Comparison of neuroprotective effects of extract and fractions from Agarum clathratum against experimentally induced transient cerebral ischemic damage

Abstract

Contexts: Agarum clathratum (Laminariaceae), a typical brown algae, has been identified by National Plant Quarantine Service in Korea. The extract of A. clathratum has antioxidant activities.

Objective: We investigated the neuroprotective effects of crude-extract, ethyl acetate (EA)-, n-butanol (BU)-, dichloromethane (DCM)- and n-hexane (Hx)-fractions from A. clathratum on ischemic damage in the gerbil hippocampal CA1 region (CA1) after 5 min of transient cerebral ischemia.

Materials and methods: Agarum clathratum was collected in Kangwon province (South Korea) and treated with 95% ethanol. The ethanol extract was suspended in distilled water and subjected to a series of partitions with EA, BU, DCM and Hx. Each of extract and fraction was orally administered with 50 mg/kg once a day for one week before ischemia--reperfusion (I-R).

Result: In the crude-extract-, EA- and BU-fraction-treated ischemia groups, we found strong neuroprotection in the CA1 – about 80–89% of CA1 pyramidal neurons survived. However, in the DCM- and Hx-fraction-treated ischemia groups, we did not find any significant neuroprotection. In addition, we observed changes in astrocytes and microglia in the ischemic CA1. In the crude-extract, EA- and BU-fraction-treated ischemia groups, the distribution pattern and activity of the glial cells were similar to that found in the sham group.

Discussion: Repeated supplements of crude-extract, EA- and BU-fractions of A. clathratum could protect neurons from I-R injury in the hippocampal CA1 induced by transient cerebral ischemia via decrease of glial activation.

• Gerbil hippocampus
• gliosis
• ischemia--reperfusion injury
• neuroprotection
• pyramidal neurons

Introduction

Conditions in which the brain temporarily becomes hypoxic, such as stroke, cardiac arrest, cardiovascular surgery and neurosurgical procedures, can cause death and disability (Olson & McKeon, 2004). Transient cerebral ischemia is induced by temporary deprivation of blood flow to the brain. It results in the insidious delayed degeneration of specific vulnerable neurons in some specific regions such as the hippocampus, neocortex and striatum. In particular, pyramidal neurons in the hippocampal CA1 region are vulnerable to transient cerebral ischemia (Kirino, 1982). Neuronal damage of the hippocampal CA1 region following ischemic injury occurs very slowly for several days. This phenomenon is called “delayed neuronal death (DND)” (Kirino & Sano, 1984). The death of the CA1 pyramidal neurons induced by transient cerebral ischemia can cause various neurological dysfunctions such as depression and memory deficits (Provinciali & Coccia, 2002; Schwartz et al., 1998).

In the central nervous system, astrocytes play an important role in several processes, such as neurotransmission and neuronal functions (Horner & Palmer, 2003). In addition, microglia, one of the main immune cells in the central nervous system, play important roles in defending the brain against various pathological events. It is well known that microglia participate in the regeneration and repair of damaged neurons and cause neuronal death or dysfunction (Boje & Arora, 1992; Nakajima & Kohsaka, 1993). Whenever it is injured, the central nervous system undergoes a damage response of glial cells, usually called reactive gliosis or glial scarring (Fawcett & Asher, 1999).

Marine algae are classified into brown, red and green algae, on the basis of their pigmentation. Brown algae are a common food all over the world, especially in Asia (Pangestuti & Kim, 2011). Extracts of various edible seaweeds display pharmacologic activities, including antioxidant, anti-inflammatory, antitumor and antimicrobial activities (Bocanegra et al., 2009; MacArtain et al., 2007; Wijesekara et al., 2010). For example, Laminaria japonica (Laminariaceae), which is a kind of brown algae that inhabits the temperate seaside areas of the northwest Pacific including Korea, Japan and China, has high nutritional value (Park et al., 2011a). Fucoidan, extracted from L. japonica, has many biological actions such as anti-inflammatory and antioxidative effects, and it shows potent neuroprotective effects (Cui et al., 2012; Ha et al., 2009; Park et al., 2011a).

