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Neuroprotective effects of a traditional herbal prescription on transient cerebral global ischemia in gerbils
Journal of Ethnopharmacology 138 (2011) 723–730
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Neuroprotective effects of a traditional herbal prescription on transient cerebral global ischemia in gerbils Mudan Cai a,1 , Bum Young Shin b,1 , Dong Hyun Kim a , Jong Min Kim a , Se Jin Park a , Chan Sung Park c , Do Hee Won c , Nam Doo Hong c , Dong Hyo Kang d , Yamamoto Yutaka d , Jong Hoon Ryu a,b,∗ a
Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemoon-ku, Seoul 130-701, Republic of Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemoon-ku, Seoul 130-701, Republic of Korea c R&D Center, Kwang Dong Pharmaceutical Co., Ltd., 621-1 Jangdang-dong, Pyongtaek-si, Kyonggi-do 459-020, Republic of Korea d Tochimoto Tenkaido Co., Ltd., 3-21, Suehiro-cho, Kita-ku, Osaka-city, Osaka 530-0053, Japan b
a r t i c l e
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Article history: Received 22 March 2011 Received in revised form 8 October 2011 Accepted 10 October 2011 Available online 14 October 2011 Keywords: Kyung-Ok-Ko Global ischemia Hippocampal cell death Inflammation
a b s t r a c t Aim of the study: Kyung-Ok-Ko (KOK), a traditional herbal prescription composed of Rehmannia glutinosa var. purpurae, Panax ginseng, Poria cocos, Lycium chinense, Aquillaria agallocha and honey, has been used to treat age-related symptoms, such as amnesia or dementia, and has been shown to ameliorate scopolamine-induced memory impairment in mice. However, the effects of KOK on transient cerebral global ischemia-induced brain damage are unclear. Materials and methods: Transient cerebral global ischemia was induced by occluding the bilateral common carotid artery for 5 min followed by reperfusion for 7 days. KOK (0.25, 0.5, 1, or 2 g/kg) was administered orally immediately after reperfusion and once a day over the next 7 days. Y-maze or novel object recognition tasks were to analyze learning and memory capabilities at 4 or 5 days after reperfusion, respectively. Histochemistry and immunohistochemistry were used for evaluation of the effect of KOK on neuronal degeneration. Results: Histochemical studies showed that KOK increased the number of viable cells detected by Nissl staining and decreased the number of degenerated neuronal cells detected by Fluoro-Jade B staining in the hippocampal CA1 region. In the immunohistochemical study, the sub-chronic KOK administration attenuated the ischemia-induced activation of microglia and astrocytes and the increase of cytokine IL-1 (P < 0.05). In addition, KOK administration significantly attenuated the ischemia-induced cognitive impairments observed in the Y-maze and novel object recognition tasks (P < 0.05). Conclusion: These findings suggest that the neuroprotective effects of KOK may be mediated by its antiinflammatory activities, resulting in the attenuation of memory impairment. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cerebral ischemic injury resulting from either focal or global circulatory arrests in the brain, is one of the major causes of death and disability in adults (Pinkston et al., 2009). Global cerebral ischemia is a clinical outcome occurring as a consequence of cardiac arrest, reversible severe hypotension or other situations that deprive the brain of oxygen and glucose (Nedergaard and Diemer, 1988; White et al., 1993). Multiple factors, including
∗ Corresponding author at: Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, #1 Hoeki-dong, Dongdaemoon-ku, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 9230; fax: +82 2 966 3885. E-mail address: [email protected] (J.H. Ryu). 1 1 These authors contributed equally to this paper. 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.016
excitotoxicity, oxidative stress, and inflammatory cytokines (tumor necrosis factor-␣, interleukin (IL)-1 or IL-6), are responsible for hippocampal neuronal damage (Endoh et al., 1994; Kasparova et al., 2005; Pinkston et al., 2009; Ikonomidou and Kaindl, 2010a,b, 2011), especially the pyramidal neurons of the CA1 region in the hippocampus (Kirino, 2000). Among these factors, the inflammatory response is a delayed process and could be a potential target for the treatment of brain ischemia (Yrjanheikki et al., 1999; Tuttolomondo et al., 2009). In line with inflammation in ischemic brain, glial cells including microglia and astrocytes are also involved in neuronal degeneration (Petito et al., 1990; Denes et al., 2007). Based on these observations, many drugs have been tested for their abilities to delay neuronal death, including cyclooxygenase2 inhibitors (Gackowski et al., 2008; Hamel et al., 2008), nuclear factor-B inhibitors (Ridder and Schwaninger, 2009), inducible nitric oxide synthase inhibitors (Cai et al., 2008; Shin et al., 2010),
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and inhibitors against glial cell activation (Muramatsu et al., 2004; Lee et al., 2011). However, when selective anti-inflammatory or anti-excitatory agents were applied in ischemic brain damage, satisfactory outcomes were not obtained (Guo et al., 2009). Kyung-Ok-Ko (KOK; Qiong-yu-gao in Chinese; Kei-gyoku-kou in Japanese) is a traditional herbal prescription that contains six ingredients: Rehmannia glutinosa var. purpurae, Panax ginseng, Poria cocos, Lycium chinense, Aquillaria agallocha and honey. It has been used for age-related symptoms, such as amnesia and dementia in East Asia (Hur, 1999). Owing to the traditional use of KOK, we observed that KOK ameliorated scopolamine-induced memory impairments in mice (Shin et al., 2009). Some studies on the medicinal properties of KOK have also focused on immunological activities (Lee et al., 2002), inflammation (Lee et al., 2008), or gastric ulcers (Whang et al., 1994). From these studies, we hypothesized that KOK may be effective on inflammation-related brain disorders which exhibit memory impairment. However, no attempts have been made to investigate whether KOK has neuroprotective or ameliorative effects against ischemic brain damage-induced memory impairment. In recent years, various tries to develop antiischemic drugs from natural products are being conducted (Gupta et al., 2010). Therefore, the purpose of this study was to investigate whether KOK has neuroprotective effects on transient cerebral global ischemia-induced neuronal damage and whether it ameliorates ischemia-induced learning and memory deficits.
