ISSN 0030- 6096

O S A K A
C I T Y

Vol.

65

No.2

M E D I C A L

2019

J O U R N A L
V o l . 6 5 ,N o . 22 0 1 9PUBLISHED BYOSAKA CITY MEDICAL ASSOCIATIONOSAKA JAPANOsaka City Medical Journal
Vo l . 6 5 , N o . 2 ,December 2019

CONTENTS
page
Influence of Dose-volume Prescription in Three-dimensional Conformal
Radiotherapy for Patients with Stage Ⅲ Non-small-cell Lung Cancer
TOMOHIRO NISHIKAWA, SHINICHI TSUTSUMI, TAKUHITO TADA,NAOKI MUKUMOTO, MASARU MAKIHARA, MASAHIRO TOKUNAGA,NORIKO TANAKA, YOSHIKAZU HASEGAWA, MASAKO HOSONO,KENTARO ISHII, and YUKIO MIKI･･････････････････････････････････････････････････････････････････････ 77
Clinical Response to Palliative Radiotherapy for Gastric Cancer
MASARU MAKIHARA, TAKUHITO TADA, MASAHIRO TOKUNAGA,NORIKO TANAKA, HIROSHI TSUKUDA, TAKAYO OTA,TOMOHIRO NISHIKAWA, KOSUKE AMANO, and KENTARO ISHII ･･････････････････････････････ 85
The Neural Effects of Inadequate Pauses in Speech on the Comprehension of
Logical and Illogical Speeches: A Magnetoencephalography Study
YUKI KODA, and KISHIKO SUNAMI･････････････････････････････････････････････････････････････････････ 93
Microcuff ® Pediatric Endotracheal Tube Decreases the Tube Exchange Ratio
without Increasing Postoperative Complications in JapaneseChildren: A Single Center Retrospective Cohort StudyYOSUKE INADA, YUSUKE FUNAI, TAKASHI SHUTOU, TAKASHI MORI,
and KIYONOBU NISHIKAWA･･････････････････････････････････････････････････････････････････････････････ 109
Time-lapse Observation of Stabilized Mitochondrial Membrane Potential in
Chemotherapy-induced Granulosa Cell Damage Treatedwith L-carnitine
AKI TAKASE, DAISUKE TACHIBANA, YUKIMI KIRA, AKIHIRO HAMURO,TAKUYA MISUGI, and MASAYASU KOYAMA ････････････････････････････････････････････････････････ 119― Case Report ―Bilateral Ovarian Fibromatosis Diagnosed with Magnetic Resonance Imaging:
A Case Report
NORIKO TAKESHITA, TAKUHITO TADA, MASAHIRO TOKUNAGA,
AKIRA OKIMURA, MASARU MAKIHARA, and TOHRU TAKESHITA･･････････････････････････････ 135

Osaka City Med. J. Vol. 65, 77-83, 2019

Influence of Dose-volume Prescription in Three-dimensional
Conformal Radiotherapy for Patients with Stage Ⅲ
Non-small-cell Lung Cancer
TOMOHIRO NISHIKAWA1), SHINICHI TSUTSUMI2), TAKUHITO TADA3), NAOKI MUKUMOTO4),
MASARU MAKIHARA3), MASAHIRO TOKUNAGA3), NORIKO TANAKA3), YOSHIKAZU HASEGAWA5),
MASAKO HOSONO2), KENTARO ISHII6), and YUKIO MIKI1)
Departments of Diagnostic and Interventional Radiology1)
and Radiation Oncology2), Osaka City University Graduate School of Medicine;
Departments of Radiology3) and Oncology5), Izumi City General Hospital;
Department of Radiation Oncology4), Yodogawa Christian Hospital; andDepartment of Radiation Oncology6), Tane General Hospital

Abstract
BackgroundDose-volume prescription (DVP) is usually used in intensity-modulated radiation therapy (IMRT)
and point-dose prescription (PDP) is usually used in three-dimensional conformal radiotherapy(3DCRT) in Japan. This study aimed to retrospectively evaluate the impact of DVP instead of PDP
on the doses and outcomes of 3DCRT for patients with lung cancer.
Methods
Since 2011, the DVP has been used in place of the PDP in routine 3DCRT for patients with lungcancer in our institution. Twenty-one patients with stage Ⅲ non-small-cell lung cancer who
underwent definitive chemoradiotherapy using DVP were included this study. The patients received aprescribed dose of either 60 Gy or 66 Gy, both of which covered 95% of the planning target volume(PTV). The clinical target volume (CTV) was defined as the gross tumor volume plus a 5 mm or moremargin, and the PTV was defined as the CTV plus a 5 mm or more margin.
Results
The median ratios of the dose in the DVP to that in the PDP were 1.059 and 1.077 in the actual
treatment and in the planning study using unreduced PTVs, respectively. The median follow-up was
16.2 months. The overall 2-year Kaplan-Meier survival rate was 57%. The 2-year in-field control
rates for the 66 Gy group and for the 60 Gy group were 100% and 0%, respectively.
Conclusions
Although the PTVs were reduced, the DVP induced a 1.059-fold overdose. Meanwhile, treatment
Received October 26, 2018; accepted January 15, 2019.
Correspondence to: Shinichi Tsutsumi, MD.
Department of Radiation Oncology, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan
Tel: +81-6-6645-3831; Fax: +81-6-6645-6655
E-mail: ttm@ttm.jp- 77 -

Nishikawa et al

outcomes using the 66 Gy dose were satisfactory.
Key Words: ‌Non-small cell lung cancer; Radiotherapy; Chemotherapy; Dose-volumeprescription

Introduction
Recent clinical trials using three-dimensional conformal radiotherapy (3DCRT) for non-small-celllung cancer (NSCLC) adopted Dose-volume prescription (DVP)1-3). However, in several countries,
including Japan, intensity-modulated radiation therapy (IMRT) remains an uncommon treatment
modality, and the use of 3DCRT usually involves point-dose prescription (PDP) using heterogeneity
correction. In this paper, a retrospective study was performed to evaluate the influence of DVP on thedoses and outcomes of 3DCRT.

Materials and Methods
This study was approved by the institutional review board at Izumi Municipal Hospital and was
conducted in accordance with the Declaration of Helsinki. All patients gave informed consent toparticipate.
Since 2011, DVP has been used instead of PDP in routine 3DCRT for patients with lung cancer in
our institution. In this paper, a retrospective study was performed to evaluate the influence of DVP
on the doses and outcomes of 3DCRT.
Patients
Between October 2011 and March 2015, 33 patients with stage Ⅲ NSCLC underwent definitive
radiotherapy in our institution. Of the 33 patients, 5 patients who underwent radiotherapy with PDP
following protocols of other multicenter clinical trials and 7 patients who underwent radiotherapy
alone were excluded. The remaining 21 patients who underwent concurrent chemoradiotherapy with
DVP were enrolled in this retrospective study. Patients who showed relapse after surgery were
excluded in this study group.
Radiotherapy
Irradiation was performed using involved radiation fields. The clinical target volume (CTV) was
defined as the gross tumor volume (GTV) plus a 5 mm or greater margin, and the planning target
volume (PTV) was defined as the CTV plus a 5 mm or greater margin. 3DCRT was delivered in 10MV photons. A dose of 60 Gy was prescribed to 7 patients with the following risk factors: advancedage (80 years or more; n＝3), a large PTV resulting in more than 35% of the total lung volume
receiving 20 Gy or more (n ＝3), and severe emphysema (n＝1). Meanwhile, a dose of 66 Gy wasprescribed for the remaining 14 patients. The prescribed doses covered 95% of the PTV (D95
prescription). For treatment planning, a commercially available superposition-based algorithm wasused.
Except for the first 2 patients, the PTV margin was reduced using heterogeneity correction to
avoid inconsistency of radiation dose with historical dosing methods, that is, PDP. However, in thesepatients, the reduced PTVs contained at least the CTVs. The radiation field consisted of the PTV
plus 5 mm or grater leaf margin. When the PTV margin was reduced, the leaf margin was expanded
to 8 mm or more in most patients.
Chemotherapy
Chemotherapy was concurrently performed with radiotherapy in all 21 patients. Of these, 9, 6, 5,- 78 -

Influence of Dose-volume Prescription

and 1 received a chemotherapeutic regimen consisting of carboplatin plus paclitaxel, carboplatinalone, cisplatin plus navelbine, and TS-1 alone, respectively.
End points
The end points were correction factors (CFs) that were defined as the ratios of the dose in the DVP
to that in the PDP, in-field control, clinical response, overall survival, and acute adverse events. To
obtain the CFs, PDP planning was also performed using heterogeneity correction. In the planning
procedure, the same radiation field and dose weighting with those in the DVP were used. Therefore,
an isodose line of A Gy in the DVP was consistent with that of A X CF Gy in the PDP. Principally, thereference points were to be in the mediastinum, where the dose gradient was minimal. Furthermore,
for comparison, planning with use of the DVP using unreduced PTVs was also performed. In-field
control and overall survival were evaluated using the Kaplan-Meier method. The clinical response
was evaluated according to the Response Evaluation Criteria in Solid Tumor (RECIST), version 1.14).
Meanwhile, acute adverse events were evaluated according to the National Cancer Institute s
Common Terminology Criteria for Adverse Events version 4.05).

Results
The patient characteristics are listed in Table 1. A total of 14 and 7 patients had squamous cell
carcinoma and adenocarcinoma, respectively, and stage ⅢA and stage ⅢB disease were seen in 14
and 7 patients, respectively. The CFs are summarized in Table 2. For all cases, the correction factor
is greater than 1. This indicates that DVP is superior to PDP in PTV dose distribution. The median
CFs in the actual treatment and in the planning study using the unreduced PTVs were 1.059 and1.077, respectively. In the present study, the PTVs ranged from 19 cm3 to 472 cm3 (median, 186 cm3),

Table 1. Patient‌characteristics
Characteristic

Value

Age (y)Range
Median

38-85
71

Sex
Male
Female15
6Clinical stage
ⅢA
ⅢB

14
7

Histology
Squamous cell carcinoma
Adenocarcinoma

14
7

＊

ECOG Performance status012ECOG; Eastern Cooperative Oncology Group.