Agarum clathratum (Laminariaceae) is a typical representative of brown algae (Choi & Kim, 2012). Some researchers have reported that many brown algae including A. clathratum and L. japonica have a common chemical composition and active constituents, such as lipids, sterols, terpenes and polysaccharides (Kamenarska et al., 2002; Obluchinskaia, 2008). However, no study regarding its neuroprotective effects in in vivo models of ischemic cerebral stroke has been reported. In the present study, we investigate the neuroprotective effects of five different extracts and fractions from A. clathratum on the gerbil following 5 min of transient cerebral ischemia. This is a good model of transient cerebral ischemia because the animal does not have interconnections between the carotid and vertebro-basilar circulation (Horiguchi et al., 2002; Jig et al., 2007).

Materials and methods

Preparation of A. clathratum extract and fractions

Agarum clathratum was collected by Professor Sang Guan You during a one-week period in Kangwon province (South Korea), in March 2011; it was identified and authenticated by Professor Il-Jun Kang. The voucher specimen was deposited at the Regional Innovation Center, Hallym University, Chuncheon, Korea. Briefly, A. clathratum was placed in 95% ethanol at 60 °C for 2 h. This procedure was repeated three times. After centrifugation, the supernatant was collected and concentrated using a rotary evaporator and vacuum drier. The ethanol extract was suspended in distilled water and subjected to a series of partitioning with n-hexane, dichloromethane, ethyl acetate and n-butanol. The mass of crude extract (95% EtOH) and fractions was 840.46 mg.

Experimental animals

We used the progeny of male adult Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Kang won University, Chuncheon, South Korea. Mongolian gerbils were used at 6 months (adult, B.W. 65–75 g) of age. The animals were housed in a conventional cage under adequate temperature (23 °C) and humidity (60%) control, 12 h light/dark cycle, and free access to water and food. Animal handling and care followed the guidelines of current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985, revised 1996) and were approved (approval no.: Hallym-1-35) by the Hallym’s Medical Center Institutional Animal Care and Use Committee (IACUC). All the experiments were conducted to minimize the number of animals used and avoid animal suffering.

Treatment of A. clathratum extract and fractions

The animals were divided into seven groups (n = 7 in each group): sham-operated group (sham group); vehicle-treated ischemia group; 50 mg/kg crude-extract-treated ischemia groups; 50 mg/kg EA-fraction-treated ischemia groups; 50 mg/kg BU-fraction-treated ischemia groups; 50 mg/kg DCM-fraction-treated ischemia groups; 50 mg/kg Hx-fraction-treated ischemia group. Each tested extract and fraction was dissolved in 7% ethyl alcohol. The drugs were intraperitoneally administered once a day for one week before ischemic surgery: the last treatment was at 30 min before the surgery. Our preliminary study with 25, 50 and 100 mg/kg of each extract and fraction showed significant effects in animals treated with 50 and 100 mg/kg, not with 25 mg/kg; therefore, we administered 50 mg/kg of each extract and fraction.

Induction of transient cerebral ischemia

Transient cerebral ischemia was developed following our previous method (Kim et al., 2013). The experimental animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. A midline ventral incision was then made in the neck, and bilateral common carotid arteries were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. The complete interruption of blood flow was confirmed by observing the central artery in retinae under an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. Restoration of the blood flow (reperfusion) was directly observed under the ophthalmoscope. Body (rectal) temperature was maintained under free-regulating or normothermic (37 ± 0.5 °C) conditions with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA) and a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. Thereafter, animals were kept on the thermal incubator (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature until the animals were euthanized. Sham groups were subjected to the same surgical procedures except that the common carotid arteries were not occluded.