fibrillary acidic protein (GFAP) antibody, cresyl violet acetate and Fluoro-Jade B (FJ-B) were purchased from Chemicon (Temecula, CA). Biotinylated secondary antibody and avidin–biotin-peroxidase complex (ABC) kits were purchased from Vector (Burlingame, CA). MK-801, bovine serum albumin (BSA), and 3,3 -diaminobenzidine tetrahydrochloride (DAB) were purchased from Sigma Chemical Co. (St. Louis, MO). Zoletil 50® was purchased from Virbac laboratory (Carros, France). All other materials were of the highest grade commercially available. 2.3. Sample preparation Kyung-Ok-Ko (100 g) was prepared following method. Juice of root of Rehmannia glutinosa Liboschitz var. purpurae Makino (Scrophulariaceae) (32.0 g), powder of dried root of Panax ginseng C.A. Meyer (Araliaceae) (2.8 g), powder of cortex of Poria cocos Wolf (Polyporaceae) (8.0 g), powder of dried fruit of Lycium chinense Miller (Solanaceae) (0.9 g), powder of resin of Aquillaria agallocha Roxburgh (Thymelaeaceae) (0.1 g), honey (38.5 g), and simple syrup (17.7 g) were mixed and heated at 80 ◦ C in the water bath for 72 h. Viscous extract was obtained and used for present study. KOK (Lot No., OV30) standardized with 5-hydroxymethyl furaldehyde (9.4%) for consistency of quality was donated by Kwang Dong Pharmaceutical Co. (Pyongtaek, Korea). 2.4. Surgical procedure and drug administration
2. Materials and methods 2.1. Animals Mongolian gerbils (60–80 g) were purchased from the Orient Co., Ltd., a branch of Charles River Laboratories (Seoul, Korea). Animals were housed 4 per cage, allowed access to water and food ad libitum, and maintained under a constant temperature (23 ± 1 ◦ C) and humidity (60 ± 10%) under a 12-h light/dark cycle (light on 07:30–19:30 h). Animal treatment and maintenance were conducted in accordance with the Principle of Laboratory Animal Care (NIH Publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea. For the experiments, gerbils were divided into six groups [sham, n = 5; ischemia, n = 5; ischemia + KOK (0.25 g/kg), n = 5; ischemia + KOK (0.5 g/kg), n = 6; ischemia + KOK (1 g/kg), n = 6; ischemia + KOK (2 g/kg), n = 6; ischemia + MK-801, n = 6; a total of 39 animals were used]. 2.2. Materials The goat anti-ionized calcium-binding adaptor molecule-1 (Iba1) antibody and rabbit anti-interleukin-1 (IL-1) antibody were purchased from Abcam (Cambridge, UK). The mouse anti-glial
Animals were anesthetized with isoflurane (2.5% for induction, 1% for maintenance) in a mixture of nitrous oxide and oxygen (70:30), and the duration of anesthesia was no longer than 5 min. Transient cerebral ischemia was induced by the bilateral common carotid artery occlusion (BCCAO) as following. After making a median incision in the neck skin, both common carotid arteries were exposed and occluded with aneurysm clips for 5 min. Body temperature was maintained at 37 ± 0.5 ◦ C with a heating pad throughout surgery (Biomed S.L., Alicante, Spain). Circulation was restored by removing the clips. Animals receiving the same surgical operation without clipping the carotid arteries served as sham-operated controls. Regional cerebral blood flow (rCBF) was monitored using laser Doppler flowmetry (LDF; Perimed, PF5010, JarFalla, Sweden). Cyanoacrylate adhesives were used to attach flexible probes (model 407, Perimed, Jarfalla, Sweden) to the intact skull ±3.5 mm of the bregma. The change in rCBF was measured for 1 min immediately after occlusion and expressed as a percentage of the baseline value. The gerbil which showed over 80% of rCBF reduction was used for further study. After reperfusion, the animals were placed in a warm incubator (32–33 ◦ C). KOK (0.25, 0.5, 1, or 2 g/kg, p.o.) was administered once a day for 7 days beginning immediately after reperfusion until designated time points (Fig. 1). A group intraperitoneally treated with MK-801
Novel object recognition task
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KOK Fig. 1. Experimental procedures. BCCAO, bilateral common carotid artery occlusion; KOK, Kyung-Ok-Ko; IHC, immunohistochemistry.
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Fig. 2. Photomicrographs of Nissl (A) and Fluoro-Jade B (FJ-B, C) staining of the hippocampus, the percentages of viable neurons (B), and the number of FJ-B-positive cells (D) in CA1 at 7 days after transient cerebral global ischemia induced by bilateral common carotid artery occlusion (BCCAO) for 5 min followed by reperfusion. Kyung-Ok-Ko (KOK) (0.25, 0.5, 1, or 2 g/kg/day, p.o.) was administered immediately after reperfusion and once a day for the next 7 days. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.05, compared with the sham control; # P < 0.05, compared with the vehicle-treated ischemic control group. Magnification: 400×. Bar = 100 m.
(3 mg/kg, i.p.), an N-methyl-d-aspartate (NMDA) receptor antagonist, immediately after reperfusion was used as a positive control group (Kim et al., 2010). The last administration was completed 1 h before each experiment. In the sham and ischemic control groups, vehicle solution (10% Tween 80 solution) was administered orally using the same time schedule.
2.5.1. Nissl staining After the sections were mounted onto gelatin-coated slides, they were stained with 0.5% cresyl violet, dehydrated through graded alcohols (70%, 80%, 90%, and 100%, 2×), placed in xylene, and coverslipped using histomount medium.
2.5. Tissue preparation
2.5.2. Fluoro-Jade B staining To determine the extent of neuronal death in the hippocampus following KOK administration, FJ-B staining was conducted, as described elsewhere (Schmued and Hopkins, 2000a,b). In brief, the sections were mounted onto gelatin-coated slides in distilled water and sequentially placed in 100% ethanol for 3 min, 70% ethanol for 1 min, 30% ethanol for 1 min and distilled water for 1 min. The slices were then oxidized for 15 min using a 0.1% KMnO4 solution followed by brief rinses in distilled water for 1 min. The slides were then immersed in a 0.0015% solution of FJ-B in 0.09% acetic acid in the dark for 30 min, rinsed with distilled water, dried for 30 min at 37 ◦ C, cleared with xylene and coverslipped using DPX medium.
Immediately after object recognition test, animals were anesthetized with an intramuscular injection of Zoletil 50® (10 mg/kg), perfused transcardially with phosphate buffer (100 mM, pH 7.4) followed by ice-cold 4% paraformaldehyde and then decapitated. The brains were removed and postfixed in phosphate buffer (50 mM, pH 7.4) containing 4% paraformaldehyde overnight, then immersed in a 30% sucrose solution (in 50 mM phosphate-buffered saline, PBS) and stored at 4 ◦ C until sectioning. Brains were cut along in the coronal plane (30 m) using a cryostat (Leica, Nussloch, Germany) and kept in storage solution at 4 ◦ C.
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Fig. 3. Photomicrographs of Iba-1 (A) and GFAP (C) staining in the hippocampus and the number of Iba-1- (B) and GFAP- (D) positive cells at 7 days after transient cerebral global ischemia induced by bilateral common carotid artery occlusion (BCCAO) for 5 min followed by reperfusion. Kyung-Ok-Ko (KOK) (0.25, 0.5, 1, or 2 g/kg/day, p.o.) was administered immediately after reperfusion and once a day for the next 7 days. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.05, compared with the sham control; # P < 0.05, compared with the vehicletreated ischemic control group. Magnification: 400×. Bar = 100 m.
2.5.3. Immunohistochemistry Free floating sections were incubated for 24 h in PBS (room temperature) containing goat anti-Iba-1 (1:1000 dilution), mouse anti-GFAP (1:1000 dilution) or rabbit anti-IL-1 (1:200 dilution) antibody with 0.3% Triton X-100, 1% BSA, and 1.5% normal rabbit serum (for Iba-1), horse serum (for GFAP) or goat serum (for IL-1) from the ABC kit. The sections were then incubated with biotinylated secondary antibody (1:200 dilution) for 90 min, treated with ABC solution (1:100 dilution) for 1 h at room temperature, and reacted with 0.02% DAB and 0.01% H2 O2 for approximately 3 min. After each incubation step, the sections were washed three times with PBS for 5 min. Finally, the sections were mounted on gelatin-coated slides, dehydrated in an ascending alcohol series, and cleared in xylene.
