Hirosaki University Repository for Academic Resources

弘前大学博士（医学）掲載誌:体力・栄養・免疫学雑誌, 28, p.166-173, 201

生嶌, 健也

 
 
 
 
 
 
 
 
 
 
 
 
 
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Japanese

1 0 代前半におけるb r a c h i a l – a n k l e p u l s e w a v e v e l o c i t y の変動とその関連因子に関する研究

弘前大学博士（医学）掲載誌:Acta Otolaryngol, 138, p.471-474, 201

後藤, 真一

 
 
 
 
 
   
 
Relationship between cognitive function and balance in a community-dwelling 
population in Japan. 
 
 
 
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English

Relationship between cognitive function and balance in a community-dwelling population in Japan

弘前大学博士（医学）掲載誌:Acta Otolaryngol, 138, p.447-451, 201

三浦, 智也


2 
 
Abstract 
Conclusion: The present study indicates that the presence of epithelium-
derived thymic stromal lymphopoietin (TSLP) in the eustachian tubes plays 
an important role in the onset of eosinophilic otitis media (EOM). 
Objective: In the present study, we aimed to clarify the expression of TSLP, a 
key trigger of Th2-type allergic disease, in the middle ear mucosa of EOM. 
Methods: An immunohistological study of TSLP was conducted in patients 
with EOM and in animal models of EOM constructed by intraperitoneal and 
intratympanic injection of ovalbumin (OVA) for 7 days and 14 days. In 
addition, the messenger RNA (mRNA) expression of TSLP in the middle ear 
mucosa of the animal models was analyzed using real-time PCR, and was 
compared with that of the control animals. 
Results: Immunoreactivities for TSLP were observed in the middle ear 
mucosa around the tympanic ostium of the eustachian tube of patients with 
EOM. In the animal model, strong immunoreactivity for TSLP was also 
observed in the eustachian tube epithelium, and mRNA expression of TSLP 
in the 7-day stimulated animals was significantly higher than that in the 
controls. 
 
Key words: eosinophil, thymic stromal lymphopoietin, middle ear mucosa, 
eustachian tube 
 
 
 
 
 
3 
 
Introduction 
Eosinophilic otitis media (EOM) is an intractable otitis media often associated 
with bronchial asthma and is characterized by eosinophil dominant, highly 
viscous middle ear effusion (MEE). Tomioka et al. identified cases of this 
disease as intractable otitis media with bronchial asthma in 1993 [1] and 
termed the expression EOM [2] based on the critical feature of the 
accumulation of eosinophils in the MEE. 
Over the next two decades, clinical studies were primarily conducted in Japan. 
Diagnostic criteria for EOM were initially proposed by Nagamine et al. in 
2002, and were confirmed by the EOM study group in 2011 [3] based on the 
clinical characteristics of EOM. In brief, the criteria were as follows: one 
major criterion, eosinophil dominant MEE, and four minor criteria, highly 
viscous MEE, resistance to conventional therapy for otitis media, association 
with bronchial asthma, and association with nasal polyposis. 
From the pathological point of view, several reports described the presence of 
IL-5 in MEE [4, 5] and IL-13 immunoreactivity in middle ear mucosa [6]. 
These Th2-type cytokines play important roles in the pathological features of 
EOM in the form of eosinophil infiltration caused by IL-5 and mucosal 
thickening of middle ear mucosa due to periostin induced by IL-13 [7]. 
Therefore, a Th2-type immune response is strongly suggested in EOM as well 
as various allergic diseases. 
In recent years, epithelium-derived cytokines, such as thymic stromal 
lymphopoietin (TSLP), have been shown to induce Th2-type responses that 
trigger several allergic diseases [8]. TSLP is an IL-7-like cytokine found in 
4 
 
the murine thymic stromal cell line as a growth factor that promotes 
lymphocyte differentiation [9, 10]. A strong correlation has been reported 
between TSLP and bronchial asthma, atopic dermatitis, and allergic rhinitis 
[8, 11]. However, there is no reported evidence of involvement of TSLP in the 
pathogenesis of EOM. In this study, we investigated the immunohistological 
localization and mRNA expression of TSLP in the middle ear mucosa of 
patients with EOM and animal models. 
 