Tissue processing for histology

The animals (n = 7 in each group) were anesthetized with pentobarbital sodium and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The brains were removed and post-fixed in the same fixative for 6 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Wetzlar, Germany) into 30 μm coronal sections, and they were then collected into six-well plates containing PBS.

Immmunohistochemistry for NeuN

To investigate the neuronal changes in the CA1 after transient cerebral ischemia, according to a previous study (Lee et al., 2013), NeuN immunoreactive staining was carried out in the present study. In brief, anti-neuronal nuclei (NeuN) were used. The sections, which were collected into six-well plates containing PBS after tissue processing, were sequentially treated with 0.3% hydrogen peroxide (H₂O₂) in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min. The sections were then incubated with diluted mouse anti-NeuN (1:1000, Chemicon, Temecula, CA) overnight at 4 °C. Thereafter, the tissues were exposed to biotinylated goat anti-mouse IgG (Vector, Burlingame, CA) and streptavidin peroxidase complex (1:200, Vector) and were visualized by staining with 3,3′-diaminobenzidine tetrahydrochloride in 0.1 M Tris-HCl buffer (pH 7.2) and mounted on gelatin-coated slides. After dehydration, the sections were mounted with Canada balsam (Kanto, Tokyo, Japan).

Fluoro-Jade B histofluorescence staining

To confirm neuronal death in the brain after transient forebrain ischemia, sham- and ischemia-operated animals (n = 7 in each group), Fluoro-Jade B histofluorescence (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration) was conducted five days after the ischemic surgery under the same conditions for all the animals. F-J B histofluorescence staining procedures were accomplished according to the method described by Candelario-Jalil et al. (2003). The sections were immersed in a solution containing 1% sodium hydroxide in 80% alcohol and then followed in 70% alcohol. Next, the sections were transferred to a solution of 0.06% potassium permanganate and then to a 0.0004% F-J C (Histochem, Jefferson, AR) staining solution. After washing, the sections were placed on a slide warmer (approximately 50 °C) and examined under an epifluorescent microscope (Carl Zeiss, Carl-Zeiss-Strasse, Germany) with blue (450–490 nm) excitation light and a barrier filter. In this method, neurons that undergo degeneration fluoresce brightly in comparison with the background.

Cell counts

All the measurements were performed to ensure objectivity in blind conditions (two observers for each experiment), control and experimental samples were assayed under the same conditions. The studied tissue sections were selected according to anatomical landmarks corresponding to AP from −1.4 to −1.8 mm in the gerbil brain atlas (Thiessen & Goar, 1970). The number of NeuN immunoreactive and F-J B-positive neurons was counted in a 250 × 250 μm square, applied approximately at the center of the CA1 region in the stratum pyramidale. Cell counts were obtained by averaging the total number of NeuN-immunoreactive and F-J B-positive neurons from each animal per group: a ratio of the count was calibrated as percent.

Immunohistochemistry for GFAP and Iba-1

To examine the change in astrocytes and microglia in the CA1 region after ischemia--reperfusion, we carried out immunohistochemical staining with rabbit anti-glial fibrillary acidic protein (GFAP, 1:800, Chemicon, Temecular, CA) for astrocytes, rabbit anti-ionized calcium-binding adapter molecule (Iba-1, 1:500, Wako, Tokyo, Japan) for microglia, and biotinylated goat anti-rabbit IgG (Vector, Burlingame, CA) for secondary antibody according to the above mentioned method (see NeuN immunohistochemistry). To quantitatively analyze GFAP and Iba-1 immunoreactivity, the corresponding areas of the hippocampal CA1 region were measured from 15 sections per animal. Images of all GFAP- and Iba-1-immunoreactive structures were taken from three layers (strata oriens, pyramidale and radiatum in the hippocampus proper) through an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss, Germany) connected to a PC monitor. Images were calibrated into an array of 512 × 512 pixels corresponding to a tissue area of 140 × 140 μm (40 × primary magnification). The densities of all GFAP- and Iba-1-immunoreactive structures were evaluated by optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log(256/mean gray level). The background OD was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the optical density of the image file was calibrated as % (relative optical density, ROD) using Adobe Photoshop version 8.0 (Adobe Systems, Inc., San Jose, CA) and then analyzed using NIH Image 1.59 software (National Institutes of Health, Bethesda, MD).