(40-cm long and 3-cm wide with 12-cm high walls) in which the arms are at 120◦ angles from each other. The maze floor and walls were constructed from dark opaque polyvinyl plastic as previously described (Kim et al., 2006). Animals were initially placed within one arm, and the sequence (i.e., ABCCAB) and number of arm entries were recorded manually for each animal over an 8-min period. A spontaneous alternation was defined as entries into all three arms on consecutive choices (i.e., ABC, CAB, or BCA, but not BAB). Maze arms were thoroughly cleaned with water between animals to remove residual odors. One hour after the last administration of each drug or vehicle, gerbils were gently placed in the maze. The percentage of alternations was defined according to the following equation: % Alternation = [(Number of alternations)/(Total arm entries − 2)] × 100. The number of arm entries serves as an indicator of locomotor activity.
2.6. Y-maze task 2.7. The novel object recognition task We conducted a Y-maze task to investigate the ameliorating effects of KOK on the hippocampal-dependent short-term memory at 4 days after BCCAO. The Y-maze is a three-arm horizontal maze
Cognitive function was tested using a novel object recognition task at 5 days after BCCAO. The novel object recognition protocol
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was specifically designed to minimize spatial learning and hippocampal involvement (Dere et al., 2005; Bevins and Besheer, 2006; Reger et al., 2009). On day 1 (habituation trial), each gerbil was exposed to the tub (41 cm × 41 cm × 41 cm high) individually for 5 min to habituate to the testing environment. On day 2 (familiarization trial), the animals were placed in the tub and given 5 min to explore two identical familiar objects. Twenty-four hours later (test trial), the gerbils were again placed in the tub with one familiar and one novel object (counter balanced across subjects) and given 5 min to investigate the items. All behavior data were recorded using Ethovision (Nodulus, Wageningen, The Netherlands). An investigator, who was blind to the experimental conditions, later scored the time spent exploring each object. Exploration was defined as direct sniffing or snout contact with the object. 2.8. Quantification and statistical analysis Cell counts in the hippocampal CA1 region were conducted using a computerized image analysis system (Leica Microsystems AG, Wetzlar, Germany). Cells were counted from three sections per animal by a person blind to the treatment group of each sample. Values are expressed as the means ± S.E.M. Data were analyzed with one-way analysis of variance followed by Newman–Keuls’s post hoc test for multiple comparisons. Statistical significance was set at P < 0.05. 3. Results 3.1. Effects of KOK on ischemia-induced delayed neuronal death in the hippocampal CA1 region Representative photomicrographs of the hippocampal CA1 regions are shown in Fig. 2A and C. Marked cell losses in this hippocampal subfield were observed in the ischemic control group. Significant group effects on surviving cell numbers were observed [F (6, 32) = 9.972, P < 0.05, Fig. 2B]. The mean number of surviving cells in the CA1 region of the ischemic control group was significantly lower than that in the sham group (P < 0.05), and a significant increase in the number of surviving cells was observed in the KOK (2 g/kg)- or MK-801-treated groups compared with the ischemic control group (P < 0.05). Moreover, significant group effects were also observed in the number of degenerating cells detected by FJ-B staining [F (6, 32) = 27.27, P < 0.05, Fig. 2D]. The increase in degenerating cells induced by ischemic insults was significantly decreased by the administration of KOK (1 or 2 g/kg, P < 0.05) or MK-801. 3.2. Effects of KOK on ischemia-induced glial activation In the sham group, microglial cells immunostained with the anti-Iba-1 antibody were scattered and had a ramified form, indicating an inactivate state (Fig. 3A). In the vehicle-treated ischemic control group, microglial cells in the CA1 region were condensed and hypertrophied, indicating an active state. The number of activated microglial cells was significantly decreased by the administration of KOK (2 g/kg, for 7 days) or MK-801 [F (6, 32) = 8.779, P < 0.05, Fig. 3B]. The activated astrocytes, which were positively immunostained with the anti-GFAP antibody, were significantly increased in the CA1 region of the ischemic control group compared with the sham group (P < 0.05, Fig. 3C). The administration of KOK (2 g/kg, 7 days) or MK-801 significantly reduced the number of activated astrocytes [F (6, 32) = 7.068, P < 0.05, Fig. 3D]. 3.3. Effects of KOK on ischemia-induced IL-1ˇ elevation Pro-inflammatory cytokines, including IL-1, promote systemic inflammation following ischemic insults (Boutin et al.,
Fig. 4. The effects of Kyung-Ok-Ko (KOK) on interleukin-1 (IL-1) expression after transient global ischemia induced by bilateral common carotid artery occlusion (BCCAO) for 5 min followed by reperfusion for 7 days. KOK (0.25, 0.5, 1, or 2 g/kg/day, p.o.) was administered immediately after reperfusion and once a day for the next 7 days. (A) Representative photomicrographs of IL-1-immunopositive cells in the hippocampal region. (B) Numbers of IL-1-immunopositive cells in CA1 at 7 days post-BCCAO. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.05, compared with the sham control; # P < 0.05, compared with the untreated ischemic control group. Magnification: 400×. Bar = 100 m.
2001). Therefore, we investigated whether KOK attenuates IL-1 expression levels in transient global ischemia. The number of IL-1immunopositive cells was significantly increased in the ischemic control group, but this decrease was rarely observed in the sham group (Fig. 4A). Administration of KOK (1 or 2 g/kg) or MK-801 significantly decreased the number of IL-1-immunopositive cells [F (6, 32) = 8.801, P < 0.05, Fig. 4B]. 3.4. Effects of KOK on ischemia-induced memory impairment There was a significant group effect in terms of spontaneous alternation behavior in the Y-maze task [F (6, 32) = 6.436, P < 0.05, Fig. 5A]. The number of spontaneous alternations in the ischemic control group was significantly lower than that in the sham group
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Fig. 5. Effects of Kyung-Ok-Ko (KOK) on transient cerebral global ischemia-induced memory deficits in the Y-maze and novel object recognition tasks. Transient global ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 5 min reperfusion. Gerbils were orally treated with KOK (0.25, 0.5, 1, or 2 g/kg) for 7 days. The final administration was performed 60 min before the behavioral task. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. During the administration of KOK, Y-maze task was conducted at 4 day after reperfusion (A, spontaneous alternation behavior; B, number of arm entries). The novel object recognition task was performed from day 5 after reperfusion (C, percentage of exploration time; D, total exploration time). Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.5, compared with the sham group in A; # P < 0.05, compared with the vehicle-treated ischemic control group; *P < 0.05, compared with the sham group; $ P < 0.05, compared with the novel object within same-treated group in C.