Materials and methods 
This study was approved by the Ethics Committee of Hirosaki University 
School of Medicine and written informed consent was obtained from all 
participants. All animal experiments followed the Guidelines for Animal 
Experimentation, Hirosaki University. 
Tissue preparation 
Middle ear specimens were obtained from patients diagnosed with EOM, 
using established diagnostic criteria [2], who attended the Hirosaki 
University Hospital. Tissue samples of the granulated middle ear mucosa 
were obtained by the biopsy through the external auditory canal in five 
outpatients with EOM (No. 1, 2, 3, 4, 5). In one inpatient (No. 6) who 
underwent radical mastoidectomy at our hospital, samples were taken from 
several regions including mucosa around the tympanic ostium of the 
eustachian tube during surgery. The samples were immediately immersed in 
a 10% formaldehyde solution and paraffin-embedded sections (3 m) were 
prepared for immunohistological analysis. 
5 
 
The animal model of EOM was constructed using Hartley guinea pigs 
(weighing 250–350 g) as previously reported [7]. Animals were 
aluminum hydroxide (alum) on day 0, and with 
alum on days 7 and 14, for general sensitization. From day 21, they were 
ears. The daily application of OVA was continued for 7 days (OVA 7-day group; 
n = 5) or 14 days (OVA 14-day group; n = 7). Animals not receiving any 
sensitization were used as the control group (n = 6). All procedures were 
carried out under anesthesia with a mixture of medetomidine hydrochrol, 
midazolam, and butorphanol tartrate (0.23 mg, kg, 
respectively). Twenty four hours after the final OVA injection, the animals 
temporal bones on one side were then removed and fixed with 10% 
formaldehyde, and decalcified with EDTA 2Na in 0.1 M TRIS (pH 7.2). 
Thereafter, paraffin-
immunostaining. The temporal bones on the other side were cut off around 
the tympanic ostium of the eustachian tube and immediately put into 
RNAlater for real-time PCR. 
Immunohistology 
The sections were deparaffinized and rehydrated through a graded series of 
ethanol to phosphate-buffered saline (PBS). Then the specimens were 
incubated in the following solutions: (1) 0.3% H2O2 for 30 min; (2) Protein 
6 
 
Block Serum-Free (Code X0909, DAKO, Carpinteria, CA, USA) for 30 min; (3) 
anti-TSLP polyclonal antibody (ab47943, Abcam, Cambridge, UK) and 1% 
bovine 
2O2 for 5 min. Sections were 
rinsed well with PBS between each step. Samples were counterstained with 
hematoxylin and examined under a light microscope. 
Real-time PCR 
Total cellular RNA was extracted from the middle-ear mucosa and purified 
using a RNeasy mini kit (Qiagen GmbH, Strasse, Germany). Cloned DNA 
(cDNA) was prepared from 500 ng of total RNA by reverse transcription at 
37°C for 60 min and 95°C for 5 min using a High Capacity RNA-to-cDNA kit 
(Applied Biosystems, Foster City, CA, USA) in accordance with the 
manufacturer’s protocols. Quantitative real-time PCR was carried out using 
the TaqMan assay and GeneAmp PCR system (Applied Biosystems). The 
quantitative real-time PCR assay was based on primers that specifically 
amplify TSLP. The primers for TSLP -actin 
(Cq03755212_g1) were purchased from Applied Biosystems. The 
-actin were validated in 
a preliminary PCR analysis. Each sample was run in duplicate in separate 
tubes to permit quantification of the TSLP gene normalized to -actin. The 
7 
 
PCR conditioning consisted of an initial denaturation step at 95°C for 10 min, 
followed by 40 cycles of amplification at 95°C for 15 sec and 60°C for 60 sec. 
 
Data analysis 
Data analysis was performed using Bio-Rad CFX Manager 3.1-CFX connect 
(Applied Biosystems). For quantitative analysis, we used the comparative 
threshold cycle (Ct) method according to the manufacturer’s instructions 
-actin 
mRNA level. 
Unpaired t-test was performed using Stat Mate ver. 4.01 (ATMS, Tokyo, 
Japan) to compare the data obtained from the animal model and control. P < 
0.05 was considered statistically significant. 
 