＊- 79 -

1
14
6

Nishikawa et al
Table 2. Correction factors
Correction factor≦11.001-1.049

1.050-1.099

1.100-1.149≧1.150

Actual treatment

0

9

8

4

0

Planning study

0

3

11

6

1

Correction factors were defined as the ratios of the dose in the dose-volume prescription to that in the point-doseprescription. The planning study was performed using unreduced planning target volumes.

Figure 1. Kaplan-Meier survival curves for all patients, patients in the 66 Gy group, and those in the 60 Gy group.

Figure 2. The in-field control curves for all patients, patients in the 66 Gy group, and those in the 60 Gy group.

- 80 -

Influence of Dose-volume Prescription

and the unreduced PTVs ranged from 52 cm3 to 557 cm3 (median, 246 cm3).
The median follow-up was 16.2 months (range, 1.6-37 months). The response rate according to
RECIST was 81% (complete response: 38%; partial response: 43%). The overall 2- and 3-year KaplanMeier survival rates were 57% and 48%, respectively (Fig. 1). The in-field control curves for all
patients, for patients in the 66 Gy group, and for patients in the 60 Gy group are shown in Figure 2.
The 2-year in-field control rates for all patients, for the 66 Gy group, and for the 60 Gy group were58%, 100%, and 0%, respectively.
Grades 3 and 5 radiation pneumonitis were observed in 2 and 1 patients, respectively; all of whomwere in the 66 Gy group. No other grade 3 or greater acute nonhematologic toxicity was observed.

Discussion
Concurrent chemoradiotherapy is the standard of care for patients with unresectable stage Ⅲ
NSCLC. However, in-field control is unsatisfactory with 3DCRT 6,7). Furthermore, considerable
underdosing occurs in the peripheral lung in the PTV due to tissue heterogeneity and build-up effect.
Therefore, several dose escalation studies have been performed. To avoid underdosing in the PTV,
the D95 prescription was adopted in the RTOG0617 study 2), which was started based on the
hypothesis that radiotherapy with a dose of 74 Gy would yield better outcomes than that using 60 Gy.
The D95 prescription was also adopted in our institution, and the prescribed dose of 66 Gy forpatients without risk factors was established as follows. When DVP was adopted in our institution,
the RTOG0617 study was yet to be concluded, and we presumed that the optimal dose would be
between 66 Gy and 74 Gy based on former phase I and phase Ⅱ studies3,8-10). The lowest dose in therange was adopted for safety.
In the first 2 patients treated with 3DCRT using the unreduced PTV, the CFs were 1.083 and1.091. At that time, we had no experience with such a large fractional dose in concurrent
chemoradiotherapy. To avoid inconsistencies with the PDP, the PTV margin was then reduced insucceeding patient treatments. As shown in the planning study using the unreduced PTV, the CFs
decreased. However, the median CF remained at 1.059. When the unreduced PTVs were used, themedian CF was 1.077 although the minimal size of the PTV following the protocol was used. In lungstereotactic body radiotherapy, Kawahara et al. compared the outcomes of DVP with PDP, and the
produced CF was as high as 1.14311). Thus, one of the aims of the present report was to determine thedifference in doses between DVP and PDP.
The risk of relapse around the margin of the radiation field increases with a decreased PTV.
However, in-field control in the 66 Gy group has been satisfactory. At the least, more doses weredelivered in the DVP than in the PDP even when the reduced PTVs were used. To avoid
inconsistency with the PDP, the DVP was considered the optimal choice.
In 3DCRT planning using DVP, beam weighting is usually adjusted to improve the minimal dosein the PTV. Therefore, the DVP minimizes underdosing more than the simple CFs. In the present
study, the 66 Gy group achieved favorable in-field control, although the follow-up time wasinsufficient. Meanwhile, in-field control for the 60 Gy group was unsatisfactory. However, a 60 Gy
dose cannot be ruled out when considering treatment limitations, such as large GTV causing large
PTV and mild chemotherapy due to advanced age.
Frank et al compared heterogeneity-corrected DVP using the D95 prescription with the classicalhomogeneous PDP and concluded that both produced equivalent PTV, CTV, and isocenter doses for- 81 -

Nishikawa et alpatients with stage Ⅰ/Ⅱ NSCLC12). Therefore, adopting the DVP in place of the PDP in recent clinical
trials was reasonable1-3). However, the PTVs for stage Ⅲ disease are generally larger than those forstage Ⅰ/Ⅱ disease. The large PTVs often resulted in overdosing in the DVP. Furthermore,
institutional protocols and clinician experience can influence treatment planning13). The overdosing
can be significant in institutions with insufficient experience. These might explain the unexpected
results of the RTOG0617 study, in which outcomes of radiotherapy with a dose of 74 Gy werecompared with those of 60 Gy using the D95 prescription, and the results indicated that 60 Gy
yielded significantly better outcomes2). Therefore, approximately 50% of patients were treated with
3DCRT. Agreeable overdosing in the 60 Gy group prolonged survival time, whereas it negatively
influenced survival time in the 74 Gy group. Notably, the favorable outcomes in the 60 Gy groupwere not those in the heterogeneity-corrected PDP. As such, the 60 Gy dose was not necessarily
optimal in such group.
In the 66 Gy group, grade 3 or higher radiation pneumonitis was observed in 3 patients. Whenthe lung dose is appropriately restricted, the risk of Clinically-significant symptomatic pneumonitis isabout 20%14). The incidence in this study is not high compared to past study. High-grade radiation
pneumonitis is inevitable to some degree. To confirm the tolerability, further data accumulation isrequired.
IMRT can provide better dose distribution in the PTV and can reduce CFs. In IMRT, reducing
PTV was not significantly necessary, and the optimal dose might be different from that in 3DCRT
when the same PTV was used.
There were several limitations to this study. First, this was a retrospective study conducted at
one institution. Any conclusion revealed here needs to be demonstrated prospectively. Additionally,
it will be difficult to find significant relationships from the data because of the small number ofpatients. Further data accumulation is required to determine its clinical benefits and limitations.
In conclusion, the DVP yielded considerable overdosing in 3DCRT for patients with stage Ⅲ
NSCLC although the PTVs were reduced. In the present study, treatment outcomes from a dose of 66
Gy were satisfactory. Further data accumulation is required to determine its clinical benefits andlimitations.
Acknowledgements
All authors have no COI to declare regarding the present study.
References
1. Senan S, Brade A, Wang LH, et al. PROCLAIM: Randomized phase Ⅲ trial of pemetrexed-cisplatin or
etoposide-cisplatin plus thoracic radiation therapy followed by consolidation chemotherapy in locally advanced
nonsquamous non-small-cell lung cancer. J Clin Oncol 2016;34:953-962.
2. Bradley JD, Paulus R, Komaki R, et al. Standard-dose versus high-dose conformal radiotherapy withconcurrent and consolidation carboplatin plus paclitaxel with or without cetuximab for patients with stage
ⅢA or ⅢB non-small-cell lung cancer (RTOG 0617): a randomised, two-by-two factorial phase 3 study. Lancet
Oncol 2015;16:187-199.
3. Socinski MA, Blackstock AW, Bogart JA, et al. Randomized phase Ⅱ trial of induction chemotherapy followedby concurrent chemotherapy and dose-escalated thoracic conformal radiotherapy (74 Gy) in stage Ⅲ nonsmall-cell lung cancer: CALGB 30105. J Clin Oncol 2008;26:2457-2463.
4. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised
RECIST guideline (version 1.1). Eur J Cancer 2009;45:228-247.
5. US Department of Health and Human Services. National Cancer Institute. Common terminology criteria for- 82 -

Influence of Dose-volume Prescription
adverse events (CTCAE) version 4.0. National Institutes of Health/National Cancer Institute, 2009. pp. 1-194.
6. Tada T, Minakuchi K, Matsui K, et al. A single institutional subset analysis of the WJTOG study comparing
concurrent and sequential chemoradiotherapy for stage Ⅲ non-small-cell lung cancer. Radiat Med 2004;22:
163-167.
7. Kong FM, Ten Haken RK, Schipper MJ, et al. High-dose radiation improved local tumor control and overall
survival in patients with inoperable/unresectable non-small-cell lung cancer: long-term results of a radiationdose escalation study. Int J Radiat Oncol Biol Phys 2005;63:324-333.
8. Bradley JD, Bae K, Graham MV, et al. Primary analysis of the phase Ⅱ component of a phase Ⅰ/Ⅱ dose
intensification study using three-dimensional conformal radiation therapy and concurrent chemotherapy forpatients with inoperable non-small-cell lung cancer: RTOG 0117. J Clin Oncol 2010;28:2475-2480.
9. Schild SE, McGinnis WL, Graham D, et al. Results of a phase Ⅰ trial of concurrent chemotherapy andescalating doses of radiation for unresectable non-small-cell lung cancer. Int J Radiat Oncol Biol Phys
2006;65:1106-1111.
10. Stinchcombe TE, Lee CB, Moore DT, et al. Long-term follow-up of a phase Ⅰ/Ⅱ trial of dose escalating threedimensional conformal thoracic radiation therapy with induction and concurrent carboplatin and paclitaxel inunresectable stage ⅢA/B non-small cell lung cancer. J Thorac Oncol 2008;3:1279-1285.
11. Kawahara D, Ozawa S, Kimura T, et al. Marginal prescription equivalent to the isocenter prescription in lungstereotactic body radiotherapy: preliminary study for Japan Clinical Oncology Group trial (JCOG1408). J
Radiat Res 2017;58:149-154.
12. Frank SJ, Forster KM, Stevens CW, et al. Treatment planning for lung cancer: traditional homogeneous pointdose prescription compared with heterogeneity-corrected dose-volume prescription. Int J Radiat Oncol Biol
Phys 2003;56:1308-1318.
13. Lee JS, Scott CB, Komaki R, et al. Impact of institutional experience on survival outcome of patients
undergoing combined chemoradiation therapy for inoperable non-small-cell lung cancer. Int J Radiat Oncol
Biol Phys 2002;52:362-370.
14. Marks LB, Bentzen SM, Deasy JO, et al. Radiation dose-volume effects in the lung. Int J Radiat Oncol Biol
Phys 2010;76:S70-S76.