(P < 0.05). Moreover, the reduced levels of spontaneous alternation induced by ischemia were significantly reversed by the administration of KOK (2 g/kg, p.o., 7 days) or MK-801 (P < 0.05, Fig. 5A). The total number of arm entries was similar across all experimental groups [F (6, 32) = 2.501, P > 0.05, Fig. 5B]. In the object recognition test, there were significant group effects in terms of the percentage of time spent exploring novel objects [F (6, 32) = 6.798, P < 0.05]. Sham animals were able to successfully discriminate between the novel and familiar objects (P < 0.05), whereas the ischemic control group was not (P > 0.05). However, the percentage of time spent exploring the novel object was significantly higher in the KOK (1 or 2 g/kg)- or MK-801-treated group than in the ischemic control group (P < 0.05, Fig. 5C). There were no significant differences between groups regarding the total exploration time [F (6, 32) = 0.623, P > 0.05, Fig. 5D]. 4. Discussion In the present study, we observed that KOK inhibited the delayed neuronal cell death in the hippocampal CA1 region induced by transient cerebral global ischemia. In addition, KOK attenuated the ischemia-induced activation of microglia and astrocytes and inhibited the expression of IL-1 in the hippocampal CA1 region. These beneficial effects of KOK on the ischemia were also confirmed by behavioral tasks. Transient cerebral ischemia causes delayed neuronal injuries in the hippocampal CA1 region (Pulsinelli et al., 1982; Hagan and Beaughard, 1990; Hartman et al., 2005). Several factors, including free radicals, eicosanoids, lipid degradation products, and immune responses, can instigate secondary detrimental reactions within the
central nervous system. These factors are released or activated over a period of time starting from within a few seconds to days after the primary ischemic insult and may act either sequentially or in parallel to cause delayed neuronal cell death (Harukuni and Bhardwaj, 2006; Kumar, 2006). In the present study, we also observed that transient global ischemia in gerbils caused marked and delayed neuronal cell death in the hippocampal CA1 region 7 days after reperfusion. In addition, treatment with KOK for 7 days was found to reduce delayed neuronal cell death in the CA1 region as measured by Nissl and FJ-B staining. Unfortunately, we did not observe any protective effects following single administration of KOK (data not shown). Inflammation is a defense reaction against several insults that serves to remove noxious agents or to limit their detrimental effects. There is increasing evidence that post-ischemic inflammation plays an important role in ischemic brain damage (Kriz, 2006). Transient cerebral global ischemia induces the activation and proliferation of glial cells, including microglia and astrocytes (Petito et al., 1990; Denes et al., 2007). Microglial cells are resident macrophages that play critical roles as resident immunocompetent and phagocytic cells in the brain. They are rapidly activated after brain damage (Lakhan et al., 2009). Astrocytes, like microglia, are capable of secreting inflammatory factors, such as cytokines, chemokines, or nitric oxide (Swanson et al., 2004). The most studied cytokine related to inflammation in acute ischemic stroke is IL-1. IL-1 is primarily neurotoxic and is involved in the pathogenesis of hypoxic ischemic brain damage (Clausen et al., 2008; Kleinig and Vink, 2009). Following the transient cerebral global ischemia, the number of activated microglial cells and astrocytes in the hippocampal CA1 region was markedly increased in the
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untreated ischemic control group. The sub-chronic administration of KOK attenuated the activation of microglial cells and astrocytes in hippocampal CA1 regions on day 7, as measured by Iba-1 and GFAP immunostaining, respectively. In addition, KOK also inhibited the expression IL-1, a pro-inflammatory cytokine. These results suggest that KOK could exert neuroprotective effects through its anti-inflammatory properties. It is well known that transient cerebral global ischemia leads to delayed neuronal cell death and results in memory impairment (Fox et al., 1998; Pullela et al., 2006). In the present study, we employed the Y-maze task to evaluate hippocampal-dependent short-term spatial memory and a novel object recognition task to test hippocampal-independent working recognition memory (Lelong et al., 2003; Thompson et al., 2005). We observed that transient global ischemia caused memory impairments in both the Y-maze and the novel object recognition tasks. In addition, KOK significantly improved the transient cerebral ischemia-induced cognitive impairments. These results suggest that KOK exhibits anti-inflammatory activity, resulting in neuroprotective effects and the amelioration of cognitive dysfunction. In the present study, KOK exerted its beneficial activities against cerebral global ischemia at a dose of 2 g/kg. This dose is likely to be high compared to the general pharmacologically effective dose of a single compound. However, KOK is a viscous extract containing a high level of water content (over 30%). Compared to the clinic dosage for adults (12 g/kg), the effective dose in mice (2 g/kg) would be acceptable based on the report by others (Reagan-Shaw et al., 2008). In conclusion, KOK significantly attenuated inflammatory responses and rescued neuronal cells from the transient global cerebral ischemia-mediated delayed neuronal cell death in the hippocampal CA1 region of gerbils. Concomitantly, KOK ameliorated the memory impairment induced by ischemia in a dose-dependent manner. Taken together, our present findings suggest that KOK has therapeutic potential for the treatment of delayed neuronal cell death following cerebral ischemia. Acknowledgement This research was supported by Grants from the Kwang Dong Pharmaceutical Co., Ltd. in Korea. References Bevins, R.A., Besheer, J., 2006. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ‘recognition memory’. Nature Protocols 1, 1306–1311. Boutin, H., LeFeuvre, R.A., Horai, R., Asano, M., Iwakura, Y., Rothwell, N.J., 2001. Role of IL-1␣ and IL-1 in ischemic brain damage. The Journal of Neuroscience 21, 5528–5534. Cai, Z.Y., Yan, Y., Sun, S.Q., Zhang, J., Huang, L.G., Yan, N., Wu, F., Li, J.Y., 2008. Minocycline attenuates cognitive impairment and restrains oxidative stress in the hippocampus of rats with chronic cerebral hypoperfusion. Neuroscience Bulletin 24, 305–313. Clausen, B.H., Lambertsen, K.L., Babcock, A.A., Holm, T.H., Dagnaes-Hansen, F., Finsen, B., 2008. Interleukin-1 and tumor necrosis factor-␣ are expressed by different subsets of microglia and macrophages after ischemic stroke in mice. Journal of Neuroinflammation 5, 46. Denes, A., Vidyasagar, R., Feng, J., Narvainen, J., McColl, B.W., Kauppinen, R.A., Allan, S.M., 2007. Proliferating resident microglia after focal cerebral ischaemia in mice. Journal of Cerebral Blood Flow & Metabolism 27, 1941–1953. Dere, E., Huston, J.P., De Souza Silva, M.A., 2005. Integrated memory for objects, places, and temporal order: evidence for episodic-like memory in mice. Neurobiology of Learning and Memory 84, 214–221. Endoh, M., Maiese, K., Wagner, J., 1994. Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia. Brain Research 651, 92–100. Fox, G.B., Fan, L., LeVasseur, R.A., Faden, A.I., 1998. Effect of traumatic brain injury on mouse spatial and nonspatial learning in the Barnes circular maze. Journal of Neurotrauma 15, 1037–1046. Gackowski, D., Rozalski, R., Siomek, A., Dziaman, T., Nicpon, K., Klimarczyk, M., Araszkiewicz, A., Olinski, R., 2008. Oxidative stress and oxidative DNA damage is characteristic for mixed Alzheimer disease/vascular dementia. Journal of Neurological Science 266, 57–62.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Neuroprotective effects of a traditional herbal prescription on transient cerebral global ischemia in gerbils Mudan Cai a,1 , Bum Young Shin b,1 , Dong Hyun Kim a , Jong Min Kim a , Se Jin Park a , Chan Sung Park c , Do Hee Won c , Nam Doo Hong c , Dong Hyo Kang d , Yamamoto Yutaka d , Jong Hoon Ryu a,b,∗ a
Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemoon-ku, Seoul 130-701, Republic of Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong, Dongdaemoon-ku, Seoul 130-701, Republic of Korea c R&D Center, Kwang Dong Pharmaceutical Co., Ltd., 621-1 Jangdang-dong, Pyongtaek-si, Kyonggi-do 459-020, Republic of Korea d Tochimoto Tenkaido Co., Ltd., 3-21, Suehiro-cho, Kita-ku, Osaka-city, Osaka 530-0053, Japan b
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 8 October 2011 Accepted 10 October 2011 Available online 14 October 2011 Keywords: Kyung-Ok-Ko Global ischemia Hippocampal cell death Inflammation
a b s t r a c t Aim of the study: Kyung-Ok-Ko (KOK), a traditional herbal prescription composed of Rehmannia glutinosa var. purpurae, Panax ginseng, Poria cocos, Lycium chinense, Aquillaria agallocha and honey, has been used to treat age-related symptoms, such as amnesia or dementia, and has been shown to ameliorate scopolamine-induced memory impairment in mice. However, the effects of KOK on transient cerebral global ischemia-induced brain damage are unclear. Materials and methods: Transient cerebral global ischemia was induced by occluding the bilateral common carotid artery for 5 min followed by reperfusion for 7 days. KOK (0.25, 0.5, 1, or 2 g/kg) was administered orally immediately after reperfusion and once a day over the next 7 days. Y-maze or novel object recognition tasks were to analyze learning and memory capabilities at 4 or 5 days after reperfusion, respectively. Histochemistry and immunohistochemistry were used for evaluation of the effect of KOK on neuronal degeneration. Results: Histochemical studies showed that KOK increased the number of viable cells detected by Nissl staining and decreased the number of degenerated neuronal cells detected by Fluoro-Jade B staining in the hippocampal CA1 region. In the immunohistochemical study, the sub-chronic KOK administration attenuated the ischemia-induced activation of microglia and astrocytes and the increase of cytokine IL-1 (P < 0.05). In addition, KOK administration significantly attenuated the ischemia-induced cognitive impairments observed in the Y-maze and novel object recognition tasks (P < 0.05). Conclusion: These findings suggest that the neuroprotective effects of KOK may be mediated by its antiinflammatory activities, resulting in the attenuation of memory impairment. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cerebral ischemic injury resulting from either focal or global circulatory arrests in the brain, is one of the major causes of death and disability in adults (Pinkston et al., 2009). Global cerebral ischemia is a clinical outcome occurring as a consequence of cardiac arrest, reversible severe hypotension or other situations that deprive the brain of oxygen and glucose (Nedergaard and Diemer, 1988; White et al., 1993). Multiple factors, including
∗ Corresponding author at: Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, #1 Hoeki-dong, Dongdaemoon-ku, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 9230; fax: +82 2 966 3885. E-mail address: [email protected] (J.H. Ryu). 1 1 These authors contributed equally to this paper. 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.016
excitotoxicity, oxidative stress, and inflammatory cytokines (tumor necrosis factor-␣, interleukin (IL)-1 or IL-6), are responsible for hippocampal neuronal damage (Endoh et al., 1994; Kasparova et al., 2005; Pinkston et al., 2009; Ikonomidou and Kaindl, 2010a,b, 2011), especially the pyramidal neurons of the CA1 region in the hippocampus (Kirino, 2000). Among these factors, the inflammatory response is a delayed process and could be a potential target for the treatment of brain ischemia (Yrjanheikki et al., 1999; Tuttolomondo et al., 2009). In line with inflammation in ischemic brain, glial cells including microglia and astrocytes are also involved in neuronal degeneration (Petito et al., 1990; Denes et al., 2007). Based on these observations, many drugs have been tested for their abilities to delay neuronal death, including cyclooxygenase2 inhibitors (Gackowski et al., 2008; Hamel et al., 2008), nuclear factor-B inhibitors (Ridder and Schwaninger, 2009), inducible nitric oxide synthase inhibitors (Cai et al., 2008; Shin et al., 2010),
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and inhibitors against glial cell activation (Muramatsu et al., 2004; Lee et al., 2011). However, when selective anti-inflammatory or anti-excitatory agents were applied in ischemic brain damage, satisfactory outcomes were not obtained (Guo et al., 2009). Kyung-Ok-Ko (KOK; Qiong-yu-gao in Chinese; Kei-gyoku-kou in Japanese) is a traditional herbal prescription that contains six ingredients: Rehmannia glutinosa var. purpurae, Panax ginseng, Poria cocos, Lycium chinense, Aquillaria agallocha and honey. It has been used for age-related symptoms, such as amnesia and dementia in East Asia (Hur, 1999). Owing to the traditional use of KOK, we observed that KOK ameliorated scopolamine-induced memory impairments in mice (Shin et al., 2009). Some studies on the medicinal properties of KOK have also focused on immunological activities (Lee et al., 2002), inflammation (Lee et al., 2008), or gastric ulcers (Whang et al., 1994). From these studies, we hypothesized that KOK may be effective on inflammation-related brain disorders which exhibit memory impairment. However, no attempts have been made to investigate whether KOK has neuroprotective or ameliorative effects against ischemic brain damage-induced memory impairment. In recent years, various tries to develop antiischemic drugs from natural products are being conducted (Gupta et al., 2010). Therefore, the purpose of this study was to investigate whether KOK has neuroprotective effects on transient cerebral global ischemia-induced neuronal damage and whether it ameliorates ischemia-induced learning and memory deficits.