Statistical analysis

Data are expressed as the mean ± SEM. The data were evaluated by a one-way ANOVA SPSS program (IBM Corp., Armonk, NY), and the means was assessed using Duncan’s multiple-range test. Statistical significance was set at p < 0.05.

Results

Neuroprotective effects

We examined neuroprotective effects in the crude-extract-, EA-, BU-, DCM- and Hx-fraction-treated ischemia groups five days after ischemia--reperfusion using NeuN immunohistochemistry and F-J B histofluorescence.

NeuN-immunoreactive cells

We observed numerous NeuN-immunoreactive cells in all the hippocampal subregions of the sham group (Figure 1A and B). In the vehicle-treated ischemia group, a few NeuN-immunoreactive cells were stained in the stratum pyramidale of the ischemic hippocampal CA1 region. The mean percentage of NeuN-immunoreactive neurons was 8.3% of that in the sham group (Figure 1C and D).

Figure 1. NeuN immunohistochemistry of the CA1 region in the sham- (A and B), vehicle- (C and D), crude-extract- (E and F), EA-fraction- (G and H), BU- fraction- (I and J), DCM- fraction- (K and L) and Hx fraction- (M and N) treated ischemia groups five days after ischemia--reperfusion. Only the crude-extract-, EA- and BU-fraction-treated ischemia groups contain a larger amount of pyramidal neurons in the stratum pyramidale (SP) than the vehicle-treated ischemia group. SO, stratum oriens; SR, stratum radiatum. Scale bar = 200 μm (low-magnification photos, A, C, E, G, I, K and M), 50 μm (high-magnification photos, B, D, F, H, J, L and N). (O) Relative analysis as percent in the number of NeuN-immunoreactive neurons in the CA1 region (n = 7 per group; *p < 0.05, significantly different from the sham group, #p < 0.05, significantly different from the vehicle-treated ischemia group). The bars indicate the means ± SEM.

In the crude-extract-, EA- and BU-fraction-treated ischemia groups, many NeuN-immunoreactive cells were well preserved in the stratum pyramidale of the CA1 region (Figure 1E–J). The mean percentages of NeuN-immunoreactive neurons in these groups were similar to the value obtained for the sham group (about 86, 88 and 78% of the sham group) (Figure 1O). However, in the DCM- and Hx-fraction-treated ischemia groups, the mean percentages of NeuN-immunoreactive neurons in the stratum pyramidale were similar to the value found in the vehicle-treated ischemia group (Figure 1K–N).

F-J B positive cells

We conducted F-J B histofluorescence staining, to examine neuronal degeneration in the CA1 region after ischemia-reperfusion (Figure 2). In the sham group, we did not note F-J B-positive cells in the stratum pyramidale; however, we detected many F-J B-positive cells in the vehicle-treated ischemia group five days after ischemia--reperfusion (Figure 2A and B).

Figure 2. F-J B staining in the CA1 region of the sham- (A), vehicle- (B), crude-extract- (C), EA-fraction- (D), BU- fraction- (E), DCM- fraction- (F) and Hx fraction- (G) treated ischemia groups five days after ischemia--reperfusion. Many F-J B-positive cells are observed in the stratum pyramidale (SP) in the vehicle-treated ischemia group; however, F-J B-positive cells are hardly found in the crude-extract-, EA- and BU-fraction-treated ischemia groups. SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm. (H) Relative analysis as percent in the number of F-J B-positive cells in the CA1 region (n = 7 per group; *p < 0.05, significantly different from the sham-group, #p < 0.05, significantly different from the vehicle-treated ischemia group). The bars indicate the means ± SEM.