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Osaka City Med. J. Vol. 65, 85-91, 2019

Clinical Response to Palliative Radiotherapy for Gastric Cancer
MASARU MAKIHARA1), TAKUHITO TADA1), MASAHIRO TOKUNAGA1), NORIKO TANAKA1),
HIROSHI TSUKUDA2), TAKAYO OTA2), TOMOHIRO NISHIKAWA3), KOSUKE AMANO3), and KENTARO ISHII4)
Departments of Radiology1) and Medical Oncology2), Izumi City General Hospital;
Department of Radiation Oncology3), Osaka City University Graduate School of Medicine; andDepartment of Radiation Oncology4), Tane General Hospital

Abstract
Background
Clinical studies on preoperative chemoradiotherapy have been performed for highly advanced
gastric cancer, and gastric cancer is suggested to be radiosensitive. In order to provide data on theradiosensitivity, a retrospective study was performed.
Methods
The study group comprised 14 patients, who underwent palliative radiotherapy for the primarysite, the regional lymph node metastasis, or disseminated lesions between 2011 and 2016. Radiation
doses were between 33 and 37.5 Gy (median, 36 Gy).
Results
The response rate was 64%. The mean reduction rate was 50% (range, 0%-100%). For 7 patients
who underwent concurrent chemoradiotherapy, the median reduction rate was 58% (p＝0.56). Fourpatients achieved 100% reduction. The maximum reduction was observed between 2.0 and 6.8
months (median 4.7 months) after radiotherapy. One-year in-field control for all patients was 59%.
Palliation of the symptoms was achieved in 92%, 100%, and 0% of the patients showing obstruction,
abdominal pain, and bleeding, respectively.
Conclusions
In conclusion, the response rate was 64% and the mean reduction rate was as high as 50%
although the patients underwent palliative radiotherapy. The median time of the maximum tumor
reduction was 4.7 months after radiotherapy.
Key Words: ‌Gastric cancer; Radiotherapy; Radio sensitivity; Chemotherapy

Introduction
Gastric cancer is the fifth most common cancer and was the second leading cause of cancer-related
deaths worldwide in 2013. In developed countries, it ranks fifth for incidence and third for mortality,
and in developing countries, it ranks third for both incidence and mortality1). Surgery has been a
Received July 10, 2018; accepted April 19, 2019.
Correspondence to: Masaru Makihara, MD.
Department of Radiology, Izumi City General Hospital,
Wake-cho 4-5-1, Izumi, 594-0073, Japan
Tel: +81-725-41-1331; Fax: +81-725-43-3350
E-mail: macky449344@yahoo.co.jp- 85 -

Makihara et almainstay in the treatment of gastric cancer. Several clinical studies on preoperative
chemoradiotherapy showed pathological complete response in 9%-26% of patients with the disease2-4).
Thus, gastric cancer is suggested to be radiosensitive.
In Japan, radiation therapy is performed mainly for palliation of symptoms from the disease, andin a limited number of the patients with unresectable, highly advanced gastric cancer. Several
reports presented favorable outcome of palliative radiotherapy5-7). In this study, the clinical response
to palliative radiotherapy for gastric cancer was evaluated using follow-up CT.

Methods
The institutional review board approved this study. This study was conducted in accordance with
the Declaration of Helsinki.
Patient population
Between November 2011 and March 2016, a total of 17 consecutive patients with gastric cancer
underwent palliative radiation therapy with a dose of 30 Gy or more for the primary site, the regional
lymph node metastasis, or disseminated lesions at our institution. Three patients were excluded
from the study since they had not undergone abdominal CT after radiotherapy. The remaining 14
patients who underwent CT comprised the study group. In all patients, measurable lesions with alongitudinal diameter of 10 mm or more were observed.
Patient characteristics are summarized in Table 1 and Table 2. All patients had a pathologicaldiagnosis of adenocarcinoma and had stage Ⅳ disease. Before radiotherapy, 12 patients had received
Table 1. Patient characteristics
Characteristics

Value

Age (years)
Range
MedianGender

47-79
70

Male
Female＊Performance status (ECOG )1
2＊＊
Presenting symptoms

104Bleeding
Abdominal pain
Obstruction
Treatment before radiotherapy
Curative gastrectomy
Chemotherapy
Concurrent chemotherapy
YesNo
Adjuvant chemotherapy
Yes
No

3
5
12

＊

Eastern Cooperative Oncology Group.

＊＊

Symptoms partly overlap.

- 86 -

6
8

3
12
7
7
10
4

- 87 -

M

M

F

M

72

74

75

66

37.5d

36

36

36

36

36

Ⅳ

Ⅳ

Ⅳ

Ⅳ

Ⅳ

36

Ⅳ

Ⅳ

36

Ⅳ

36

36

Ⅳ

Ⅳ

33

Ⅳ

36

36

Ⅳ

Ⅳ

36

Radiationdose (Gy)

Ⅳ

Stage

Primary site

Primary site

Lymph NodePrimary site

Primary site

Disseminated mass

Primary site

Primary site

Primary site

Primary site

Primary site

Disseminated mass

Primary site

Primary site

CTVa

PTX

TS1

−

HERh

−

−

−

PTXg

−

TS1

−

−

CDDP

CDDPe, 5FUf

Concurrent
chemotherapy

−

−

＋

−

−

＋

−

−

−

−

−

＋

−

−

Surgery

98.7

34.1

29.6

59.1

41.3

39.5

79.2

95.1

64.7

49.8

128

36.5

44

70.6

Diameterb
(mm)

0%

1%

18%

64%

17%

100%

65%

100%

0%

44%

35%

59%

100%

100%

Reductionrate

2 (0.8-3.3 mo)

1 (1.1 mo)

2 (1.0-3.1 mo)

2 (0.8-4.7 mo)

2 (0.2-0.8 mo)

16 (2.9-30.2 mo)

7 (0.7-10.3 mo)

7 (1.4-13.3 mo)

1 (0.9 mo)

5 (0.6-3.2 mo)

1 (1.3 mo)

4 (0.9-9.6 mo)

3 (1.6-6.5 mo)

5 (2.5-12.6 mo)Number of CTc (range of interval)aCTV, clinical target volume; bMaximal tumor diameter; cCT, computed tomography; and dThe daily dose was 2.5 Gy. For all other patients, the daily dose was 3.0 Gy.
CDDP, cis-diamminedichloridoplatinum (cisplatin); f5FU, 5-fluorouracil; gPTX, paclitaxel; and hHER, herceptin (trastuzumab).

e

F

79

F

64

M

M

70

47

M

63

M

F

47

76

M

70

M

M

61

64

GenderAge(years)Table 2. Patient characteristics and reduction rate

Radiosensitivity of Gastric Cancer

Makihara et al

chemotherapy. Among them, 3 patients had undergone curative gastrectomy. In these patients,
relapsed tumor after the treatment was irradiated.
Treatment
Radiation was administered with 10 MV photons from a linear accelerator (Mevatron Primus:
Siemens, Munich, Germany). All patients underwent three-dimensional conformal radiotherapy
using the involved field. Principally, the clinical target volume (CTV) contained the gross tumor
volume plus a 5 mm or more margin, and the planning target volume contained the CTV plus a 5 mm
or more margin. Radiation doses ranged from 33 to 37.5 Gy (median, 36 Gy). Twelve patients (86%)
received 36 Gy in 12 fractions. In 7 patients, chemotherapy was performed concurrently withradiotherapy. The contents were TS-1 (n＝2), paclitaxel (n＝2), 5-fluorouracil plus cisplatin (n＝1),
cisplatin (n＝1), and trastuzumab (n＝1). After completion of abdominal radiotherapy, 2 patients
underwent additional palliative radiotherapy for other sites.
Evaluation
In the present study, the primary endpoint was response, which was evaluated using the Response
Evaluation Criteria in Solid Tumors (RECIST) version 1.18). CT examination was performed under
diet restriction in principle. In this patient group, the number of CT ranged from 1 to 16, and theinterval ranged from 0.2 to 30.2 months (Table 2). Evaluation was performed when the maximum
reduction was obtained. The secondary endpoint was palliation of symptoms. Palliation of
obstruction and abdominal pain were evaluated based on complaint of the patients. And palliation of
bleeding was evaluated using hemoglobin value. Furthermore, toxicity was evaluated according to
the Common Terminology Criteria for adverse Effect (CTCAE) version 4.0 9). Follow-up was performed
starting from the day 1 of irradiation.
Analysis
Survival rates were calculated with the Kaplan-Meier method. Statistical analyses were
performed with the Mann-Whitney U-test.

Results
The follow-up time ranged from 9.6 to 30.2 months (median, 19.9 months). The response rate
based on the RECIST was 64%. The mean reduction rate was 50% (range, 0%-100%). For 7 patients
who underwent concurrent chemoradiotherapy, the mean reduction rate was 58% (p ＝0.56). Fourpatients achieved 100% reduction. The maximum reduction was observed between 2.0 and 6.8
months (median, 4.7 months) after radiotherapy for patients who underwent CT twice or more.
An overall survival curve is shown in Figure 1. One-year survival was 21%, and median survival
time was 5.6 months. An in-field control curve was drawn in Figure 2. When relapse was classified
into in-field relapse, out-field relapse, and marginal relapse according to the radiation field, one-year
in-field control was 59% at this dose.
Palliation of the symptoms was achieved in 92%, 100%, and 0% of the patients showing
obstruction (n＝12), abdominal pain (n＝5), and bleeding (n＝3), respectively. Palliation of obstruction
was obtained with an interval ranging from 3 to 44 days (median, 5 days) after the beginning ofradiotherapy, and that of abdominal pain was obtained with an interval ranging from 4 to 87 days
(median, 17 days) after the beginning of radiotherapy. Grade 3 hematologic toxicity and grade 1
gastrointestinal toxicity were observed in 2 and 3 patients, respectively. However, none of thepatients showed grade 4 hematologic toxicity or greater than grade 2 non-hematologic toxicity.

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Radiosensitivity of Gastric Cancer

Figure 1. Kaplan-Meier survival rate curve for all patients.

Figure 2. Kaplan-Meier in-field control rate curve for all patients.

