June 2017

Medical Mass Spectrometry Vol. 1 No. 1

Research Paper

Distinct spatial localization of three types of phosphatidyl choline
in rat buccal mucosa identiﬁed by matrix-assisted laser
desorption/ionization imaging mass spectrometry
1,2

1,2

1

3

Mariko Miyagi , Hana Fukano , Ryosuke Atsumi , Hiroyuki Suzuki ,2,4,5,6,7

1,4, Ikuko Yao *Mitsutoshi Setou
1

Department of Optical Imaging, Institute for Medical Photonics Research,
Preeminent Medical Photonics Education & Research Center,
Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431‒3192, Japan2
Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine,
Hamamatsu, Shizuoka 431‒3192, Japan
3
Hiroyuki Dental Clinic of Oral Surgery, 381‒11 Hatsuoi-cho, Kita-ku, Hamamatsu, Shizuoka 433‒8112, Japan
4
International Mass Imaging Center, Preeminent Medical Photonics Education & Research Center,
Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431‒3192, Japan
5
Department of Anatomy, The University of Hong Kong,
6/F, William MW Mong Block 21 Sassoon Road, Pokfulam, Hong Kong SAR, China
6
Division of Neural Systematics, National Institute for Physiological Sciences,
National Institutes of Natural Sciences, Okazaki, Aichi 444‒0867, Japan
7
Riken Center for Molecular Imaging Science, Kobe, Hyogo 650‒0047, Japan
Abstract

The buccal mucosa is an inner lining exposed to frequent friction caused by teeth occlusion and therefore, avulnerable site to lesions such as stomatitis, leukoplakia, lichen planus and cancer. Although it is considered an important tissue in the ﬁeld of oral surgery, not enough information regarding the buccal mucosa is available. In this study, wecharacterized the buccal mucosa in the oral cavity using imaging mass spectrometry (IMS). Tissues such as epithelium,
lamina propria and muscle were identiﬁed in rat buccal mucosa by HE staining. Moreover, IMS data of high-intensity ionswere classiﬁed into 4 groups according to their distribution. In a previous IMS analysis of mouse tongue tissue, we detected linoleic acid-containing phosphatidyl choline (PC)(diacyl 16:0/18:2), oleic acid-containing PC(diacyl 16:0/18:1) andDHA-containing PC(diacyl 16:0/22:6) as major PCs. In the present work, we analyzed buccal mucosa tissue with emphasison the aforementioned PC by IMS and showed that the investigated PCs existed in the layers of epithelium, lamina propria, and muscle at different ratios. This is the ﬁrst study to analyze by IMS the buccal mucosa. The results presented here
are likely to provide the perspective on understanding oral environments and to develop treatments for oral disorders.
Key words: imaging mass spectrometry, buccal mucosa, phosphatidylcholine, mass microscopy, matrix assisted laser desorption/ionization* Corresponding author
Ikuko Yao

Introduction

Department of Optical Imaging, Institute for Medical Pho-Oral mucosa is often afﬂicted by inﬂammatory diseases

tonics Research, Preeminent Medical Photonics Education &caused by normal oral ﬂora and exposure to external,
1)
hygiene-compromised environments . Although oral

Research Center, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒
3192, Japan
E-mail: yaoik@hama-med.ac.jp

mucosa is coated with saliva and protected by the moist

Received January 16, 2017. Accepted March 23, 2017.

external factor. As the entrance of the breathing air and

DOI: 10.24508/mms.2017.06.002

nutrition, it is considered as an important tissue.

environment, the condition is quite unstable due to the2June 2017

Medical Mass Spectrometry Vol. 1 No. 1

square conductive ITO-coated (100 Ω slides)] were pur-Imaging mass spectrometry (IMS) is a technique that candirectly analyze molecular distribution on the surface of tis-chased from Matsunami Glass Ind., Ltd. (Osaka, Japan).
2,5-dihydroxybenzoic acid (DHB) was purchased fromsues without previous extraction. In addition, mass spectrometry employing matrix-assisted laser desorption/ ion-

Bruker Daltonics (Bremen, Germany). All chemicals used2)

in this study were of the highest purity available.

ization (MALDI) can analyze with high sensitivity mostlipid classes without major fragmentation. CouplingPreparation of rat buccal mucosa tissue sections

MALDI with IMS produces the best technique to analyze
distribution of lipid molecules in tissues. As a result, a num-

Rats were anesthetized and decapitated afterward. The

3)

ber of lipid analyses by IMS have been reported to date .