In the present study, we hardly found F-J B-positive cells in the crude-extract- and EA-fraction-treated ischemia groups (Figure 2C and D); in the BU-fraction-treated ischemia group, we detected a few F-J B-positive cells in the stratum pyramidale (about 7% of the vehicle-treated ischemia group) (Figure 2E and H). However, in the DCM- and Hx-fraction-treated ischemia groups, we verified many F-J B-positive cells in the stratum pyramidale (about 71 and 77% of the vehicle-treated ischemia group, respectively) (Figure 2F–H).

Glial activation

In the present study, we observed the activation of astrocytes and microglia in the ischemic hippocampal CA1 region.

Astrocytes activation

In the sham group, we detected GFAP-immunoreactive astrocytes as a resting form consisting of a small body with thread-like thin process were detected in the stratum oriens and radiatum as well as in the stratum pyramidale of the CA1 region (Figure 3A). In the vehicle-treated ischemia group, GFAP immunoreactivity apparently increased in all the layers, and the GFAP-immunoreactive astrocytes were in the active form with thickened processes (Figure 3B).

Figure 3. GFAP immunohistochemistry in the CA1 region of the sham- (A), vehicle- (B), crude-extract- (C), EA-fraction- (D), BU- fraction- (E), DCM- fraction- (F) and Hx fraction- (G) treated ischemia groups five days after ischemia--reperfusion. GFAP immunoreactivity (arrows) in the crude-extract-, EA- and BU-fraction-treated ischemia group is similar to that detected in the sham group. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 50 μm. H: Relative optical density as percent of GFAP-immunoreactive structures in each group (n = 7 per group; *p < 0.05, significantly different from the sham group, #p < 0.05, significantly different from the vehicle-treated ischemia group). The bars indicate the means ± SEM.

In the crude-extract-treated ischemia group, a few GFAP-immunoreactive astrocytes were in the activated form (Figure 3C); the EA- and BU-fraction-treated ischemia groups gave similar results (Figure 3D and E). In these groups, the morphology of GFAP-immunoreactive astrocytes was different than that in the vehicle-treated ischemia group and their GFAP-immunoreactivity was much lower than that observed in the vehicle-treated ischemia group (Figure 3H). However, in the DCM- and Hx-fraction-treated ischemia groups, reactive GFAP-immunoreactive astrocytes were present in a larger number as compared with the sham group, and their distribution pattern was similar to that obtained for the vehicle-treated ischemia group (Figure 3F and G).

Microglia activation

In the sham group, Iba-1-immunoreactive microglia showed a resting form that is involved in processes with web-like network characteristics; these microglia were scattered throughout all the layers or the CA1 region (Figure 4A). In the vehicle-treated ischemia group, many Iba-1-immunoreactive microglia aggregated in the stratum pyramidale of the CA1 region in their activated form, which have hypertrophied bodies and thickened processes (Figure 4B).

Figure 4. Iba-1 immunohistochemistry in the CA1 region of the sham- (A), vehicle- (B), crude-extract- (C), EA-fraction- (D), BU- fraction- (E), DCM- fraction- (F) and Hx fraction- (G) treated ischemia groups five days after ischemia--reperfusion. The pattern of microglia distribution in the crude-extract-, EA- and BU-fraction-treated ischemia group is similar to that detected in the sham group. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar = 50 μm. (H) Relative optical density as percent of Iba-1-immunoreactive structures in each group (n = 7 per group; *p < 0.05, significantly different from the-sham group, #p < 0.05, significantly different from the vehicle-treated ischemia group). The bars indicate the means ± SEM.