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Makihara et al

Discussion
Saikawa et al performed a phase Ⅱ study of chemoradiation with a dose of 40 Gy in 20 fractions
for patients with inoperable advanced gastric cancer and reported that clinical response rate was
66% 3). For patients with recurrent lymph node metastasis or local recurrence after curative
gastrectomy, Ishido et al reported the response rate of 62% in chemoradiotherapy with a median doseof 48.6 Gy11). The response rate of 64% in the present study was consistent with these reports in spiteof palliative radiotherapy. Furthermore, 4 patients achieved 100% reduction. Thus, gastric cancer
showed acceptable radiosensitivity, and it was encouraging. When this radiosensitivity is taken into
account in the clinic, more aggressive treatment can be chosen for patients with lower stage disease
and the dose can be modified for patients with poor performance status.
Another finding in the study was the timing of the maximum reduction. The median time of 4.7
months was longer than what we anticipated. For patients, in whom the radiation effect wasinsufficient just after radiotherapy, further downsizing could be expected. Berrington et al reported
that annual radiation exposure was the highest in Japan10). Unnecessary CT examination should be
avoided to reduce radiation exposure for the population. However, this frequent CT examination
brought these findings in this study.
Due to the acceptable radiosensitivity of gastric cancer, relief from obstruction or abdominal pain
was obtained in a majority of patients. Although relief from bleeding was confirmed in none of thepatients, there was a possibility that bleeding was not appropriately evaluated in this retrospective
study. In addition, there were only 3 patients who suffered from bleeding in the study group. Tey etal reported that 80.6% of patients with bleeding responded to radiotherapy with a dose ranging from
8 Gy in a single fraction to 40 Gy in 16 fractions5). When appropriate evaluation was performed, therate of relief might increase.
Asakura et al concluded that a dose of 30 Gy in 10 fractions was adequate in patients with poor
prognosis7). Although a little higher doses were delivered in the present study, severe toxicity was notobserved. These doses can be chosen when the prognosis of patients is not so poor.
Although statistical significance was not observed, the patients who underwent chemoradiotherapy
showed favorable reduction rate. These results suggested existence of radiosensitizing effect ofchemotherapy in gastric cancer. Past reports with use of chemoradiotherapy also presented favorable
response rate.3,11)
There were several limitations in the present study since it was a retrospective one. With theeligibility criteria, 3 patients who did not undergo CT after radiotherapy were excluded. When theeffect of radiotherapy was insufficient, the opportunity to perform CT decreased even in Japan.
Therefore, these exclusions might cause a bias to overestimation. On the other hand, the study groupcontained 3 patients who underwent CT only once after radiotherapy. If additional CT could beobtained, their reduction rate might improve and that caused for a bias to underestimation. In
addition, 10 patients underwent adjuvant chemotherapy was after completion of radiotherapy. Thismight cause for a bias to overestimation.
In conclusion, the response rate was 64% and the mean reduction rate was as high as 50%
although the patients underwent palliative radiotherapy. The median time of the maximum
reduction was 4.7 months after radiotherapy.

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Radiosensitivity of Gastric Cancer

Acknowledgements
All authors have no COI to declare regarding the present study.
References
1. Global Burden of Disease Cancer Collaboration, Fitzmaurice C, Dicker D, et al. The global burden of cancer
2013. JAMA Oncol 2015;1:505-527.
2. Ajani JA, Winter K, Okawara GS, et al. Phase Ⅱ trial of preoperative chemoradiation in patients with localized
gastric adenocarcinoma (RTOG 9904): quality of combined modality therapy and pathologic response. J Clin
Oncol 2006;24:3953-3958.
3. Saikawa Y, Kubota T, Kumagai K, et al. phase Ⅱ study of chemoradiotherapy with S-1 and low-dose cisplatin
for inoperative advanced gastric cancer. Int J Radiat Oncol Biol Phys 2008;71:173-179.
4. Rivera F, Galán M, Tabernero J, et al. Phase Ⅱ trial of preoperative irinotecan-cisplatin followed by
concurrent irinotecan-cisplatin and radiotherapy for resectable locally advanced gastric and esophagogastricjunction adenocarcinoma. Int J Radiat Oncol Biol Phys 2009;75:1430-1436.
5. Tey J, Choo BA, Leong CN, et al. Clinical outcome of palliative radiotherapy for locally advanced symptomatic
gastric cancer in the modern era. Medicine (Baltimore) 2014;93:e118.
6. Sun J, Sun YH, Zeng ZC, et al. Consideration of the role of radiotherapy for abdominal lymph node metastases
in patients with recurrent gastric cancer. Int J Radiat Oncol Biol Phys 2010;77:384-391.
7. Asakura H, Hashimoto T, Harada H, et al. Palliative radiotherapy for bleeding from advanced gastric cancer:
is a schedule of 30 Gy in 10 fractions adequate? J Cancer Res Clin Oncol 2011;137:125-130.
8. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumors: revised
RECIST guideline (version 1.1). Eur J Cancer 2009;45:228-247.
9. National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE). Washington, DC: US
department of Health and Human Services, National Cancer Institute; 2010; Available from: http://evs.nci.nih.
gov/ftp1/CTCAE/About.html.
10. Berrington de González A, Darby S. Risk of cancer from diagnostic X-rays: estimates for the UK and 14 othercountries. Lancet 2004;363:345-351.
11. Ishido K, Higuchi K, Tanabe S, et al. Chemoradiotherapy for patients with recurrent lymph-node metastasis
or local recurrence of gastric cancer after curative gastrectomy. Jpn J Radiol 2016;34:35-42.

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Osaka City Med. J. Vol. 65, 93-107, 2019

The Neural Effects of Inadequate Pauses in Speech on theComprehension of Logical and Illogical Speeches:
A Magnetoencephalography Study
YUKI KODA, and KISHIKO SUNAMI
Department of Otolaryngology and Head & Neck Surgery, Osaka City University
Graduate School of Medicine

Abstract
BackgroundThe comprehension of speech is often affected by the missing of the parts of speech due to such as
background noise. The disturbance of the connections among sounds in speech which should be utteredcontinuously to make the speech intelligible may be a reason for the impaired comprehension of the
speech. Therefore, we aimed to clarify the neural mechanisms related to the comprehension of speechin which pauses are placed at inadequate places in sentence by using magnetoencephalography (MEG).
Methods
Twenty-four healthy male volunteers with normal hearing ability, whose native language isJapanese, participated in our present study. They listened to the speeches in Japanese based on
logically-correct and logically-incorrect stories read with inadequate pauses and the comprehension of
the speeches and the neural activity related to listening to the speeches were assessed compared withthose observed when listening to the speeches read with adequate pauses.
Results
The level of the comprehension of speech was lower when listening to logically-correct andlogically-incorrect stories read with inadequate pauses compared with when listening to the speeches
read with adequate pauses. The increase of gamma band power in the Brodmann s area (BA) 10
observed when listening to the speeches based on logically-correct stories read with inadequate
pauses was negatively associated with the deterioration of the comprehension.
Conclusions
Our findings may help understand the neural mechanisms related to the comprehension of speech,
especially in the situation that the disturbance of the connections within the semantically related
words is caused by background noise.
Key Words: ‌Comprehension of speech; Pause; Illogical sentence;
Magnetoencephalography (MEG)
Received July 22, 2019; accepted October 8, 2019.
Correspondence to: Yuki Koda, MD.
Department of Otolaryngology and Head & Neck Surgery, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3871; Fax: +81-6-6646-0515
E-mail: m1162337@med.osaka-cu.ac.jp- 93 -

Koda et al

Introduction
The comprehension of speech is often affected by the missing of the parts of speech due to such as
background noise. It has been reported that the speech discrimination in noisy environments isworse in the hearing-impaired individuals than in the normal hearing individuals 18) and that thepatients with cochlear implantation are susceptible to the effects of background noise on therecognition of speech9). Thus, clarifying the neural mechanisms of the comprehension of speech innoisy environments is beneficial to develop treatment methods for those suffering from difficulty inspeech comprehension in noisy environments.
It is known that the auditory system is capable of restoring missing sounds in speech replaced byextraneous sound and listeners feel as if they hear the missing sound as a result (i.e., phonemic
restoration)25). It has been proposed that the disability of phonemic restoration can impair speechcomprehension in noisy environment1,23). In the case of failure in restoring the missing sound in
speech, the incomplete words and/or sentences caused by the missing of the sound would deteriorate
the comprehension of the speech. In addition to the missing of the acoustic information which isnecessary to identify the words used in sentences, the disturbance of the connections within thesemantically related units caused by the extraneous sounds may be a reason for the deterioration of
the comprehension of the speech.
There have been reports that investigated the effects caused by replacing parts of continuous
speech or words with noise stimuli or temporal gaps on the comprehension of the speech and thealterations of neural activity caused by the replacements. It has been reported that the replacementof the portions of words or sentences in speech with silence caused the decrease of recognition score(i.e., intelligibility) in listeners with and without hearing impairment11,20) and that the deterioration of
the intelligibility of sentence whose portions were replaced by silence was greater in older listeners
with and without hearing impairment than in younger listeners with normal-hearing20). In a study inwhich neural activity related to the comprehension of segmented speech created by replacing parts ofspeech with noise having a spectral profile and amplitude envelope identical to those of the original
speech was assessed by using functional magnetic resonance imaging (fMRI), the activation inperiauditory brain areas was observed in relation to the comprehension of segmented speech4). In
another fMRI study, the replacement of the parts of speech with silence affected the neural activity inthe left frontal gyrus, right medical frontal gyrus, left temporal cortex, parahippocampal gyrus,
posterior cingulate cortex, caudate, thalamus, and right superior temporal gurus24). In these studies,
since parts of speech or words were replaced by noise or silence to simulate the comprehension of
speech contaminated by noise in real world20), the deterioration of the comprehension of speech may
be caused by both the missing of acoustic information and the disturbance of the connections within
the semantically related units. In fact, although the deterioration of the comprehension of speechcaused by the insertion of silent intervals into speech (i.e., parts of speech were not replaced bysilence) was reported10), there seems to be no report that directly investigated the neural mechanisms
related to the deterioration of the comprehension of speech caused by the disturbance of theconnections within the semantically related units in speech. We hypothesized that there was adifference in neural activity by the disturbance of the connections among sounds in speech (between
adequate pauses and unnatural intonation).
In our present study, we examined neural activity related to the comprehension of speech read
aloud with inadequate pauses. Logical writings in Japanese written for general population in Japan- 94 -

Neural Effects of Comprehension of Speeches

were read aloud by a professional announcer with and without adequate pauses: In the speeches read
aloud with inadequate pauses, although pauses in speech are usually placed between the semantic
units, the pauses were placed at inadequate places in the speech so as to disturb the connections
within the semantically related units in the speech. We required our participants to answer whether
they were able to follow the logic of each speech or not. In addition to the original writings, the logicof which were relatively easy to follow, the writings modified from original ones to be illogical werealso used to assess the effects caused by the inadequate pauses on the comprehension of relatively
difficult speech to follow their logic. The neural activity during listing to the speeches was assessedby using magnetoencephalography (MEG) with high temporal and spatial resolutions. It has beenreported that the neural oscillations assessed by MEG are related to the processing of sentences andspeeches13,15). The experiment of our study included a condition in which the writings were read aloudwith unnatural intonation and a set of speeches whose conclusions were modified to be wrong;
however, the data regarding these conditions and set of speeches were not analyzed in this study.
We aimed to clarify the neural activity caused by the disturbance of the connections among sounds
in speech which should be uttered continuously to make the speech intelligible.