center of the head was incised and the surface of one side

Using IMS as the analytical tool, we previously charac4)
5)
terized tongue and tooth tissues, with emphasis on phos-of the buccal mucosa was visually exposed. Next, the toothpholipids. In the study, we identiﬁed and visualized lipid
species in mouse tongue tissue by MALDI-IMS. We

buccal mucosal tissue. After those procedures, buccal

showed that three types of phosphatidyl choline (PC),namely linoleic acid-containing PC(diacyl 16:0/18:2), oleic

All samples damaged by cuts or long exposure to ambience during processing were excluded. Buccal mucosal

acid-containing PC(diacyl 16:0/18:1), and DHA-containing
PC(diacyl 16:0/22:6), were located mainly in stratiﬁed epi-

samples were embedded in 4% carboxymetyl cellulose(CMC), submerged in n-hexane and frozen with dry ice

thelium, peripheral nerve and muscle as ions at m/z 758.5,

within 5 min after decapitation.

cervix was carefully peeled off from the border to isolate
mucosa were cut and removed.

4)

Processed samples were then stored at −80°
C with no
ﬁxation. Next, tissues were sliced into 10-μm sections using

m/z 760.5, and m/z 806.5, respectively .
Buccal mucosa is the inner lining of cheeks and lips,
which are anatomic regions that include all mucous mem-a cryostat (Cryostat CM 1850; Leica Microsystems, Wet-

branes from the opposing line of contact of lips to thezlar, Germany) with the chamber temperature at −20°C.

mucosa at the line of attachment of the alveolar ridges and

For IMS analysis, the tissue sections were mounted onto
ITO-coated glass slides. All slides with mounted sections

6)

the mandibular ridge . Although the buccal mucosa is an
important tissue in the oral cavity, there is not enough infor-

were stored at −20°
C until further use.

mation about the buccal mucosa, and no IMS analysis of

Matrix application

buccal mucosa has been previously conducted. Here we forthe ﬁrst time attempted to build the basis of IMS for buccal

A matrix solution was prepared by dissolving 50 mg of

mucosal tissue, especially focused our analysis on the three
types of PCs we previously visualized in mouse tongue tis-

DHB in 1 mL of methanol/water (7:3, v/v) with 125 mM
ammonium sulfate. DHB matrix solution was sprayed uni-sue [i.e., PC(diacyl 16:0/18:2), PC(diacyl 16:0/18:1) andPC(diacyl 16:0/22:6)].

formly at a distance of 15 cm from the tissue surface withan airbrush equipped with a 0.2-mm nozzle. In total, 2 mL

Material and Methodsof DHB matrix solution was sprayed on each slide. DHB isa common matrix used to ionize lipids by MALDI‒IMS in

Animals

the positive ion mode. Ammonium sulfate was added to the＋

Adult female Wister rats were purchased from SLC

matrix solution in order to form positive ions of [M＋H] ,＋(Hamamatsu, Japan) and given ad libitum standard diet andwater. The care and use of laboratory animals were con-＋but not of [M＋Na] and [M＋K] , which helped reduceoverlapping of signals from different molecules. After glass
slide-mounted tissue sections were dried, they were ana-

ducted in accordance with the Animal Experiment Regulations issued by the Hamamatsu University School of Medi-lyzed by attaching the glass slides to the MS microscopecine, which follows the Guidelines for the proper Conduct

holder.

of Animal Experiments approved by the Science Council of

Imaging mass spectrometry

Japan.

MALDI‒IMS analysis was couducted using iMScope, aChemicals and reagents

MS microscope (Shimadzu, Kyoto, Japan). Data wereacquired in the positive ion mode with a 50-μm pitch. The

Indium-tin-oxide (ITO)-coated Glass slides [13-mm3

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analysis conditions were: laser irradiation time, 200 shots;

Results and Discussion

frequency, 1000 Hz; illumination diameter, minimum (5 μMorphology and histology of rat mucosa tissuem); laser power, 30%; and mass range, 600 to 1000. A mass

Prior to IMS analysis, we validated the morphology andrange setting of 400 to 1000 resulted in failure, due to noise
signals in the data. Imaging MS Solution software (Shi-histology of rat mucosal tissue (Fig. 1). An image of rat
buccal tissue prior to separation of the mucosa and othersurrounding tissues is shown in Fig. 1a. The surface of buc-madzu) was used to create two-dimensional ion-density
maps. After analysis by MALDI‒IMS, glass slide-mounted

cal mucosa tissue was smooth and with a gray-purple color.

tissue sections were immersed in acetone for 5 s to washthe matrix off tissues, and stained with hematoxylin-eosin

After removal, the buccal mucosal tissue and the muscle
shrunk and thickened due to loss of attachment to the sur-(HE) afterward.

rounding tissues. In the HE-stained buccal mucosal tissue,
layers of epithelium, lamina propria, and muscle wereobserved, which were histologically similar to that ofhuman, although the epithelium was thicker than that foundFig. 1.