Results 
Six patients with EOM were included in this study. Gender, age, and 
complications are listed in Table 1.  
TSLP immunoreactivities were detected in the epithelial cells of the middle 
ear mucosa around the tympanic ostium of the eustachian tube in one patient 
(No. 6; Fig.1A), and in the inflammatory cells in three patients (No. 3, 5, 6; 
Fig. 1B). TSLP-positive cells were also observed in the vascular endothelial 
cells in the sample from No. 6 (data not shown). No immunoreactivity for 
TSLP was shown in other three cases (No. 1, 2, 4). Strong immunoreactivity 
for TSLP was observed in the middle ear tissue near the eustachian tube in 
8 
 
the specimen collected during surgery (No. 6). 
In the middle ear mucosa from the OVA-stimulated animal models, a strong 
immunoreactivity for TSLP was observed in many eustachian tube epithelial 
cells (Fig. 2: A-1, B-1) in both the OVA 7-day and OVA 14-day groups. In the 
middle ear mucosa, TSLP-positive cells were detected around the tympanic 
ostium of the eustachian tube (Fig. 2; A-2, B-2). However, fewer cells showed 
TSLP immunoreactivity in sites farther from the tympanic ostium of 
eustachian tube (Fig. 2; A-3, B-3). When comparing the topical stimulation 
days, the TSLP immunoreactivity in the OVA 14-day group appeared stronger 
than that in the OVA 7-day group. 
No immunoreactivity for TSLP was observed in the control group (Fig. 2 C). 
In real-time PCR, the expression of TSLP mRNA was increased in the 
stimulated animal model OVA 7-day and OVA 14-day groups compared with 
the control group (Fig. 3). There is no significant difference between the OVA 
7-day and OVA 14-day groups (Fig. 3). 
 
Discussion  
EOM is well known as a high-risk disease for hearing loss. The EOM study 
group reported that 81 out of 138 cases of EOM showed bone conductive 
hearing loss (BCHL), and 8 cases experienced complete hearing loss [2]. High-
tone BCHL was also progressive, worsening as the period to appropriate 
treatment of the EOM became longer [12, 13]. These results indicate that it 
is important to understand the pathology of EOM and to promptly start 
appropriate treatment. Previous studies have shown that the presence of Th2 
9 
 
cytokines, such as IL-5 and IL13 [4, 5, 6], and the pathology of EOM are most 
likely to result from a Th2-type immune response. The presence of 
epithelium-derived cytokines such as TSLP is a key trigger for a Th2-type 
immune response; however, the presence of TSLP in EOM had not been 
reported.  
In this study, we found the immunoreactivity for TSLP in inflammatory cells 
of the middle ear mucosa in three cases of EOM patients. With regard to the 
epithelial layer, no positive staining was found in the biopsy specimens 
through the external auditory canal, but positive staining was detected in the 
specimens around eustachian tube who underwent radical mastoidectomy. It 
is difficult to clarify the correlation between TSLP expression and clinical 
features of EOM patients from the present results, because the number of 
EOM patients is small. Therefore, basic experiments using model animals are 
important to find out the pathogenesis of EOM. 
In our model animals, TSLP immunoreactivities were also detected around 
the tympanic ostium of the eustachian tube, and the molecular biological 
study also supported these immunohistological results in the animal model. 
As we described in the previous reports of our animal model of EOM, there 
is a significant difference of eosinophils infiltrating in the middle ear 
mucosa between the control group and the OVA 14-day group (the number of 
2; mean ± SD: 0.28 ± 0.23 and 1.88 ± 0.78 respectively), 
but not the OVA 7-day group (mean ± SD: 0.44 ± 0.41) [7, 14], and 
immunoreactivity of eotaxin has also been found in the OVA 7-day group 
[15]. In the present study, we detected the immunoreactivity and gene 
10 
 