Methods
Participants
Twenty-four healthy male volunteers, whose native language are Japanese, with normal hearing
ability [23.1±2.6 years of age (mean±SD)] participated in this study. The participants were allundergraduate students. All participants were right-handed according to the Edinburgh Handedness
Inventory16). Current smokers, individuals with a history of mental illness, brain injury, or upper
extremity disorder, and individuals taking chronic medications that affect the central nervous systemwere excluded. The Ethics Committee of Osaka City University approved the study protocol(approval number, 3850) and this study was conducted in accordance with the Declaration of Helsinki
and the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects.
All participants provided written informed consent for participation in this study.
Experimental design
In our present study, the participants lay on a bed placed in a magnetically shielded room andlistened to 45 different stories in a row with their eyes closed (Fig. 1A). The neural activity duringlistening to the stories was recorded by MEG. Just after listening to each story, they were asked
whether they were able to follow the logic of the story and to choose their answer from three options:first option, there was no leap in logic in the story and I was able to comprehended the points of thestory ; second option, there was a leap in logic in the story; however, I was able to comprehend eachsentence in the story ; third option, I was not able to comprehend the story even at sentence level
and thus, I was not able to figure out whether there was a leap in logic or not . They were instructedto call the number of the options (i.e, first, second, or third) they chose.
Stories
Fifteen stories in Japanese were selected from logical essays written for Japanese general
population by Japanese philosophers. Another 15 stories were created by modifying original stories
to be logically incorrect but the conclusions of the story were not changed (illogical stories; Fig. 1B).
The other 15 stories were created by modifying the conclusions of the original stories to becontradictory to the original ones (wrong stories; Fig. 1B). Each story was read aloud by a

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Koda et al

professional announcer with adequate pauses, with an unnatural falling intonation (i.e., a segment of
a sentence which was read without pause was read with high tone at the beginning and the tone
gradually decreased toward the end of the segment, irrespective of the meaning the segment conveys),
and with inadequate pauses (i.e., pausing regardless of semantic unit including the pauses such thatplaced at the middle of a word). The voice was recorded by using Wave Pad (NCH Software). It is ofnote that the data regarding the speeches whose conclusions were contradictory to the original ones(i.e., wrong stories) and the speeches read aloud with unnatural intonation were not analyzed in ourpresent study. Forty-five stories were randomly selected for each participant to include same numberof stories in each of nine story-categories (i.e., original, illogical, and wrong stories read with adequate
pauses, those read with inadequate pauses, and those read with unnatural intonation; Fig. 1B). The
numbers of letters and the reading rates (letters/min) of the speeches each participant listened towere summarized in Table 1 (n＝19, as described in the Results). The reading rates for the speeches
based on the original (p＝0.306, t18＝1.0) and illogical (p＝0.105, t18＝1.7) stories read with inadequate
pauses and the speeches based on the illogical stories read with adequate pauses (p＝0.8689, t1＝0.2)
were not different from that for the speeches based on the original stories read with adequate pauses(paired t-test). The recorded stories were played on a Windows Media player (2010) using a speaker

Figure 1. Experimental procedure.
A. It is written in the methods (Experimental design) in the text.
B. It is written in the method (stories) in the text. In our present study, the data regarding the speeches read withunnatural intonation and the speeches with modified conclusions (i.e., wrong stories) were not analyzed. The speeches
whose data were not analyzed were marked with asterisk.

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Neural Effects of Comprehension of Speeches
Table 1. ‌The summary of the numbers of letters and the reading rates (letters/min) of the speeches which
each participant listened to during the MEG measurements
Adequate pauses

Inadequate pauses

Original stories

Illogical stories

Original stories

Illogical stories

Mean

227.2

223.2

226.3

222.1

SD

14.7

11.9

12.1

17.2

Mean
SD

400.6
34.7

403.2
39.6

418.0
42.9

420.3
29.5

Letters

Reading rate
(letters/min)

MEG, magnetoencephalography.

system (SANWA SUPPLY INC., Okayama, Japan) placed outside the shield room during the MEG
recordings. The extent to which each participant was able to follow the logic of the speeches based on
the original stories was assessed by the percentage of the number of the speeches for which theparticipants selected the first option out of the three choices and the extent to which each participant
was able to follow the logic of the speeches based on the illogical stories was assessed by thepercentage of the number of the speeches for which the participants selected the second option out of
the three choices. All the participants declared that they had not read or listened to the original
stories presented during the experiments.
MEG recording
MEG was recorded using a 160-channel whole-head-type MEG system (MEG vision; Yokogawa
Electric Corporation, Tokyo, Japan) with a magnetic field resolution of 4 fT/Hz1/2 in the white-noise
region. The sensor and reference coils were gradiometers with 15.5 mm diameter and 50 mm baseline,
and the two coils were separated by 23 mm. The sampling rate was 1000 Hz and data were high-passfiltered at 0.3 Hz.
MEG analyses
Before processing the MEG data, the magnetic noise that originated from outside the magneticallyshielded room was eliminated by subtracting the data obtained from reference coils using specialized
software (MEG160; Yokogawa Electric Corporation, Tokyo, Japan). Epochs of the raw MEG data that
included artifacts were visually identified and were excluded before averaging. Spatial filtering
analysis of the MEG data was performed to identify changes in oscillatory brain activity that reflected
time-locked cortical activities8,17) caused by listening to the speeches based on the original and illogicalstories read with adequate and inadequate pauses. The MEG data were bandpass filtered at 1-4 Hz,
4-8 Hz, 8-13 Hz, 13-25 Hz, and 25-58 Hz by a finite impulse response filtering method using Brain
Rhythmic Analysis for MEG software (BRAM; Yokogawa Electric Corporation, Tokyo, Japan) toobtain delta, theta, alpha, beta, and gamma signals, respectively. After the bandpass filtering, thelocation and intensity of the cortical activities were estimated using BRAM, which uses a narrowband adaptive spatial filtering algorithm 2,19). Voxel size was set at 5.0×5.0×5.0 mm. For eachfrequency band, the oscillatory power of the MEG data during listening to one type of speech wascompared with that during listening to another type of speech (i.e., the beamformer images as
oscillatory power ratio). To minimize any effects caused by the initiations and cessations of the- 97 -

Koda et al

auditory stimuli, oscillatory power ratio was calculated for the MEG data from 2000 ms after thestart of each speech to 2000 ms before the end of the speech.
Data were then analyzed using statistical parametric mapping (SPM8, Wellcome Department of
Cognitive Neurology, London, UK), implemented in Matlab (Mathworks, Natick, MA). The individual
structural MRI image was transformed into the Montreal Neurological Institute T1-weighed image
template5). The beamformer images registered to the individual MRI image was normalized applying
the same parameters used when the individual structural MRI was normalized. The anatomicallynormalized beamformer images were filtered with a Gaussian kernel of 20 mm (full-width at halfmaximum) in the x-, y-, and z-axes. To enable inferences to be made at a population level, individual
data were summarized and incorporated into a random-effect model 7). The weighted sum of theparameters estimated in the individual analysis was used to create contrast images that were used
for group analyses7). The resulting set of voxel values for each comparison constituted a statisticalparametric map (SPM) of the t statistic [SPM(t)]. The SPM(t) was transformed to the units of normal
distribution [SPM(Z)]. The significance of any difference in the oscillatory power caused by listening
to the speeches based on the original and illogical stories read with inadequate pauses and those
based on the illogical stories read with standard intonation, compared with those based on theoriginal stories read with standard pauses, was assessed using t statistics (one sample t test) on avoxel-by-voxel basis7). The height threshold for the SPM(t) of the one sample t test was set at p＜0.0017 (family-wise-error corrected for multiple comparisons), considering the number of contrasts(i.e., the original stories read with inadequate pauses vs the original stories read with adequate
pauses, the illogical stories read with adequate pauses vs the original stories read with adequate
intonation, and the illogical stories read with inadequate pauses vs the original stories read withadequate pauses) and frequency bands (i.e., delta, theta, alpha, beta, and gamma bands). In addition,
the peak voxels located within the cluster larger than 1 voxel was reported. Localization of the brain
regions was performed using WFU_PickAtras, Version 3.0.4 (http://fmri.wfubmc.edu/software/
pickatlas) and Talairach Client, Version 2.4.3 (http://www.talairach.org/client.html).
Magnetic resonance (MR) image overlay
Anatomical MR imaging was performed using a Philips Achieva 3.0 TX (Royal Philips Electronics,
Eindhoven, The Netherlands) to permit registration of magnetic source locations with their respective
anatomical locations. Before MR scanning, five adhesive markers (Medtronic Surgical Navigation
Technologies Inc., Broomfield, CO.) were attached to the skin of the head: two markers 10 mm infront of the left and right tragus, one marker 35 mm above the nasion, and two markers 40 mm toeither side of the marker above the nasion. The MEG data were superimposed on MR images using
information obtained from these markers and MEG localization coils.
Statistical analyses
Values are presented as mean and SD unless otherwise stated. A paired t-test with Bonferroni
correction was used to compare the correction rates of the questions for speeches based on the original
stories read with adequate pauses with those based on the original and illogical stories read withinadequate pauses and those based on the original stories read with adequate pauses. Relationships
between the correction rates for the questions and the alterations of oscillatory band power were
evaluated using Pearson s correlation analyses. All p values were two-tailed and values less than
0.05 were considered statistically significant. These statistical analyses were performed using the
IBM SPSS 21.0 software package (IBM, Armonk, NY).

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Neural Effects of Comprehension of Speeches

Figure 2. Closed column indicates the data corresponding to the speeches based on the original stories and open
column indicate the data corresponding to the speeches based on the illogical stories. Data are presented as mean±
SD. *p＜0.05, paired t-test with Bonferroni correction.