Optical and ion mages of rat buccal mucosal tissue.

(a) Image of rat buccal tissue prior to separation of the mucosa tissue and other surrounding tissues. (b) HE-stained tissue section of
rat buccal mucosa. After imaging mass spectrometry (IMS) analysis, the same section was subjected to HE staining. The right panel
shows a high magniﬁcation image, and the epithelium, lamina propria, and muscle can observed. Corniﬁed layer can be seen on
the edge. (c) Optical image of the same section analyzed by IMS. Muscle (M), Lamina propria (L), and Epithelium (E) areas wereTM
determined by visual observation for re-analysis by Imaging MS Solution . (d‒g) Reconstructed ion images with high intensity ineach layer of buccal mucosa.

4June 2017

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Table 1.
No.
1

List of major ions and their intensities detected in each analyzed region of interest

MuscleLaminapropria

Epithelium

Matrixm/z

Intensitym/zIntensitym/zIntensitym/zIntensity

782.5

1,349,435

782.5

1,204,431

718,973681.1

2,760,6342

806.5§

1,217,318

806.5§

871,982782.5
760.6*

676,456

637.0

1,468,609

3

810.6

1,134,766

758.5#

868,018758.5#

660,100

682.1

1,088,642

4

758.5#

875,041810.6

700,867681.1

626,310817.1

865,2825

918.6

711,437760.6*

674,228734.5

506,647

698.1

760,5906

786.6

671,377

918.6

599,040896.6

461,728

639.0

684,4437

783.5

652,758783.5

567,330

894.6

412,153

619.0

668,6208

946.6

649,585894.6

520,215806.5§

360,392

638.0

604,3809

942.6

634,826734.5

504,391810.6

353,496716.1

601,66710

807.5

601,907786.6

457,348786.6

353,244692.9

548,10211

760.6*

599,772896.6

453,831870.6

347,305

624.9

529,043

The top 11 ions in each regions of interest (ROI) are listed. The ions investigated in this study are underlined and ions at m/z 758.5, 760.5,
and 806.5 are indicated by #, *, and§, respectively.

in human oral cavity (Fig. 1b). In addition, keratinocytes or

m/z 806.5 and 942.6 observed in the muscle were classiﬁedsomewhat corniﬁed cells were observed on the edge where

into yet another group (Fig. 1f). Lastly, ions at m/z 758.5,

lower molars touched (Fig. 1b). Corniﬁcation is believed to786.5, 810.6, 894.6, 918.6, and 946.6, as well as the ion athave been caused by daily friction because no other causes

m/z 782.5, with the highest signal intensity in all three ROI,

6,7)

were

were classiﬁed into the ﬁnal group. All ions in this ﬁnal

detected. The tissue section contained part of the salivarygroup were widely distributed throughout the entire ROI ofduct (Fig. 1b), Thus, we believed the tissue section also

the tissue section (Fig. 1g).

of inﬂammation such as lymphocytes inﬁltration

Based on data from previously reported studies, includ-

contained the parotid gland which is a major salivary gland
8)

4,9)

located along the cheek muscle .

ing our previous IMS analysis of mouse tongue tissue,

ions at m/z 758.5, 760.6, and 806.5, detected on buccal

Distributions of major ions detected in the buccal mucosa

mucosal tissue (Table 1), were regarded as [PC(diacyl

The region of interest (ROI) corresponding to the threelayers of muscle, lamina propria, and epithelium were cho-(m/z 760.6), and [PC(diacyl 16:0/22:6)＋H]＋(m/z 806.5).

sen in images of matrix-applied samples, based on theidentiﬁed by their m/z values and data from previously

image of HE staining (Fig. 1c). In the three ROIs, the 16

reported IMS analyses. For example, the ion at m/z 782.5

ion species were included in the top 11 ions (Table 1) andwith a broad distribution was identiﬁed as PC(16:0/20:4)＋

images of 13 ion species out of 16 were reconstructed (Fig.
1c‒f). Ions at m/z 783.5 and 807.6 were omitted because

