Effect of the Edible Mushroom Mycoleptodonoides aitchisonii on Transient Global Ischemia-Induced Monoamine Metabolism Changes in Rat Cerebral Cortex

Introduction

S ome nutrients and food components are now known to have neuroprotective effects, although it was previously thought that food components did not influence the central nervous system; however, only a few studies have examined the effects of mushrooms on brain functions. The edible and medicinal mushroom Dictyophora indusiata contains neuroprotective compounds against excitatory neurotoxins. ¹ Armillaria mellea, a fungus, had an effect on complete ischemia in mice. ² Recently we reported that the edible mushroom Mycoleptodonoides aitchisonii increases nerve growth factor and catecholamines in the rat brain. ³ In addition, an aqueous extract of M. aitchisonii has been shown to lower blood pressure in spontaneously hypertensive rats. ^4,5 These results suggest that other mushrooms may also impact important brain functions.

There are many diseases of the brain, but a significant cause of death and disability is stroke, a reduction in blood flow to the brain; however, an effective neuroprotective treatment has not yet developed for this disease. There are two main types of stroke, hemorrhagic and ischemic, with the former being a rupture of blood vessels in the brain and the latter a disruption of blood flow to the brain. Increased oxidative stress and inflammation are key components for neural cell death following ischemia. Several brain regions are vulnerable to ischemic damage, such as hippocampus, striatum, and cerebral cortex. ^6,7 Levels of some neurotransmitters such as dopamine (DA), norepinephrine, serotonin, ^8,9 acetylcholine, ^10,11 and glutamate ¹⁰ change following cerebral ischemic damage in brain regions; in addition, levels of monoamines ^9,12 and related enzymes ¹² change in the cortex following cerebral ischemia. Therefore, in this study, we investigated whether feeding rats an aqueous extract of M. aitchisonii affects functional changes in monoaminergic neurons of the cerebral cortex following ischemic damage.

Materials and Methods

M. aitchisonii, belonging to the Climacodontaceae family, is an edible mushroom mainly found in the Kashmir region of India and in Japan. The cap diameter size is approximately 3–8 cm, the color is white to yellow with a smooth surface, and it has a very short stem. M. aitchisonii grows on the trunk of dead beech trees. M. aitchisonii was cultured by Kirin Brewery Co., Ltd. (Tokyo, Japan) for this experiment, and aqueous extracts of M. aitchisonii were also obtained from Kirin. Freeze-dried powder of the fungus was added to a 20-fold volume of distilled water and heat-extracted for 1 hour. The extract was filtered through gauze, and a threefold volume of ethanol was added. Then sedimentation was allowed for 10 minutes with stirring followed by overnight without stirring at 4°C. Finally, the alcohol was evaporated to yield the final extract.

Thirteen-week-old male Wistar rats were purchased from Japan SLC (Shizuoka, Japan). Rats of all groups were raised on the specified experimental diet and were kept at 23±1°C room temperature with 12 hours of light. During the experimental period, tap water and feed were freely available. This experiment was carried out in accordance with the guidelines for the care and use of laboratory animals of the University of Shizuoka, which refer to those of the American Association for Laboratory Animal Science.

Bilateral occlusion of the common carotid arteries was performed by a modified method as previously described. ^{12, 13} Rats were anesthetized with intraperitoneally administered 50 mg/kg pentobarbital sodium. The bilateral common carotid arteries were gently separated from the carotid sheath and vagal nerves and occluded with small clamps (NAPOX C-17; Natsume Seisakusho, Tokyo) for 15 minutes, and after transient occlusion the clamps were removed for reperfusion. The sham-operated rats also had their bilateral common carotid arteries exposed.

Rats in the body weight range 300±10 g were divided into four experimental groups: control feed and sham-operated (CS) (n=7), control feed and occlusion operation on the bilateral common carotid vessels (CV) (n=11), aqueous extract of M. aitchisonii feed and sham-operated (MS) (n=7), and aqueous extract of M. aitchisonii feed and occlusion operation on the bilateral common carotid vessels (MV) (n=11). The experimental diet consisted of 20% casein, 21.28% sucrose, 42.57% starch, 5% cellulose, 5% corn oil, 5% minerals, 1% vitamins, and 0.15% choline chloride. When powdered experimental diet was administered to the rats, an adequate amount of tap water was added to create a pellet. The test diet feed group was given a pellet containing aqueous extract corresponding to 1 g of M. aitchisonii powder per rat. The test diet was started right after the operation and provided for 17 days. Seventeen days after the surgery, the rats were sacrificed, and cerebral cortex was isolated from the brain.

Wet brain tissue (1 g) was homogenized (Sonifier^® 250; Branson, Danbury, CT, USA) in 4 mL of 0.2 M perchloric acid buffer (pH 2.0) including 0.1 mM disodium EDTA and dl-isoproterenol hydrochloride as an internal standard and kept on ice for 1 hour. The homogenates were centrifuged at 20,000 g (model SCR20B centrifuge; Hitachi, Tokyo) for 15 minutes at 0°C. Then, 1 M sodium acetate was added to the supernatant, and the solution was passed through a cellulose acetate filter (pore size, 0.45 μm) (Advantec Toyo, Tokyo). The concentrations of the monoamines in the collected sample solutions were analyzed by high-performance liquid chromatography under the following conditions: the mobile phase was 0.1 M sodium acetate–citric acid buffer and 15% methanol containing disodium EDTA and 1-octanesulfonic acid sodium salt. The high-performance liquid chromatography system was equipped with a reversed-phase column (SC-5ODS, 3.0×150 mm; EICOM, Kyoto, Japan) and electrochemical detector (model ECD-300; EICOM). Recordings of chromatograms and all calculations were performed using an integrator (Borwin; Nihonbunkoh, Tokyo).