In the crude-extract-, EA- and BU-fraction-treated ischemia group, the distribution pattern of Iba-1-immunoreactive microglia was similar to that obtained in the sham group, and the activation of Iba-1-immunoreactive microglia was much lower than that verified in the vehicle-treated ischemia group (Figure 4C–E and H). However, we found many activated Iba-1-immunoreactive microglia in all the layers in the case of the DCM- and Hx-extract-treated ischemia groups; this finding has very similar results achieved for the vehicle-treated ischemia group (Figure 4F–H).

Discussion

In the present study, we used NeuN immunohistochemistry and Fluoro-Jade B histofluorescence to examine the neuroprotective effects of pre-treatment with five A. clathratum extracts against ischemic damage on the gerbil hippocampal CA1 region five days after ischemia--reperfusion.

We found a significant loss of NeuN-immunoreactive cells and a significant appearance of F-J B-positive cells in stratum pyramidale of the ischemic CA1 region in the vehicle-treated ischemia group five days after ischemia-reperfusion. This finding is consistent with previous studies reporting delayed neuronal death in the same animal model (Lee et al., 2012; Park et al., 2012; Yu et al., 2012). We observed neuroprotective effects on the ischemic CA1 region according to the sort of extract and fractions from A. clathratum. Among them, CA1 pyramidal neurons were well protected from ischemic damage in the crude-extract-, EA- and BU-fraction-treated ischemia groups. Previously, brown algae extracts have been reported to inhibit leukocyte recruitment, aid re-vascularization, and display antioxidant, anti-apoptosis and antitumor activities (Aisa et al., 2005; Wang et al., 2010). In particular, one of their extracts named as fucoidan has been reported to exhibit neuroprotective effect against N-methyl-d-aspartate (NMDA)-induced excitotoxicity, which is a continuous stimulation of nerve cells by glutamates (Ha et al., 2009). On the other hand, we found that treatment with the DCM- and Hx-fractions did not exert neuroprotective effects on the ischemic CA1 region. This finding may be related with the kinds of extract and fractions from A. clathratum.

In addition, in this study we examined degree of gliosis in the ischemic CA1 region treated with five A. clathratum extracts five days after ischemia--reperfusion using GFAP and Iba-1 immunohistochemistry.

Changes in astrocytes in the ischemic CA1 region are associated with neuronal death (Ordy et al., 1993; Petito & Halaby, 1993; Steward et al., 1992; Stoll et al., 1998). We have reported that a reduction in astrocyte activation in the ischemic CA1 region contributes to neuronal survival during transient cerebral ischemic damage (Park et al., 2011b; Yoo et al., 2010). In our present study, treatment with the crude-extract, EA- and BU-fractions markedly reduced astrocyte activation compared with the other fractions. This finding shows that the neuroprotective effects of the crude-extract, EA- and BU-fractions of A. clathratum may be closely related to attenuation of astrocyte activation.

Morphological and functional changes in microglia are involved in response to various neural environments (Hailer et al., 1996; Schwartz et al., 2006). Microglia activation is significantly increased in the infarct region induced by focal ischemia and in the CA1 region following transient cerebral ischemia (Mabuchi et al., 2000; Stoll et al., 1998; Sugawara et al., 2002). In the present study, Iba-1 immunoreactivity in microglia was apparently attenuated only in the crude-extract-, EA- and BU-fraction-treated ischemia groups. Therefore, our present finding indicates that the neuroprotective effects of the crude-extract, EA- and BU-fractions of A. clathratum may also be associated with an attenuation of microglia activation.

Conclusion

In the present study, we found that the crude-extract, EA- and BU-fractions from A. clathratum potentially protect CA1 pyramidal neurons from transient ischemic damage, and that the neuroprotective effects may be closely associated with a decrease in glial activation. Therefore, we suggest that extracts and fractions from A. clathratum must be useful in protecting neurons from ischemic injury in the brain.

Declaration of interest

The authors report no conflict of interest.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0007307 and 2011-0022812).

Acknowledgements

The authors would like to thank Mr Seung Uk Lee for his technical help in this study.