H . Moreover, we considered ions at m/z 734.5, 786.6 and16:0/18:2)＋H]＋(m/z 758.5), [PC(diacyl 16:0/18:1)＋H]＋
Similarly, other ions among those listed in Table 1 were

4)

810.5

they were isotopes of ions at m/z 782.5 and 806.5, respec-as

PC(16:0/16:0)＋H,

PC(18:0/18:2)＋H,

and

4)

PC(18:0/20:4)＋H, respectively .

tively. The ion at m/z 681.1 was not analyzed because it

Ion images of three PCs in buccal mucosa

was derived from the matrix (Table 1). Ion images wereroughly classiﬁed into four groups judged by the distribution. For example, ions at m/z 734.5 and 870.6 observed in

Reconstructed images of [PC(diacyl 16:0/18:2)＋H]＋(m/z758.5), [PC(diacyl 16:0/18:1)＋H]＋(m/z 760.6), and [PC(di-the parotid gland tissue and the surface area with a gradualacyl 16:0/22:6)＋H]＋(m/z 806.5) are shown in Fig. 2a‒dincrease in intensity of their signals on the surface wereclassiﬁed into one group (Fig. 1d). Ions at m/z 760.6 andand their higher magniﬁcation of these images can be seenin Fig. 2e‒h. PC(16:0/18:2) was broadly distributed

896.6 widely distributed in the salivary duct and the surface

throughout the analyzed tissue and partly accumulated inlayer were allocated into another group (Fig. 1e). Ions at

the layer around the intraoral surface (Fig. 2a, e).
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PC(16:0/18:1) produced signals on the surface layer of thespectrum were observed in this study because the analyzed

buccal mucosa and glandular area with a particularly strong

area selected in buccal mucosa was smaller than that insignal in the salivary duct (Fig. 2b, f). PC(16:0/22:6) was
homogeneously distributed, except for the surface and glan-

tongue and the small ROI contained more than one kind oftissue structures.

dular tissue areas (Fig. 2c, g). Finally, a merged image
showed that the aforementioned PCs were differently dis-Comparison of major ions in buccal mucosal tissue
with those of previous reports

tributed in the same tissue section (Fig. 2d, h). These IMS
results clearly showed that the investigated PCs were differ-

Ions detected in buccal mucosal tissue (Table 1) werecompared with molecules reported in past IMS analyses.
Warthin tumor (War-T), the second most common benign

ently distributed in each layer.
PC(16:0/18:2) at m/z 758.5, which had the highest intensity in the stratiﬁed epithelium of mouse tongue analysis ,4)parotid gland tumor, consists mainly of neoplastic epithe-was widely distributed in the ROI of the buccal mucosa

lium and lymphoid stroma . We found that ions detected(Fig. 2). PC(16:0/18:1) at m/z 760.6, which was abundantin the peripheral nerve of the mouse tongue , was observedin the present study at m/z 758.5, 760.6, 806.5, 734.5,
782.5, 786.6, and 810.6 were also reported in War-T tissue

on the surface area of buccal mucosa, salivary duct, andsections as K＋adducts at m/z 796.5, 798.5, 844.5, 772.5,

glandular area. It is worth mentioning that the signal
detected in the salivary duct was particularly high, which

820.5, 824.5, and 848.5, respectively . Of these, ion at m/z
796.5 in regions of War-T tissue had a lower level than didsuggests that PC(16:0/18:1) at m/z 760.6 is a major PC in

those detected in non-War-T tissue, but the ion at m/z 897.5

the nervous system and gland tissues. PC(16:0/22:6) at m/z

had almost matching levels in both tissues. In contrast,

806.5 was located in the muscle tissue in both in the tongue

other ions, especially m/z 772.5 and m/z 824.5, were higher
10)
in War-T tissue than in non-War-T tissue . The signal of

10)

4)

10)

and buccal mucosa, suggesting that it is a common PC not
only in oral tissue but also in other organs.

PC(16:0/16:0)＋K at m/z 772.5, which was localized
mainly in lymphoid stroma with a high intensity at the fol-Comparison of the distributions of three PCs in rat
buccal mucosa

liculus lymphaticus in the War-T tissue , corresponded to
the H＋adduct at m/z 734.6, which was detected in glandu-

The average intensity of the aforementioned PCs in rat

lar tissue in the present study. This result may indicate that

buccal mucosa was calculated (Fig. 2i). As shown in Fig.