expression of TSLP in the epithelial cells of the eustachian tube and around 
the tympanic ostium of the eustachian tube in the OVA 7-day group. These 
results suggest that, in the OVA stimulation model, TSLP produced by the 
epithelial cells of the eustachian tube may play an important role in allergic 
inflammation of the middle-ear mucosa prior to eosinophil infiltration, as 
eosinophil infiltration occurs in the lower layer of the Th2-type immune 
response cascade. For instance, both mast cells and dendritic cells are 
reported to release high levels of IL-5, which is key to eosinophil activation 
and infiltration through TSLP stimulation [16]. 
Research of middle ear allergy using animal models was conducted about 
three decades ago. At that time, the eustachian tube was reported to act as a 
“gatekeeper,” limiting the entry of most substances into the tympanic cavity, 
although type-I allergic reactions in the middle ear were reported [17]. 
However, Iino et al. identified more cases of patulous eustachian tube, which 
allows the entry of antigenic substances into the tympanic cavity, in patients 
with EOM compared with the controls [18]. Indeed, a recent report described 
the presence of antigen-specific IgE for house dust mite, fungi, and 
staphylococcus enterotoxins in the MEE [19]. Morphologically, the epithelium 
cilia is present in the eustachian tube and around the tympanic ostium, 
similar to airway epithelium, but the tympanic epithelium loses the cilia and 
takes a different form farther away from the eustachian tube. In the present 
study, TSLP immunoreactivity was found in the epithelium around the 
eustachian tube, indicating that EOM is a kind of airway allergy and 
supporting the concept of “one airway, one disease.” 
11 
 
We used OVA stimulation as an allergen in the present study and identified 
TSLP in the epithelial cells around the eustachian tube. Since TSLP is 
released from epithelial cells in response to not only allergens but also 
microbial products [16], and pathogens in MEE are risk factors for BCHL and 
EOM severity [13, 19], the eustachian tube is deeply involved in the pathology 
of EOM from a functional and immunological point of view. 
For the treatment of EOM, we use a combination of anti-allergic drugs, such 
as Cys-LTs receptor antagonists, phosphodiesterase inhibitors, and systemic 
or topical steroids. If TSLP acts as a trigger to eosinophil infiltration and the 
activation of a Th2-type immune response, the development of anti-TSLP 
modalities can be expected as a target for new treatments. Further study is 
required to identify more effective treatments for EOM. 
 
Acknowledgments 
We would like to thank Professor A. Kurose for his helpful comments. 
 
Disclosure statement 
The authors report no conflicts of interest. The authors alone are responsible 
for the content and writing of the paper. 
 
Funding 
This study was supported by a Grant-in-aid Scientific Research (C: 16K11169 
to AM) from the Ministry of Education, Science, Sports, and Culture of Japan. 
 
12 
 
Figure legends 
Table 1. Clinical features, allergic background, and immunohistological 
findings in six patients with EOM. Note that TSLP positive immnostaining 
in the inflammatory cells was observed in three cases, and that in the 
epithelial cells was detected in only one case whose specimens were taken 
during mastoidectomy. 
AR: allergic rhinitis, BA: bronchial asthma. 
 
 
Figure 1. Immunoreactivity for TSLP in patients with EOM 
TSLP was detected in epithelial cells (arrows, case No. 6 in Table 1) and 
inflammatory cells (arrow heads, case No. 3 in Table 1). 
 
Figure 2. Immunoreactivity for TSLP in animal models of EOM 
OVA 14-day group (A), OVA 7-day group (B), and control group (C). 
Eustachian tube (-1), around the tympanic ostium (-2), and distant site (-3). 
 
Figure 3. Comparison of mRNA level of TSLP expression in the animal model 
t-test, * P < 0.05 
 
 
 
 
 
13 
 
References 
 [1] Tomioka S, Yuasa R, Iino Y. Intractable otitis media in cases with 
bronchial asthma. Recent advances in otitis media. In: Mogi G, Honjo I, Ishii 
T, Takasaka T, editors. Proceedings of the second extraordinary international 
symposium on recent advances in otitis media. Amsterdam, New York: Kugler 
Publications 1993;183-186 
[2] Tomioka S, Kobayashi T, Takasaka T. Intractable otitis media in patients 
with bronchial asthma (eosinophilic otitis media). In:Sanna M, editor. 
Cholesteatoma and mastoid surgery. Rome: CIC Edizioni Internazionali 
1997;851-853 
[3] Iino Y, Tomioka-Matsutani S, Matsubara A, Nakagawa T, Nonaka M. 
Diagnostic criteria of eosinophilic otitis media, a newly recognized middle ear 
disease. Auris Nasus Larynx 2011;38:456-461 
[4] Nonaka M, Fukumoto A, Ozu C, Mokuno E, Baba S, Ruby P, Yagi T. IL-5 
and Eotaxin levels in middle ear effusion and blood from asthmatics with 
otitis media with effusion. Acta Otolaryngol 2003;123:383-387 
[5] Iino Y, Kakizaki K, Katano H, Saigusa H, Kanegasaki S. Eosinophil 
chemoattractants in middle ear of patients with eosinophilic otitis media. 
Clon Exp Allergy 2005;35:1370-1376 
[6] Ohta N, Ishida A, Kurakami K, Suzuki Y, Kakehata S, Ono J, Ikeda H, 
Okubo K, Izuhara K. Expressions and roles of periostin in otolaryngical 
disease. Allergology 2014;63:171-180 
[7] Nishizawa H, Matsubara A, Nakagawa T, Ohta N, Izuhara K, Shirasaki T, 
Abe T, Takeda I, Shinkawa H. The role of periostin in eosinophilic otitis media. 
14 
 