Results
The extent to which participants followed the logic of the speeches
The data from five participants were excluded from the analysis: The MEG data from threeparticipants were contaminated with magnetic noise that originated from outside the shielded roomand the MEG data form the other two participants were excluded because of their head motion during
the MEG recording.
The extent to which participants were able to follow the logic of the speeches (i.e., the comprehensionof logic) were calculated for the speeches based on the original stories read with standard intonation(hereinafter refer to as original A ), those based on the original stories read with inadequate pauses(hereinafter refer to as original I ), and those based on the illogical stories read with inadequate
pauses (hereinafter refer to as illogical I ) (Fig. 2). The comprehension of logic for the speeches
based on the original I was lower than that for the speeches based on the original A (p＜0.011, t18＝3.6,
paired t-test with Bonferroni s correction). Both in the speeches read with adequate and inadequate
pauses, the comprehension of logic for the speeches based on the illogical stories was lower than that- 99 -

Koda et al

Figure 3. Statistical parametric maps of the brain regions where oscillatory power was lower when listening to the
speeches read with inadequate pauses and those based on illogical A compared with when listening to the speeches
based on the original A. The decreases of oscillatory band power caused by listening to the speeches based on theoriginal stories read with inadequate pauses A, the decreases of oscillatory band power caused by listening to the
speeches based on the illogical stories read with inadequate pauses B, and the decreases of oscillatory band power
caused by listening to the speeches based on the illogical stories read with adequate pauses C were observed comparedwith when listening to the speeches based on the original A. Random-effect analyses of 19 participants, p＜0.0017,
family-wise-error corrected for the entire search volume (A, anterior; P, posterior; L, left; and R, right).

for the speeches based on the original stories (p＝0.020 and t18＝3.4 for the adequate pauses and p＝0.039 and t 18＝3.1 for the inadequate pauses, paired t-test with Bonferroni s correction). The
comprehension of logic for the speeches based on the illogical I was lower than that for the speeches
based on the original A (p＜0.001, t18＝6.7, paired t-test with Bonferroni s correction).
The number of the speeches which our participants thought to be incorrect did not differ between
the speeches based on the original I and those based on the original A (p＝0.450, t18＝0.8, paired t-test
without correction for multiple comparisons) and between the speeches based on the illogical I and
those based on the original A (p ＝0.450, t 18＝0.8, paired t-test without correction for multiple
comparisons).
Spatial filtering analyses of MEG data
To identify the alterations in the oscillatory power caused by the speeches based on the original I,
oscillatory powers in delta, theta, alpha, beta, and gamma bands caused by listening to the speeches- 100 -

Neural Effects of Comprehension of Speeches

Figure 4. Maximum intensity projections of the statistical parametric maps of the brain regions where oscillatory
power was higher when listening to the speeches read with inadequate pauses and those based on illogical A comparedwith when listening to the speeches based on the original A. The increases of oscillatory band power caused bylistening to the speeches based on the original stories read with inadequate pauses A, the increases of oscillatory
band power caused by listening to the speeches based on the illogical stories read with inadequate pauses B, andthe increases of oscillatory band power caused by listening to the speeches based on the illogical stories read withadequate pauses C were observed compared with when listening to the speeches based on the original A. Randomeffect analyses of 19 participants, p＜0.0017, family-wise-error corrected for the entire search volume (A, anterior; P,
posterior; L, left; and R, right).

based on the original I were compared with those caused by listening to the speeches based on theoriginal A. There were brain regions in which the oscillatory power caused by listening to the
speeches based on the original I was smaller (Fig. 3A) or greater (Fig. 4A) than that caused bylistening to the speeches based on the original A (Table 2).
To identify the effects caused by the speeches based on the illogical I, the oscillatory powers caused
by listening to the speeches based on the illogical I were compared with those caused by listening to
the speeches based on the original A. There were brain regions in which the oscillatory power caused
by listening to the speeches based on the illogical I was smaller (Fig. 3B) or greater (Fig. 4B) thanthat caused by listening to the speeches based on the original A (Table 3).
In addition, to identify the effects caused by the speeches based on the illogical stories read withadequate pauses (hereinafter refer to as illogical A ), the oscillatory powers caused by listening to- 101 -

Koda et al

Figure 5. The relationship between the decrease of the extent to which participants were able to follow the logicof speech caused by the speeches based on the original I and the increase of the 25-58 Hz band power in the BA 10
when listening to the speeches. (A) The maximum intensity projections of the statistical parametric maps of the brain
regions including the left BA 10 (arrow head) where the increase of the 25-58 Hz band power was associated with thedecrease of the comprehension caused by the speeches based on the original I was shown. Random-effect analyses of
19 participants, p＜0.0017, family-wise-error corrected for the entire search volume (A, anterior; P, posterior; and L,
left; R, right). (B) The relationship between the decrease of the extent to which participants were able to follow the
logic of speech caused by the speeches based on the original I and the increase of the 25-58 Hz band power in the BA
10 when listening to the speeches was shown. The linear regression line, Pearson s correlation coefficient, and the pvalue are shown.

the speeches based on the illogical A were compared with those caused by listening to the speeches
based on the original A. There were brain regions in which the oscillatory power caused by listening
to the speeches based on the illogical A was smaller (Fig. 3C) or greater (Fig. 4C) than that caused bylistening to the speeches based on the original A (Table 2). Whether each brain regions listed in Table
3 was included in the brain areas in which alterations in oscillatory brain activity were observed
when the speeches based on the original I and when the speeches based on the illogical A wereindicated in Table 3.
To examine the neural activity related to the comprehension of logic affected by listening to the
speeches based on the original I , the correlations between the alterations of oscillatory brain activity
caused by listening to the speeches based on the original I (Figs. 3A and 4A) and the decrease of theextent to which participants were able to follow the logic of the speeches were examined. The
increase of the 25-58 Hz band power in the Brodmann s area (BA) 10 (Fig. 5A) caused by listening to
the speeches based on the original I was negatively associated with the decrease of the extent towhich participants were able to follow the logic in the speeches based on the original I (p＝0.027, r＝−0.507; Fig. 5B). To examine the neural activity related to the comprehension of logic affected bylistening to the speeches based on the illogical I , the correlations between the alterations ofoscillatory brain activity caused by listening to the speeches based on the illogical I (Figs. 3B and 4B)
and the decrease of the extent to which participants were able to follow the logic of the speeches were
examined. However, there were no brain regions in which the alterations of oscillatory brain activity
were associated with the decrease of the extent to which participants were able to follow the logic of
the speeches.

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Neural Effects of Comprehension of Speeches
Table 2. ‌Brain regions that showed a decrease or increase in oscillatory power caused either by theoriginal stories read with inadequate pauses or by the illogical stories read with adequate
pauses compared with those caused by the original stories read with adequate pauses
Contrast

Frequency (Hz)

LocationBA

4-8 HzInferior Frontal Gyrus
Middle Frontal Gyrus
Middle Frontal Gyrus
Supramarginal Gyrus
Middle Frontal Gyrus
Superior Frontal Gyrus
Inferior Frontal Gyrus
Medial Frontal Gyrus
Precuneus
Angular Gyrus
InsulaSuperior Frontal Gyrus
Medial Frontal Gyrus
Precentral Gyrus

45
46
11
40
10
9
46
10
19
39
13
10
10
6

Postcentral Gyrus
Middle Frontal Gyrus
Middle Frontal Gyrus
Superior Frontal Gyrus
Middle Frontal Gyrus
Middle Temporal Gyrus
Middle Frontal Gyrus
Middle Frontal Gyrus

2
6
8
9
46
39
9
47

Superior Frontal Gyrus
Inferior Parietal Lobule
CuneusPrecentral Gyrus
Middle Frontal Gyrus
Inferior Frontal Gyrus
Inferior Parietal Lobule
Inferior Frontal Gyrus
Medial Frontal Gyrus
Precuneus

8
40
18
6
6
9
40
47
11
19

Inferior Parietal Lobule
Precentral Gyrus
CuneusInferior Parietal Lobule
Middle Temporal Gyrus
Superior Frontal Gyrus
Superior Parietal Lobule
Middle Frontal Gyrus
Inferior Frontal Gyrus
Superior Parietal Lobule
Inferior Frontal Gyrus
Postcentral Gyrus
Inferior Parietal Lobule
Inferior Parietal Lobule
Middle Frontal Gyrus
Middle Frontal Gyrus

40
6
19
40
21
8
7
10
46
7
9
1
40
40
6
46

MNI (mm)
y

z

22
52
2
−38
52
37
−33
7
−38

33
28
38
−52
58
53
43
58
−77
−72
8
53
63
−7

5
30
−10
35
25
30
5
0
40
35
20
30
5
40

5.6
5.1
5.0
4.9
5.1
4.9
4.7
4.7
5.4
5.2
5.1
5.1
5.0
4.8

57

−27

−43

13
23
38
48
−72
18
38

45
50
45
40
20
20
35
−5

5.6
5.2
4.9
4.9
5.0
5.0
4.8
4.7

50
30
5
40
45
25
55
−15
−10
40

5.4
5.2
5.1
5.1
4.9
5.3
5.1
5.1
5.0
4.8

50
30
30
30
−10
55
45
25
5
55
30
65
60
30
45
30

5.5
5.4
5.4
5.3
5.2
5.2
5.0
4.9
4.9
4.9
5.4
5.3
5.2
5.0
5.4
4.9

x

Z-value

Original/inadequate＞Original/adequate

25-58 Hz

1-4 Hz

−58
−58
−18

57
−28

Original/inadequate＜Original/adequate
4-8 Hz8-13 Hz13-25 Hz

42
17
−38
−58
−48
−33

Illogical/adequate＞Original/adequate
1-4 Hz4-8 Hz8-13 Hz

−3

33

−63

−27

17
62
−53
62
52
−53
−8
−18

−102

42
52
17
−58
−63
−33
−38
−43
52
22
−58
−43
47
−63
−58
52

−37

−7

3
18
−47
28
53
−87

Illogical/adequate＜Original/adequate
1-4 Hz4-8 Hz8-13 Hz

−2
−92
−27

3
23
−72
43
43
−72
13
−32
−42
−27
3
43

Data were obtained from random-effect analyses. Only significant changes are shown (one-sample t test, p＜0.0017,
family-wise error rate). x, y, z: Stereotaxic coordinate. BA, Brodmann area; and MNI, Montreal Neurological Institute.