fibrillary acidic protein (GFAP) antibody, cresyl violet acetate and Fluoro-Jade B (FJ-B) were purchased from Chemicon (Temecula, CA). Biotinylated secondary antibody and avidin–biotin-peroxidase complex (ABC) kits were purchased from Vector (Burlingame, CA). MK-801, bovine serum albumin (BSA), and 3,3 -diaminobenzidine tetrahydrochloride (DAB) were purchased from Sigma Chemical Co. (St. Louis, MO). Zoletil 50® was purchased from Virbac laboratory (Carros, France). All other materials were of the highest grade commercially available. 2.3. Sample preparation Kyung-Ok-Ko (100 g) was prepared following method. Juice of root of Rehmannia glutinosa Liboschitz var. purpurae Makino (Scrophulariaceae) (32.0 g), powder of dried root of Panax ginseng C.A. Meyer (Araliaceae) (2.8 g), powder of cortex of Poria cocos Wolf (Polyporaceae) (8.0 g), powder of dried fruit of Lycium chinense Miller (Solanaceae) (0.9 g), powder of resin of Aquillaria agallocha Roxburgh (Thymelaeaceae) (0.1 g), honey (38.5 g), and simple syrup (17.7 g) were mixed and heated at 80 ◦ C in the water bath for 72 h. Viscous extract was obtained and used for present study. KOK (Lot No., OV30) standardized with 5-hydroxymethyl furaldehyde (9.4%) for consistency of quality was donated by Kwang Dong Pharmaceutical Co. (Pyongtaek, Korea). 2.4. Surgical procedure and drug administration
2. Materials and methods 2.1. Animals Mongolian gerbils (60–80 g) were purchased from the Orient Co., Ltd., a branch of Charles River Laboratories (Seoul, Korea). Animals were housed 4 per cage, allowed access to water and food ad libitum, and maintained under a constant temperature (23 ± 1 ◦ C) and humidity (60 ± 10%) under a 12-h light/dark cycle (light on 07:30–19:30 h). Animal treatment and maintenance were conducted in accordance with the Principle of Laboratory Animal Care (NIH Publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea. For the experiments, gerbils were divided into six groups [sham, n = 5; ischemia, n = 5; ischemia + KOK (0.25 g/kg), n = 5; ischemia + KOK (0.5 g/kg), n = 6; ischemia + KOK (1 g/kg), n = 6; ischemia + KOK (2 g/kg), n = 6; ischemia + MK-801, n = 6; a total of 39 animals were used]. 2.2. Materials The goat anti-ionized calcium-binding adaptor molecule-1 (Iba1) antibody and rabbit anti-interleukin-1 (IL-1) antibody were purchased from Abcam (Cambridge, UK). The mouse anti-glial
Animals were anesthetized with isoflurane (2.5% for induction, 1% for maintenance) in a mixture of nitrous oxide and oxygen (70:30), and the duration of anesthesia was no longer than 5 min. Transient cerebral ischemia was induced by the bilateral common carotid artery occlusion (BCCAO) as following. After making a median incision in the neck skin, both common carotid arteries were exposed and occluded with aneurysm clips for 5 min. Body temperature was maintained at 37 ± 0.5 ◦ C with a heating pad throughout surgery (Biomed S.L., Alicante, Spain). Circulation was restored by removing the clips. Animals receiving the same surgical operation without clipping the carotid arteries served as sham-operated controls. Regional cerebral blood flow (rCBF) was monitored using laser Doppler flowmetry (LDF; Perimed, PF5010, JarFalla, Sweden). Cyanoacrylate adhesives were used to attach flexible probes (model 407, Perimed, Jarfalla, Sweden) to the intact skull ±3.5 mm of the bregma. The change in rCBF was measured for 1 min immediately after occlusion and expressed as a percentage of the baseline value. The gerbil which showed over 80% of rCBF reduction was used for further study. After reperfusion, the animals were placed in a warm incubator (32–33 ◦ C). KOK (0.25, 0.5, 1, or 2 g/kg, p.o.) was administered once a day for 7 days beginning immediately after reperfusion until designated time points (Fig. 1). A group intraperitoneally treated with MK-801
Novel object recognition task
BCCAO
Y-maze task
Habituation trial
Familiarization trial
Testt trial and Tes perfusion for IHC
Day 0
Day 4
Day 5
Day 6
Day 7
KOK Fig. 1. Experimental procedures. BCCAO, bilateral common carotid artery occlusion; KOK, Kyung-Ok-Ko; IHC, immunohistochemistry.
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Fig. 2. Photomicrographs of Nissl (A) and Fluoro-Jade B (FJ-B, C) staining of the hippocampus, the percentages of viable neurons (B), and the number of FJ-B-positive cells (D) in CA1 at 7 days after transient cerebral global ischemia induced by bilateral common carotid artery occlusion (BCCAO) for 5 min followed by reperfusion. Kyung-Ok-Ko (KOK) (0.25, 0.5, 1, or 2 g/kg/day, p.o.) was administered immediately after reperfusion and once a day for the next 7 days. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.05, compared with the sham control; # P < 0.05, compared with the vehicle-treated ischemic control group. Magnification: 400×. Bar = 100 m.
(3 mg/kg, i.p.), an N-methyl-d-aspartate (NMDA) receptor antagonist, immediately after reperfusion was used as a positive control group (Kim et al., 2010). The last administration was completed 1 h before each experiment. In the sham and ischemic control groups, vehicle solution (10% Tween 80 solution) was administered orally using the same time schedule.
2.5.1. Nissl staining After the sections were mounted onto gelatin-coated slides, they were stained with 0.5% cresyl violet, dehydrated through graded alcohols (70%, 80%, 90%, and 100%, 2×), placed in xylene, and coverslipped using histomount medium.
2.5. Tissue preparation
2.5.2. Fluoro-Jade B staining To determine the extent of neuronal death in the hippocampus following KOK administration, FJ-B staining was conducted, as described elsewhere (Schmued and Hopkins, 2000a,b). In brief, the sections were mounted onto gelatin-coated slides in distilled water and sequentially placed in 100% ethanol for 3 min, 70% ethanol for 1 min, 30% ethanol for 1 min and distilled water for 1 min. The slices were then oxidized for 15 min using a 0.1% KMnO4 solution followed by brief rinses in distilled water for 1 min. The slides were then immersed in a 0.0015% solution of FJ-B in 0.09% acetic acid in the dark for 30 min, rinsed with distilled water, dried for 30 min at 37 ◦ C, cleared with xylene and coverslipped using DPX medium.
Immediately after object recognition test, animals were anesthetized with an intramuscular injection of Zoletil 50® (10 mg/kg), perfused transcardially with phosphate buffer (100 mM, pH 7.4) followed by ice-cold 4% paraformaldehyde and then decapitated. The brains were removed and postfixed in phosphate buffer (50 mM, pH 7.4) containing 4% paraformaldehyde overnight, then immersed in a 30% sucrose solution (in 50 mM phosphate-buffered saline, PBS) and stored at 4 ◦ C until sectioning. Brains were cut along in the coronal plane (30 m) using a cryostat (Leica, Nussloch, Germany) and kept in storage solution at 4 ◦ C.
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Fig. 3. Photomicrographs of Iba-1 (A) and GFAP (C) staining in the hippocampus and the number of Iba-1- (B) and GFAP- (D) positive cells at 7 days after transient cerebral global ischemia induced by bilateral common carotid artery occlusion (BCCAO) for 5 min followed by reperfusion. Kyung-Ok-Ko (KOK) (0.25, 0.5, 1, or 2 g/kg/day, p.o.) was administered immediately after reperfusion and once a day for the next 7 days. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.05, compared with the sham control; # P < 0.05, compared with the vehicletreated ischemic control group. Magnification: 400×. Bar = 100 m.
2.5.3. Immunohistochemistry Free floating sections were incubated for 24 h in PBS (room temperature) containing goat anti-Iba-1 (1:1000 dilution), mouse anti-GFAP (1:1000 dilution) or rabbit anti-IL-1 (1:200 dilution) antibody with 0.3% Triton X-100, 1% BSA, and 1.5% normal rabbit serum (for Iba-1), horse serum (for GFAP) or goat serum (for IL-1) from the ABC kit. The sections were then incubated with biotinylated secondary antibody (1:200 dilution) for 90 min, treated with ABC solution (1:100 dilution) for 1 h at room temperature, and reacted with 0.02% DAB and 0.01% H2 O2 for approximately 3 min. After each incubation step, the sections were washed three times with PBS for 5 min. Finally, the sections were mounted on gelatin-coated slides, dehydrated in an ascending alcohol series, and cleared in xylene.