Acta Otolaryngol 2012;132:838-844 
[8] Ito T, Liu YJ, Arima K. Cellular and molecular mechanisms of TSLP 
function in human allergic disorders--TSLP programs the "Th2 code” in 
dendritic cells. Allergology 2012;61(1):35-43 
[9] Friend SL, Hosier S, Nelson A, Foxworthe D, Williams DE, Farr A. 
A thymic stromal cell line supports in vitro development of surface IgM+ B 
cells and produces a novel growth factor affecting B and T lineage cells. Exp 
Hematol 1994;22:321-328 
[10] Sims JE, Williams DE, Morrissey PJ, Garka K, Foxworthe D, Price V, 
Friend SL, Farr A, Bedell MA, Jenkins NA, Copeland NG, Grabstein K, 
Paxton RJ. Molecular cloning and biological characterization of a novel 
murine lymphoid growth factor. J Exp Med. 2000;192:671-680 
[11] Kamekura R, Kojima T, Koizumi J, Ogasawara N, Kurose M, Go M, 
Harimaya A, Murata M, Tanaka S, Chiba H, Himi T, Sawada N. Thymic 
stromal lymphopoietin enhances tight-junction barrier function of human 
nasal epithelial cells. Cell Tissue Res 2009;338:283-293 
[12] Nakagawa T, Matsubara A, Shiratsuchi H, Kakazu Y, Nakashima T, 
Koike K. Intractable otitis media with eosinophils: importance of diagnosis 
and validity of treatment for hearing preservation. ORL 2006;68:118–22 
[13] Iino Y, Usubuchi H, Kodama K, Kanazawa H, Takizawa K, Kanazawa T, 
Ohta Y. Eosinophilic inflammation in the middle ear induces deterioration of 
bone-conduction hearing level in patients with eosionophilic otitis media. Otol 
Neurotol 2009;31:100-104 
[14] Matsubara A, Nishizawa H, Kurose A, Nakagawa T, Takahata J, Sasaki 
15 
 
A. An experimental study of inner ear injury in an animal model of 
eosinophilic otitis media. Acta Otolaryngol 2014;134:227-232 
[15] Kudo N, Mastubara A, Nishizawa H, Miura T. Immunohistological 
analysis of eotaxin and RANTES in the model animal of eosinophilic otitis 
media. Acta Otolaryngol 2016;137(5):476-481 
[16] Allakhverdi Z, Comeau MR, Jessup HK, Yoon BR, Brewer A, Chartier S, 
Paquette N, Ziegler SF, Sarfati M, Delespesse G. Thymic stromal 
lymphopoietin is released by human epithelial cells in response to microbes, 
trauma, or inflammation and potently activates mast cells. J Exp Med. 
2007;204(2):253-258 
[17] Tomonaga K, Chaen T, Kurono Y, Mogi G. Type I allergic reactions of the 
middle ear and eustachian tube: an experimental study. Auris Nasus Larynx 
1990;17:121-131 
[18] Iino Y, Kakizaki K, Saruya S, Katano H, Komiya T, Kodera K, Ohta K. 
Eustachian tube function in patients with eosinophilic otitis media associated 
with bronchial asthma evaluated by sonotubometry. Arch Otolaryngol Head 
Neck Surg 2006;132:1109-1114 
[19] Kanazawa H, Yoshida N, Shinnabe A, Iino Y. Antigen-specific IgE in 
middle ear effusion of patients with eosinophilic otitis media. Ann Allergy 
Asthma Immunol 2014;113(1): 88-92 
 
 
Case
No. Gender Age AR BA
immunoreactivity
Epithelial
cell
Endothelial
cell
Inflammatory
cell
1 Female 69 + + - - -
2 Male 26 - - - - -
3 Male 49 + - - - +
4 Female 53 + + - - -
5 Male 77 + + - - +
6 Female 29 + + + + +
Table 1





The expression of thymic stromal lymphopoietin in patients and animal models with eosinophilic otitis media

弘前大学博士（医学）掲載誌 Biochemical and Biophysical Research Communications, 469, p.897-902, 201

韓, 沖

Modified sympathetic nerve regulation in AKAP5-null mice.