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Koda et al
Table 3. ‌Brain regions that showed a decrease or increase in oscillatory power caused by the illogicalstories read with inadequate pauses compared with those caused by the original stories readwith adequate pauses
Contrast

Frequency(Hz)

MNI (mm)
LocationBA

Precentral Gyrus
Postcentral Gyrus
CuneusZ-value

x

y

z

4
7
19

27
12
32

−22

50
70
30

5.0
4.9
4.8

Cingulate Gyrus
Inferior Frontal Gyrus
Superior Parietal Lobule
Inferior Frontal Gyrus
Superior Frontal Gyrus
Inferior Frontal Gyrus

31
45
7
46
11
46

−18

−57

62
37
−48
−28
47

13
−57
43
58
38

65
20
50
5
−10
15

5.8
5.1
5.1
5.0
4.9
4.8

Inferior Frontal Gyrus
CuneusInferior Frontal Gyrus
Middle Frontal Gyrus
Middle Frontal Gyrus
Superior Temporal Gyrus
Superior Parietal Lobule
Precuneus
Superior Parietal Lobule
Middle Frontal Gyrus

44
19
47
10
46
22
7
7
7
46

−58

13

22
−53
−23
−48
67
22
7
32
−48

−97

15
20
−15
10
20
5
55
45
65
25

5.8
5.4
5.0
4.9
5.1
5.0
5.0
4.9
5.5
5.1

Included in
Original/
Illogical/
inadequate
adequate

Illogical/inadequate＞Original/adequate
1-4 Hz4-8 Hz25-58 Hz

−57
−82

○
○

Illogical/inadequate＜Original/adequate
1-4 Hz4-8 Hz8-13 Hz

38
48
48
−37
−72
−57
−57
38

Data were obtained from random-effect analyses. Only significant changes are shown (one-sample t test, p＜0.0017,
family-wise error rate). x, y, z: Stereotaxic coordinate. 〇 : brain activities observed. BA, Brodmann area; and MNI,
Montreal Neurological Institute.

Discussion
In our present study, the extent to which participants were able to follow the logic in the speeches
based on original I were lower than that in speeches based on original A, confirming that thecomprehension of speech is impaired by the disturbance of the connections within the semanticallyrelated units in the speech. The extent to which participants were able to follow the logic in the
speeches read with inadequate pauses was further deteriorated when the speeches were modified tobe illogical (i.e., the extent to which participants were able to follow the logic in the speeches based on
the illogical I was lower than that in the speeches based on the original I).
In relation to listening to the speeches based on the original I, alteration of oscillatory brain
activity in several brain regions, such as the inferior, middle, medial, and superior frontal gyrus, thesupramarginal gyrus, the angular gyrus, and so on, were observed (Figs. 3A, 3B, 4A, and 4B). The
alterations of the level of activations caused by listening to the speeches in which parts of continuous
speech sound were replaced by silence have been reported in an fMRI study: The increase of neuralactivity was observed in the left postcentral gyrus and the right medial frontal gyrus, and thedecrease of neural activity was observed in the left temporal cortex, the parahippocampal gyrus, theposterior cingulate gyrus, the caudate and thalamus, and the right superior temporal gyrus24). Thereseems to be considerable overlap between the brain regions in which alterations of oscillatory brain- 104 -

Neural Effects of Comprehension of Speeches

activity caused by listening to the speeches based on the original I were observed in our present study
and those in which the alterations of the level of activations observed in the previous fMRI study.
However, since the alterations of oscillatory brain activity assessed by MEG is not necessarily parallelto the alterations of hemodynamic responses measured by fMRI, it is difficult to draw a conclusion onwhether the neural mechanism of listening to the speeches read with inadequate pauses is different
from that of listening to the speeches in which parts of continuous speech sound were replaced bysilence.
Since the comprehension of speech is thought to be impaired by the disturbance of the connections
within the semantically related units in the speech and the increase of the 25-58 Hz (i.e., gamma)band power in the BA 10 caused by listening to the speeches based on the original I was associatedwith the decrease of the level of the comprehension of speech, the BA 10 may be a brain region thatplays an important role in the comprehension of speech affected by the disturbance of the connections
within the semantically related units in the speech. It has been reported that the alteration ofoscillatory brain activity in each frequency band (i.e., delta, theta, alpha, beta, and gamma bands)
was related to various aspects of information processing such as sensory, motor, and cognitive
processes (Klimesch 1996; Pfurtscheller and Lopes da Silva 1999). As for the neural activity related
to language processing, it has been reported that the increase of gamma (36-76 Hz) and beta (14-30
Hz) oscillations are related the prediction of the upcoming word 13,15). It has been thought that the
anticipation of the upcoming words of a sentence by using lexical-semantic knowledge is an essential
process for language comprehension and the gamma oscillation (＞35 Hz) is involved in the process tocheck the incoming lexical-semantic representations with the predicted upcoming words15). Therefore,
it is plausible that the increase of gamma band oscillation in the BA 10 observed in our present study
is related to the prediction of the upcoming words, which seems to be more crucial in the speeches
read with inadequate pauses than in the speeches read with adequate pauses. In fact, the individuals
with higher beta band power in the BA 10 showed lesser decrement of the extent t o which
participants were able to follow the logic of the speeches caused by the speeches read with inadequate
pauses in our present study.
The alterations of oscillatory brain activity observed when listening to the speeches based on
illogical I did not include the increase of gamma band power in the BA 10. This may reflect the factthat the comprehension of the speeches based on the illogical I was significantly worse compared withthat of the speeches based on the original A: The prediction of the upcoming words was impaired
when listening to the speeches based on the illogical I, resulting in the deteriorated comprehension of
the speeches. In addition, among the brain regions in which the alterations of oscillatory brain
activity were observed when listening to speeches based on illogical I, the increases of theta band
power in the BA 45 and BA 46 were the only brain activities observed when listening to the stories
based on the illogical A and the stories based on the original I, respectively. These findings show thatthe neural processing of the speeches based on the illogical I was different from that of the speeches
based on the original I and that of the speeches based on the illogical A rather than the extreme
version of these neural processing (i.e., the neural processing of the speeches based on the original I
and that of the speeches based on the illogical A).
There are limitations to our study. First, the participants of our present study were healthy maleindividuals. To generalize our results, studies with female and the individuals with hearing loss arenecessary. Second, all the speeches based on the original stories were logically correct and all the- 105 -

Koda et al

speeches based on the illogical stories were logically incorrect. For example, it has been reported thatthe processing of false sentences is related to the activation of the BA 46 and the BA10 in an fMRI
study14). However, in our present study, because the number of the speeches which our participants
thought to be incorrect did not differ between the speeches based on the original I and those based on
the original A, the increase of gamma band power observed in the BA 10 when listening to the
speeches based on the original I seems not to be due to the fact that all the speeches based on theillogical stories were logically incorrect while those based on the original stories were logicallyincorrect. Third, cognitive capacity of our participants was not assessed. However, since theparticipants were all undergraduate students and elderly individuals were not included, it is assumed
that their cognitive capacities were similar among the participants.
In conclusion, we demonstrated that the comprehension of speech is impaired by the disturbanceof the connections within the semantically related units in speech caused by placing pauses atinadequate places in the speech and that the BA 10 is involved in the comprehension of the speecheswith inadequate pauses. Our findings may help understand the neural mechanisms related to thecomprehension of speech, especially in the situation that the disturbance of the connections within
the semantically related units may be caused by such as extraneous sounds.
We believe to clarify the neural activity caused by the disturbance of the connections amongsounds in speech is beneficial to develop treatment methods for those suffering from difficulty inspeech comprehension depending on how to speak and intonation. We want to use our present study
for the development of auditory rehabilitation. We hope to clarify the neural activity caused by the
disturbance of the connections among sounds in speech may be connected to rehabilitation for the
hearing impaired and the patients with cochlear implantation.
Acknowledgements
The All authors have no COI to declare regarding the present study.
We thank Forte Science Communications for editorial help with the manuscript and Manryoukai
Imaging Clinic for MRI scans.
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Osaka City Med. J. Vol. 65, 109-118, 2019

Microcuff ® Pediatric Endotracheal Tube Decreases the Tube
Exchange Ratio without Increasing Postoperative Complications inJapanese Children: A Single Center Retrospective Cohort StudyYOSUKE INADA, YUSUKE FUNAI, TAKASHI SHUTOU, TAKASHI MORI, and KIYONOBU NISHIKAWA
Department of Anesthesiology, Osaka City University Graduate School of Medicine

Abstract
BackgroundThe usefulness of the Microcuff ® pediatric endotracheal tube (Microcuff ® PET) has been reported
from other countries, but few reports include Japanese children because Microcuff ® PET was recently
introduced in Japan in 2015. Therefore, we compared the tube exchange ratio and postoperativecomplications between Microcuff ® PET and uncuffed tubes in Japanese children.
Methods
We conducted a single center retrospective cohort study with approval from our ethics committee.
Patients aged 2 to 8 years, who underwent general anesthesia with endotracheal intubation between
October 2015 and September 2017, were included. Patients who were intubated with either uncuffed
tube (UC group) or Microcuff ® PET (MC group) were retrospectively analyzed. The primary outcome
was the tube exchange ratio, and the secondary outcome was the incidence of perioperativecomplications. Data are presented as numbers or medians.
Results
We analyzed 250 patients (UC vs MC, 127 vs 83). There were no differences between groups inbaseline characteristics. The tube inner diameter was narrower (5 vs 4.5 mm, p ＜0.01), the tube
depth was shallower (15 vs 14.5 cm, p＝0.011), and the tube exchange ratio was smaller (23.6% vs
1.2%, p＜0.01) in the MC group. There were no severe air leakage cases in the MC group. In a
multiple logistic regression analysis, usage of Microcuff ® PET was the only independent negative risk
factor for tube exchange (p ＜0.01). There were no significant differences in postoperativecomplications between the groups.
Conclusions
Compared to uncuffed tube, Microcuff ® PET significantly reduced the tube exchange ratio without
increasing postoperative complications in Japanese children.
Key Words: ‌Cuffed endotracheal tube; Microcuff ®; Pediatrics

Received August 16, 2019; accepted October 8, 2019.
Correspondence to: Yusuke Funai, MD, PhD.
Department of Anesthesiology, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8586, Japan
Tel: +81-6-6645-2186; Fax: +81-6-6645-2489
E-mail: yusuke.funai@gmail.com- 109 -