(40-cm long and 3-cm wide with 12-cm high walls) in which the arms are at 120◦ angles from each other. The maze floor and walls were constructed from dark opaque polyvinyl plastic as previously described (Kim et al., 2006). Animals were initially placed within one arm, and the sequence (i.e., ABCCAB) and number of arm entries were recorded manually for each animal over an 8-min period. A spontaneous alternation was defined as entries into all three arms on consecutive choices (i.e., ABC, CAB, or BCA, but not BAB). Maze arms were thoroughly cleaned with water between animals to remove residual odors. One hour after the last administration of each drug or vehicle, gerbils were gently placed in the maze. The percentage of alternations was defined according to the following equation: % Alternation = [(Number of alternations)/(Total arm entries − 2)] × 100. The number of arm entries serves as an indicator of locomotor activity.
2.6. Y-maze task 2.7. The novel object recognition task We conducted a Y-maze task to investigate the ameliorating effects of KOK on the hippocampal-dependent short-term memory at 4 days after BCCAO. The Y-maze is a three-arm horizontal maze
Cognitive function was tested using a novel object recognition task at 5 days after BCCAO. The novel object recognition protocol
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was specifically designed to minimize spatial learning and hippocampal involvement (Dere et al., 2005; Bevins and Besheer, 2006; Reger et al., 2009). On day 1 (habituation trial), each gerbil was exposed to the tub (41 cm × 41 cm × 41 cm high) individually for 5 min to habituate to the testing environment. On day 2 (familiarization trial), the animals were placed in the tub and given 5 min to explore two identical familiar objects. Twenty-four hours later (test trial), the gerbils were again placed in the tub with one familiar and one novel object (counter balanced across subjects) and given 5 min to investigate the items. All behavior data were recorded using Ethovision (Nodulus, Wageningen, The Netherlands). An investigator, who was blind to the experimental conditions, later scored the time spent exploring each object. Exploration was defined as direct sniffing or snout contact with the object. 2.8. Quantification and statistical analysis Cell counts in the hippocampal CA1 region were conducted using a computerized image analysis system (Leica Microsystems AG, Wetzlar, Germany). Cells were counted from three sections per animal by a person blind to the treatment group of each sample. Values are expressed as the means ± S.E.M. Data were analyzed with one-way analysis of variance followed by Newman–Keuls’s post hoc test for multiple comparisons. Statistical significance was set at P < 0.05. 3. Results 3.1. Effects of KOK on ischemia-induced delayed neuronal death in the hippocampal CA1 region Representative photomicrographs of the hippocampal CA1 regions are shown in Fig. 2A and C. Marked cell losses in this hippocampal subfield were observed in the ischemic control group. Significant group effects on surviving cell numbers were observed [F (6, 32) = 9.972, P < 0.05, Fig. 2B]. The mean number of surviving cells in the CA1 region of the ischemic control group was significantly lower than that in the sham group (P < 0.05), and a significant increase in the number of surviving cells was observed in the KOK (2 g/kg)- or MK-801-treated groups compared with the ischemic control group (P < 0.05). Moreover, significant group effects were also observed in the number of degenerating cells detected by FJ-B staining [F (6, 32) = 27.27, P < 0.05, Fig. 2D]. The increase in degenerating cells induced by ischemic insults was significantly decreased by the administration of KOK (1 or 2 g/kg, P < 0.05) or MK-801. 3.2. Effects of KOK on ischemia-induced glial activation In the sham group, microglial cells immunostained with the anti-Iba-1 antibody were scattered and had a ramified form, indicating an inactivate state (Fig. 3A). In the vehicle-treated ischemic control group, microglial cells in the CA1 region were condensed and hypertrophied, indicating an active state. The number of activated microglial cells was significantly decreased by the administration of KOK (2 g/kg, for 7 days) or MK-801 [F (6, 32) = 8.779, P < 0.05, Fig. 3B]. The activated astrocytes, which were positively immunostained with the anti-GFAP antibody, were significantly increased in the CA1 region of the ischemic control group compared with the sham group (P < 0.05, Fig. 3C). The administration of KOK (2 g/kg, 7 days) or MK-801 significantly reduced the number of activated astrocytes [F (6, 32) = 7.068, P < 0.05, Fig. 3D]. 3.3. Effects of KOK on ischemia-induced IL-1ˇ elevation Pro-inflammatory cytokines, including IL-1, promote systemic inflammation following ischemic insults (Boutin et al.,
Fig. 4. The effects of Kyung-Ok-Ko (KOK) on interleukin-1 (IL-1) expression after transient global ischemia induced by bilateral common carotid artery occlusion (BCCAO) for 5 min followed by reperfusion for 7 days. KOK (0.25, 0.5, 1, or 2 g/kg/day, p.o.) was administered immediately after reperfusion and once a day for the next 7 days. (A) Representative photomicrographs of IL-1-immunopositive cells in the hippocampal region. (B) Numbers of IL-1-immunopositive cells in CA1 at 7 days post-BCCAO. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.05, compared with the sham control; # P < 0.05, compared with the untreated ischemic control group. Magnification: 400×. Bar = 100 m.
2001). Therefore, we investigated whether KOK attenuates IL-1 expression levels in transient global ischemia. The number of IL-1immunopositive cells was significantly increased in the ischemic control group, but this decrease was rarely observed in the sham group (Fig. 4A). Administration of KOK (1 or 2 g/kg) or MK-801 significantly decreased the number of IL-1-immunopositive cells [F (6, 32) = 8.801, P < 0.05, Fig. 4B]. 3.4. Effects of KOK on ischemia-induced memory impairment There was a significant group effect in terms of spontaneous alternation behavior in the Y-maze task [F (6, 32) = 6.436, P < 0.05, Fig. 5A]. The number of spontaneous alternations in the ischemic control group was significantly lower than that in the sham group
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Fig. 5. Effects of Kyung-Ok-Ko (KOK) on transient cerebral global ischemia-induced memory deficits in the Y-maze and novel object recognition tasks. Transient global ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 5 min reperfusion. Gerbils were orally treated with KOK (0.25, 0.5, 1, or 2 g/kg) for 7 days. The final administration was performed 60 min before the behavioral task. MK-801 (MK, 3 mg/kg) was administered immediately after reperfusion. During the administration of KOK, Y-maze task was conducted at 4 day after reperfusion (A, spontaneous alternation behavior; B, number of arm entries). The novel object recognition task was performed from day 5 after reperfusion (C, percentage of exploration time; D, total exploration time). Data represent means ± SEM (n = 5–6), assessed by one-way ANOVA and Newman–Keuls’s post hoc test. *P < 0.5, compared with the sham group in A; # P < 0.05, compared with the vehicle-treated ischemic control group; *P < 0.05, compared with the sham group; $ P < 0.05, compared with the novel object within same-treated group in C.