弘前大学博士（保健学）掲載誌：Parasitology research, 117:2895-2900, 2018
DOI：10.1007/s00436-018-5980-

山内, 可南子

細則様式第４号 
論 文 審 査 及 び 最 終 試 験 結 果 報 告 書                        
氏   名 山内 可南子 
入 学 年 度      平成 25 年度 学 籍 番 号     13GG707 
領   域 医療生命科学領域 分   野 生体機能科学分野 
審 査 委 員      
主  査 高見 秀樹 
副  査 木田 和幸 
副  査 敦賀 英知 
副  査 伊藤 巧一 
 
 
論文題目： 
「北日本地域におけるバラムチア・マンドリラリスの初めての土壌分離報告」 
 
審査結果要旨： 
本研究では、髄膜脳炎を引き起こすことで知られるバラムチア・マンドリラリスを青森
県内の土壌を用いて日本で初めて分離した。自由生活性アメーバのバラムチアは、現在ま
でに、罹患者報告の集積を認める熱帯気候や砂漠気候地域を中心に、世界で 8 例の環境下
分離報告がなされている。日本国内の豪雪地域において、バラムチアが分離されたことは、
バラムチア感染症が温暖な気候区分に特有の疾病ではなく、気候の差異に関わらず多様な
環境下で発生しうる可能性を示した。 
本研究の一部は、査読を経て国際学術雑誌 Parasitology research（IF:2.56）に掲載さ
れている。また、本審査過程において申請者は主査および副査による質疑応答に的確に応
答し、研究内容を十分に理解していると共に、研究課題に関連する専門知識の習得を確認
した。以上の結果から、審査員一同は申請者・山内可南子氏が博士の学位を授与されるの
に十分な資格を有していると判断した。 
 
 
 
最終試験   平成 30 年  7 月  24 日 
 
試験の結果は    合  格  ・  不 合 格   と判定する。 


北日本地域におけるバラムチア・マンドリラリスの初めての土壌分離報告

弘前大学博士（医学）掲載誌:弘前医学, 68, p.135-141, 201

小路, 祥紘

Use of wearable cardioverter defibrillator shortens the ICU stay and enables safe management in a general ward

弘前大学博士（医学）掲載誌:Onco Targets Ther, 10, p.4329-4336, 201

浅利, ゆう子

Inhibitory effects of a selective Jak2 inhibitor on adrenocorticotropic hormone production and proliferation of corticotroph tumor AtT20 cells

弘前大学博士（医学）掲載誌:Oncol Lett, 15, p.6195-6202, 201

西下, 理恵

Expression of cancer-associated fibroblast markers in advanced colorectal cancer

弘前大学博士（医学）掲載誌 体力・栄養・免疫学, 28, p.157-165, 201

大沼, 由香

 
 
 
 
 
 
 
 
 
 
Relationship between the intake balance of energy -providing nutrients and 
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1 0 代前半におけるb r a c h i a l – a n k l e p u l s e w a v e v e l o c i t y の変動とその関連因子に関する研究

Relationship between cognitive function and balance in a community-dwelling population in Japan

The expression of thymic stromal lymphopoietin in patients and animal models with eosinophilic otitis media

Modified sympathetic nerve regulation in AKAP5-null mice.

北日本地域におけるバラムチア・マンドリラリスの初めての土壌分離報告

Use of wearable cardioverter defibrillator shortens the ICU stay and enables safe management in a general ward

Inhibitory effects of a selective Jak2 inhibitor on adrenocorticotropic hormone production and proliferation of corticotroph tumor AtT20 cells

Expression of cancer-associated fibroblast markers in advanced colorectal cancer

エネルギー産生栄養素摂取バランスと内臓脂肪との関連　ｰ岩木健康増進プロジェクトでの横断研究 -

Proposal of stream pH mapping as a new geothermal exploration method

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