increased in the chronic cerebral hypoperfusion rat brains for 24 hours postoperatively, peaked by 7 days, decreased by 21 days, and returned to basal levels by 90 days after operation. Also, VEGF protein was expressed at low levels in the control brains, whereas expression increased in the model brains from 24 hours to 21 days postoperatively, but returned to control levels 90 days after operation (Hai et al., 2003). Presently, at 7 weeks after BCCAO, the level of BDNF protein was slightly and non-significantly decreased in the untreated, SIV, and MIP groups compared with sham operation groups.

Both VEGF and BDNF increase in ischemic brain with other neurorestorative treatments of stroke (Chen et al., 2005). Presently, there were no increases in BDNF and VEGF protein level between the EGCG treated group and the untreated group. On the contrary, VEGF protein was expressed at a low level in the EGCG treated group (SIV group) compared with the untreated group. Although EGCG reportedly inhibits angiogenesis in various cancers, an anti-angiogenic effect of EGCG in the brain has not yet been reported (Jung & Ellis, 2001; Jung et al., 2001; Masuda et al., 2002). This may create the erroneous impression that EGCG is not useful in situations requiring angiogenesis for tissue repair or restoration. The precise mechanisms of reduction of VEGF expression by EGCG in brain remain to bestudied.

NMDA receptors are considered as crucial synaptic elements for the induction of activity-dependent synaptic plasticity. Long-term potentiation is a widely-used paradigm for increasing synaptic efficacy, and its induction requires, in at least one of its forms, the activation of NMDA receptors (Bliss & Lomo, 1973; Tsien, Huerta, & Tonegawa, 1996). The NMDA receptor consisting of subunit 1 (NR1), subunit 2 (NR2 A, B, C, and D) and subunit 3 (NR3 A and B) is primarily expressed in the CA1 region of the hippocampus-dependent long-term potentiation formation and in spatial learning and memory (Goebel & Poosch, 1999). The presence of NR2B subunits together with NR1 appears to be an important contributor to synaptic responses in the hippocampus (Kirson & Yaari, 1996; Kohr et al., 2003; Zhou & Baudry, 2006). NR1 and NR2B mRNA and protein expression is decreased in chronic cerebral hypoperfused rats (He et al., 2008; Huang, He, Guo, & Wang, 2008; H. Sun et al., 2010; Tang, Wang, Feng, Kyin, & Tsien, 2001). Presently, protein expression of NR1 and NR2B were increased significantly after multiple EGCG injection compared with the untreated group. EGCG has favorable effects on synaptogenesis by NR1 and NR2B protein expression. Protein expression of NR1 and NR2B were not significantly different in the SIV group compared with the untreated group. Thus, synaptogenesis may be positively associated with multiple injection.

5 Conclusion

EGCG administration at 6 weeks after BCCAO in rats was neurorestorative due to an anti-oxidant effect and synaptogenesis, and impeded angiogenesis. EGCG may be multifunctional in neurorestoration as shown in the BCCAO rat model. This should be considered when medicines extracted from EGCG are researched and developed.

Footnotes

Acknowledgments

This study was supported by the Convergence of Conventional Medicine and Traditional Korean Medicine R&D program funded by the Ministry of Health & Welfare through the Korea Health Industry Development Institute (HI14C0862).

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