Data for the individual groups are expressed as mean±SEM values. Statistical analyses used GraphPad Prism 5 for Windows (GraphPad Software, San Diego, CA, USA), and data were analyzed by one-factor analysis of variance followed by Dunnett's multiple comparison test. A value of P<.05 was considered significant.

Results

During the experiment, body weight gains and food intakes were not significantly different among the four groups. The level in the cerebral cortex of the monoamine DA precursor 3,4-dihydroxyphenylalanine (DOPA) was significantly reduced in the ischemia CV group compared with the CS group (Fig. 1). The concentration of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) was affected like that of DOPA, and DOPAC concentrations were significantly higher in the MS group than in the CV group (Fig. 1). Homovanillic acid (HVA) is a metabolite of DOPAC, and its concentration was increased in the MV group compared with the CV group similar to those of DOPA and DOPAC. The turnover rate of DA, indicated by (DOPAC+HVA)/DA, in the CV group was the lowest among the four groups; in addition, it was significantly higher in the MV group than in the CV group, and the level of the MV group was almost the same as those in the CS and MS groups (Fig. 1). Concentrations of serotonin and its metabolite 5-hydroxyindole-3-acetic acid were significantly decreased in the CV group compared with the CS group; however, there was no significant difference between the CV and MV groups (Fig. 1). There were no differences in norepinephrine among the four groups.

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FIG. 1.

Neurotransmitter concentrations related to dopaminergic and serotoninergic neuron in rat cerebral cortex measured by high-performance liquid chromatography with electrochemical detection, 17 days after 15-minute ischemia-reperfusion of the common carotid arteries. Data are mean±SEM values. *P<.05, significantly different between groups. CS, sham operation and control feed; CV, bilateral occlusion of common carotid vessels operation and control feed; DA, dopamine; DOPA, 3,4-dihydroxyphenylalanine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5HIAA, 5-hydroxyindole-3-acetic acid; 5HT, serotonin; HVA, homovanillic acid; MS, sham operation and M. aitchisonii feed; 3MT, 3-methoxytyramine; MV, bilateral occlusion of common carotid vessels operation and M. aitchisonii feed. The turnover rate of DA is defined as (DOPAC+HVA)/DA.

Discussion

Concentrations of several neurotransmitters, including DA, norepinephrine, serotonin, ^8,9 acetylcholine, ^{10, 11} and glutamate ¹⁰ are changed in the brain for short or long periods after ischemia. The monoamines measured in our experiment are involved with dopaminergic and serotoninergic neurons in the cerebral cortex, and levels of monoamine-related molecules were dramatically changed after the ischemia. Changes in DOPA, DOPAC, and DA turnover rate (DOPAC+HVA)/DA suggested that dopaminergic neurons were damaged following ischemia, but the aqueous extract of M. aitchisonii prevented the reduction of dopaminergic neuron activity.

Cvejic et al. ¹² clearly showed that alterations in monoamine metabolism persist in the cerebral cortex and basal ganglia for a long period following a brief 15-minute brain ischemia. Following 15 minutes of ischemia, monoamine oxidase and catechol-O-methyl transferase activities were reduced in the cerebral cortex and basal ganglia after 7 days. DA is metabolized into DOPAC by monoamine oxidase, and DOPAC is metabolized into HVA by catechol-O-methyl transferase. Following reestablishment of brain recirculation, neurological deficits persist in spite of improving cerebral energy metabolism, which is partly attributable to the altered monoamine metabolism. Our results showed that the significantly decreased DOPA concentrations in the CV group suggested damage in dopaminergic neurons following the brief ischemia and also indicated that the synthesizing capacity was reduced. Furthermore, the DOPAC concentration and turnover rate of DA in the CV group were also reduced compared with the CS group, but those of the MV group were significantly higher than in the CV group. These data reflected that M. aitchisonii affected monoamine oxidase activity, or it may have influenced DA release or DA reuptake at the synapses of the dopaminergic neurons. In other words, M. aitchisonii has potential for protective or recovery effects on metabolic and synaptic functions of dopaminergic neurons following ischemia injury.

Huntington's disease is a hereditary polyglutamine disease that induces involuntary movement, dementia, impaired locomotion, and other complications. This disease is thought to be caused by excessive production of the source gene, the products of which then accumulate in neuronal cells, inducing neuronal cell death and abnormal functioning. Tanaka et al. ¹⁴ showed retardation in the onset of polyglutamine disease using a mouse model and the administration of drinking water with trehalose. Usually fungi carry a large amount of trehalose, and therefore they may help in preventing the onset of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and other neuronal diseases.

In conclusion, the aqueous extract of the edible mushroom M. aitchisonii affected dopaminergic neuron homeostasis in the cerebral cortex following ischemia. Only a few studies have examined the effects of edible mushrooms on brain function, and this study is the first experiment to show that M. aitchisonii impacts damaged brain. This research suggests the possibility that other edible mushrooms may influence brain function and protect against some diseases. However, the detailed mechanisms of this action and what kinds of compounds in M. aitchisonii are responsible for the protection against brain injury still need to be elucidated.