PC(16:0/16:0) is abundant in cells that are involved in

2i, the correlation of values were in agreement with thesecretion. The ion at m/z 884.5 was observed in regions ofboth neoplastic epithelium and lymphoid stroma in War-T

10)

images of PCs detected in the same tissue section as shownin Fig. 2i, in particular in images taken at higher magniﬁca-

tissue and it was identiﬁed as PC(18:2/20:4) by MS/MS

tion. For example, PC(16:0/18:2) at m/z 758.5 had a relatively uniform intensity in all tissues except for the epithe-analysis. Nonetheless, although the m/z value corresponded

lium where the signal decreased. The intensity ofto the H＋adduct at m/z 806.5, the PC species detected in
War-T tissue may be different from those detected in pres-PC(16:0/18:1) at m/z 760.6 increased on the surface of theent study. In this study, we theorize that the ion at m/z 806.5

epithelium and was the strongest. The intensity ofis PC(16:0/22:6), which was previously identiﬁed by MS/

PC(16:0/22:6) at m/z 806.5 was notably high in the muscle,

MS in tongue tissue and it is also commonly found in

markedly reduced in the lamina propria, and the lowest in
the epithelium area (Fig. 2c). Focusing on global patterns,

muscle tissue. As it was in the present study, the ion at m/z
782.5 was reported in a previous IMS analysis of tooth tis-

we compared the average mass spectrum of each ROI (Fig.

sue , although PC(diacyl 16:0/18:2), PC(diacyl 16:0/18:1)

2j).

and PC(diacyl 16:0/22:6) were not detected. The ion at m/z

In the previous our report, it was found that the spectral
patterns obtained in the analysis of muscle, stratiﬁed epithe-782.5 was also detected in tongue tissue . We theorize that
this molecule commonly exists in large amounts in oral tis-

lium, and the peripheral nerve of tongue tissue were quite

sues. In the present study we did not detect the ion at m/z

4)

5)

4)

4)

different from each other . In contrast, no large differences
between spectral patterns were observed in the present anal-

469.3, perhaps due to noise signals detected in the analysis

ysis of buccal mucosa. It can be suggested that averaged

Materials and Methods section). While lysoPC was foundat a m/z range of 400 to 1000 (data not shown; see also the6June 2017

Fig. 2.

Medical Mass Spectrometry Vol. 1 No. 1

Speciﬁc distributions of the investigated PCs in the buccal mucosal tissue.

(a‒c) Images of ions at m/z 758.5, 760.5, and 806.5. (d) Merged image; Green, ion at m/z 758.5 for PC(diacyl 16:0/18:2)＋H; Red,
ion at m/z 760.5 for PC(diacyl 16:0/18:1)＋H; Blue, ion at m/z 806.5 for PC(diacyl 16:0/22:6)＋H. PC showed different distributions. (e‒h) High magniﬁcation images of a‒e. Hatched areas were zoomed out. The ion at m/z 758.5 was broadly distributed, theion at m/z 760.5 was found throughout most surface layer with a strong signal at the salivary duct, although fewer signals of theion at m/z 806.5 were observed on the outer layer. (i) Signal intensity of ions of the investigated PC in each area in tissues from six
different rats. Investigated PC were distributed with different populations in each layer. Values indicating signal intensity are themeans±SEM (n＝6) (j) Comparison of mass spectra obtained from each area of muscle; lamina propria; and epithelium. The peaksof ions at m/z 758.5, 760.5, and 806.5 are indicated by #, * and§, respectively. M, Muscle; L, Lamina propria; E, Epithelium.

7June 2017

Medical Mass Spectrometry Vol. 1 No. 1

Acknowledgements

at m/z 469.3 in inﬂamed tissues of a rat periodontal disease5)

model , the reason for the lack of detection of this ion inThis study was supported by MEXT KAKENHI Grant

the investigated tissues in the present study may be due toour usage of buccal mucosal tissue of healthy rats. Detec-

Number JP 24111547 and JSPS KAKENHI Grant Numbers

tion of such inﬂammation markers will contribute to diagnose or conﬁrm the effect of remedy and periodontal sur-

JP15H05898B1, Imaging Platform Grant Number 967,

JP26460388,

JP16KT0134C1,

JP26670461C1,

CREST from AMED Grant Number 921910520.

gery.
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