(P < 0.05). Moreover, the reduced levels of spontaneous alternation induced by ischemia were significantly reversed by the administration of KOK (2 g/kg, p.o., 7 days) or MK-801 (P < 0.05, Fig. 5A). The total number of arm entries was similar across all experimental groups [F (6, 32) = 2.501, P > 0.05, Fig. 5B]. In the object recognition test, there were significant group effects in terms of the percentage of time spent exploring novel objects [F (6, 32) = 6.798, P < 0.05]. Sham animals were able to successfully discriminate between the novel and familiar objects (P < 0.05), whereas the ischemic control group was not (P > 0.05). However, the percentage of time spent exploring the novel object was significantly higher in the KOK (1 or 2 g/kg)- or MK-801-treated group than in the ischemic control group (P < 0.05, Fig. 5C). There were no significant differences between groups regarding the total exploration time [F (6, 32) = 0.623, P > 0.05, Fig. 5D]. 4. Discussion In the present study, we observed that KOK inhibited the delayed neuronal cell death in the hippocampal CA1 region induced by transient cerebral global ischemia. In addition, KOK attenuated the ischemia-induced activation of microglia and astrocytes and inhibited the expression of IL-1 in the hippocampal CA1 region. These beneficial effects of KOK on the ischemia were also confirmed by behavioral tasks. Transient cerebral ischemia causes delayed neuronal injuries in the hippocampal CA1 region (Pulsinelli et al., 1982; Hagan and Beaughard, 1990; Hartman et al., 2005). Several factors, including free radicals, eicosanoids, lipid degradation products, and immune responses, can instigate secondary detrimental reactions within the
central nervous system. These factors are released or activated over a period of time starting from within a few seconds to days after the primary ischemic insult and may act either sequentially or in parallel to cause delayed neuronal cell death (Harukuni and Bhardwaj, 2006; Kumar, 2006). In the present study, we also observed that transient global ischemia in gerbils caused marked and delayed neuronal cell death in the hippocampal CA1 region 7 days after reperfusion. In addition, treatment with KOK for 7 days was found to reduce delayed neuronal cell death in the CA1 region as measured by Nissl and FJ-B staining. Unfortunately, we did not observe any protective effects following single administration of KOK (data not shown). Inflammation is a defense reaction against several insults that serves to remove noxious agents or to limit their detrimental effects. There is increasing evidence that post-ischemic inflammation plays an important role in ischemic brain damage (Kriz, 2006). Transient cerebral global ischemia induces the activation and proliferation of glial cells, including microglia and astrocytes (Petito et al., 1990; Denes et al., 2007). Microglial cells are resident macrophages that play critical roles as resident immunocompetent and phagocytic cells in the brain. They are rapidly activated after brain damage (Lakhan et al., 2009). Astrocytes, like microglia, are capable of secreting inflammatory factors, such as cytokines, chemokines, or nitric oxide (Swanson et al., 2004). The most studied cytokine related to inflammation in acute ischemic stroke is IL-1. IL-1 is primarily neurotoxic and is involved in the pathogenesis of hypoxic ischemic brain damage (Clausen et al., 2008; Kleinig and Vink, 2009). Following the transient cerebral global ischemia, the number of activated microglial cells and astrocytes in the hippocampal CA1 region was markedly increased in the
M. Cai et al. / Journal of Ethnopharmacology 138 (2011) 723–730
untreated ischemic control group. The sub-chronic administration of KOK attenuated the activation of microglial cells and astrocytes in hippocampal CA1 regions on day 7, as measured by Iba-1 and GFAP immunostaining, respectively. In addition, KOK also inhibited the expression IL-1, a pro-inflammatory cytokine. These results suggest that KOK could exert neuroprotective effects through its anti-inflammatory properties. It is well known that transient cerebral global ischemia leads to delayed neuronal cell death and results in memory impairment (Fox et al., 1998; Pullela et al., 2006). In the present study, we employed the Y-maze task to evaluate hippocampal-dependent short-term spatial memory and a novel object recognition task to test hippocampal-independent working recognition memory (Lelong et al., 2003; Thompson et al., 2005). We observed that transient global ischemia caused memory impairments in both the Y-maze and the novel object recognition tasks. In addition, KOK significantly improved the transient cerebral ischemia-induced cognitive impairments. These results suggest that KOK exhibits anti-inflammatory activity, resulting in neuroprotective effects and the amelioration of cognitive dysfunction. In the present study, KOK exerted its beneficial activities against cerebral global ischemia at a dose of 2 g/kg. This dose is likely to be high compared to the general pharmacologically effective dose of a single compound. However, KOK is a viscous extract containing a high level of water content (over 30%). Compared to the clinic dosage for adults (12 g/kg), the effective dose in mice (2 g/kg) would be acceptable based on the report by others (Reagan-Shaw et al., 2008). In conclusion, KOK significantly attenuated inflammatory responses and rescued neuronal cells from the transient global cerebral ischemia-mediated delayed neuronal cell death in the hippocampal CA1 region of gerbils. Concomitantly, KOK ameliorated the memory impairment induced by ischemia in a dose-dependent manner. Taken together, our present findings suggest that KOK has therapeutic potential for the treatment of delayed neuronal cell death following cerebral ischemia. Acknowledgement This research was supported by Grants from the Kwang Dong Pharmaceutical Co., Ltd. in Korea. References Bevins, R.A., Besheer, J., 2006. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ‘recognition memory’. Nature Protocols 1, 1306–1311. Boutin, H., LeFeuvre, R.A., Horai, R., Asano, M., Iwakura, Y., Rothwell, N.J., 2001. Role of IL-1␣ and IL-1 in ischemic brain damage. The Journal of Neuroscience 21, 5528–5534. Cai, Z.Y., Yan, Y., Sun, S.Q., Zhang, J., Huang, L.G., Yan, N., Wu, F., Li, J.Y., 2008. Minocycline attenuates cognitive impairment and restrains oxidative stress in the hippocampus of rats with chronic cerebral hypoperfusion. Neuroscience Bulletin 24, 305–313. Clausen, B.H., Lambertsen, K.L., Babcock, A.A., Holm, T.H., Dagnaes-Hansen, F., Finsen, B., 2008. Interleukin-1 and tumor necrosis factor-␣ are expressed by different subsets of microglia and macrophages after ischemic stroke in mice. Journal of Neuroinflammation 5, 46. Denes, A., Vidyasagar, R., Feng, J., Narvainen, J., McColl, B.W., Kauppinen, R.A., Allan, S.M., 2007. Proliferating resident microglia after focal cerebral ischaemia in mice. Journal of Cerebral Blood Flow & Metabolism 27, 1941–1953. Dere, E., Huston, J.P., De Souza Silva, M.A., 2005. Integrated memory for objects, places, and temporal order: evidence for episodic-like memory in mice. Neurobiology of Learning and Memory 84, 214–221. Endoh, M., Maiese, K., Wagner, J., 1994. Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia. Brain Research 651, 92–100. Fox, G.B., Fan, L., LeVasseur, R.A., Faden, A.I., 1998. Effect of traumatic brain injury on mouse spatial and nonspatial learning in the Barnes circular maze. Journal of Neurotrauma 15, 1037–1046. Gackowski, D., Rozalski, R., Siomek, A., Dziaman, T., Nicpon, K., Klimarczyk, M., Araszkiewicz, A., Olinski, R., 2008. Oxidative stress and oxidative DNA damage is characteristic for mixed Alzheimer disease/vascular dementia. Journal of Neurological Science 266, 57–62.
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