Inada et al

Introduction
Traditionally, uncuffed endotracheal tubes have been commonly used for children under 8 to 10
years old to reduce the risks of: (1) airway mucosal injury by the cuff, especially at the cricoid ringthat surrounds the narrowest portion of the pediatric trachea, and (2) increasing airway resistancebecause of the narrower inner diameter of cuffed tubes1). Conversely, an uncuffed endotracheal tube
is sometimes necessary to be exchanged for a different size because of ventilation failure owing to airleakage or overtightened seal. Multiple trials of laryngoscopy and reintubation may cause airway
injury and increase postoperative airway complications such as laryngeal edema, vocal cord injury,
hoarseness, or sore throat. In recent years, many studies have reported the usefulness of cuffed
endotracheal tubes for pediatric patients that appear to have the same benefits as that in adults2-8).
Specifically, several studies have demonstrated the superior efficacy and safety of the Microcuff ®
pediatric endotracheal tube (Microcuff ® PET; Avanos Medical, Inc. Alpharetta, GA, USA), which hasa high-volume low-pressure cuff designed especially for children 9-18). Microcuff ® PET has apolyurethane thin-wall cuff that improves sealing characteristics with lower cuff pressure, resulting
in a reduction in the number of endotracheal tube exchanges8,14,18). Additionally, its short cuff helps to
avoid subglottic compression by the cuff thus preventing perioperative airway complications 19).
However, Microcuff ® PET has only been commercially available in Japan since June 2015, and thus,
previous reports about its usefulness in Japanese children are very limited. We conducted a single
center retrospective cohort study to elucidate the usefulness and safety of Microcuff ® PET for
Japanese children aged 2 to 8 years by comparing it to the uncuffed tracheal tube (Portex® Tracheal
Tube; Smiths Medical ASD, Inc. Minneapolis, MN, USA) that has been mainly used in our hospital.
The aim of this study was to compare the tube exchange ratio and perioperative complications
between the Microcuff ® PET and Portex® Tracheal Tube in Japanese children.

Methods
A single center (Osaka City University Hospital) retrospective cohort study was conducted. This
study was approved by the Ethics Committee of Osaka City University (approval number: 3978) andregistered in the UMIN Clinical Trials Registry (No: 000035767). All patients were informed about
this study on our website (http://www.med.osaka-cu.ac.jp/medinf/rinri/public/koukai-jisshi.html) andgiven the opportunity to opt-out at any time.
Patients (American Society of Anesthesiologists Physical Status; ASA-PS 1-2) aged 2 to 8 years,
who underwent general anesthesia with tracheal intubation surgery in a supine or lateral position
between October 2014 and September 2017 at Osaka City University Hospital were included.
Patients exclusion criteria were as follows: known or suspected difficult airway, known hoarseness or
airway infection, and the usage of endotracheal tubes other than Portex ® or Microcuff ® PET.
Although the selection of each tube size was specified in the product documents (Portex®: ID 4.0 mm
for 3 to 6 months, ID 4.5 mm for 6 to 12 months, ID 5.0 mm for 2 or 3 years, ID 5.5 mm for 4 or 5
years, ID 6.0 mm for 6 or 7 years; Microcuff ® PET: ID 4.0 mm for 2 to 4 years, ID 4.5 mm for 4 to 6
years, and ID 5.0 mm for 6 to 8 years), the final decision of the tube size was made by eachanesthesiologist. The tube insertion depth was also decided by each anesthesiologist; the tube waswithdrawn from bronchial intubation depth under auscultation or the ring mark was adjusted
according to the vocal cord in certain cases of Microcuff ® PET. There were no limitations with respect
to performing general anesthesia, but all patients were intubated by the oral route under direct- 110 -

The Usefulness of Microcuff ® Tube in Japanese.

laryngoscopy after administration of rocuronium. Anesthesia was maintained by inhaled anesthetics(sevoflurane or desflurane) in all patients. Each anesthesiologist was responsible for determining thenecessity for exchanging tubes. All patients were extubated at the end of the operation. All data
were retrospectively extracted from our electronic anesthesia record system (ORSYS; Philips
Electronics Japan, Tokyo, Japan) and electronic medical records (EGMAIN-EX; Fujitsu Co., Ltd.,Tokyo, Japan). We extracted information about each patient (age, sex, height, weight, and ASA-PS),
the operation (surgical procedure, operative time, anesthesia time, blood loss volume, urinary volume,and infusion volume), intubation (tube type, tube size, insertion depth, cuff pressure, number ofintubation trial times, and Cormack-Lehane grade), and perioperative complications. The primary
outcome was the number of patients who needed tube exchange, and the secondary outcome was theincidence of perioperative complications, including respiratory complications (strider, increased
sputum, bronchospasm, atelectasis, and hoarseness), sore throat, postoperative nausea and vomiting(PONV), and others.
Statistical Analysis
Data are presented as median and interquartile ranges [IQR, 25%-75%] or numbers (percentages).
Baseline patient characteristics were analyzed using the Mann-Whitney U test for continuous
variables and the Fisher s exact test or χ 2 analysis for nominal data. A multivariate logistic
regression analysis was performed to identify the significant covariates associated with therequirement of an endotracheal tube exchange after the first intubation. As explanatory variables,
we chose factors with p＜0.3 in the univariate analysis and excluded factors that were too closely
related to tube exchange and tube type. Results are presented as odds ratios (OR) with 95%
confidence intervals (CI). Statistical significance was determined at p ＜0.05 in all cases. Based on
previous studies of other facilities, the tube exchange ratio was assumed to be 10% for the MC groupand 30% for the UC group. When performing a power analysis with α error＝0.05 and detection
power＝0.8, 72 cases were required in each group for a total of 144 cases. Statistical analysis was
performed using EZR software (version 1.30; Saitama Medical Center, Jichi Medical University,
Saitama, Japan)20).

Results
A total of 250 children (ASA-PS 1-2, aged 2 to 8 years) who underwent general anesthesia at
Osaka City University Hospital between October 2014 and September 2017 were eligible for ouranalysis (Fig. 1). Seven patients were excluded owing to incomplete data (no record of the selected
tube type). Thirty-three patients were excluded because a cuffed endotracheal tube other than theMicrocuff ® PET was used. There were no cases of known or suspected difficult airway, hoarseness, or
airway infection. Among the remaining 210 patients, 127 (UC group) intubated with uncuffed
endotracheal tubes (Portex®) and 83 (MC group) intubated with the Microcuff ® PET were finallyanalyzed (Fig. 1).
Baseline patient characteristics and information about general anesthesia for both groups are
summarized in Table 1. In brief, the median age of our participants was 4 years [IQR, 3-5 years], andthe sex distribution among the groups was comparable (males, n＝111, 52.9%). Most patients had
ASA-PS 1 (n＝175, 83.3%). The median operative and anesthesia times were 39 [IQR, 25-63.8]
minutes and 82 [IQR, 64-118] minutes, respectively. The median infusion volume during anesthesia
was 110 [80-200] mL. There was little blood loss and urinary volume during anesthesia in most- 111 -

Inada et al

Figure 1. Study synopsis. Abbreviations: UC, uncuffed tube (means Portex®); and MC, Microcuff ® PET.

cases. A total of 31 patients (14.8%) underwent endotracheal tube exchange after the first intubation.
The inner diameter of the intubated tubes was 4.0 to 6.0 mm. The median cuff pressure in the MCgroup was 20 [11.25-20] mm Hg (n＝71, 12 cases were excluded due to absence of records). Most
patients were intubated once (n＝172, 81.9%); easy success was achieved in most cases (CormackLehane grade 1, n＝185, 92.0%; 9 cases were excluded due to absence of records). In the univariate
analysis, the insertion depth was significantly shallower (UC vs MC, 15 [14-16] vs 14.5 [13-15] cm, p＝0.011), and the inner diameter of the selected tube was significantly narrower (5 [5-5.5] vs 4.5 [4-5]mm, p＜0.001) in the MC group. The number of patients needing tracheal tube exchange after the
first intubation was 30 (23.6%) in the UC group, although there was only 1 (1.2%) such case in the
MC group (p＜0.01). Table 2 shows the details of cases in which endotracheal tubes were exchanged
after the first intubation. Most of the cases required a tube size larger because of ventilation failure
due to air leakage in the UC group (n＝23, 18.1%), although no patients experienced severe airleakage in the MC group. Conversely, 7 cases in the UC group and 1 in the MC group required a tube
size smaller because of resistance to intubation or overly tight sealing pressure.
Surgical sites of the patients are summarized in Table 3. The most frequent surgical site was theabdominal region (n＝95, 45.2%), followed by head and neck (n＝51, 24.3%) and skin (n＝48, 22.9%).
Most cases of abdominal surgery were laparoscopic inguinal hernia repair, and 45% of the head andneck surgeries involved laryngopharyngeal surgery. All cases categorized under laryngopharyngeal
surgery were tonsillectomies, adenoid resections, or both. Operations included under the skin
category comprised laser therapy for nevus, skin tumor resection, and repair of cicatricial contracture.
There was no statistical difference between the groups in the distribution of any surgical sites.
Next, we performed a multivariate logistic regression analysis to identify the risk factors for therequirement of endotracheal tube exchange after the first intubation. Tube type, sex, and ASA-PS
were chosen as explanatory variables because their p values were ＜0.3 in the univariate analysis(Table 4). Tube insertion depth and inner diameter were voided as explanatory variables unless theirp values were ＜0.3 because their statistical relationship with the selection of Microcuff ® PET was too- 112 -

The Usefulness of Microcuff ® Tube in Japanese.
Table 1. ‌Baseline characteristics of study population, overall and by tube type
Overall (n＝210)

UC group (n＝127)

MC group (n＝83)

p valueAge (yr)

4 [3-5]

4 [3-5]

4 [3-5]

0.7

Male sex

111 (52.9%)

63 (49.6%)

48 (57.8%)

0.26

Height (cm)101.9 [92.3-110.7]

101.9 [93.4-110.0]

101.8 [91.6-111.8]

0.94

Weight (kg)15.8 [13.2-18.5]

15.9 [13.5-18.2]

15.5 [13.2-19.2]

0.92

1

175(83.3%)

110 (86.6%)

65 (78.3%)

2

35 (16.7%)

17 (13.4%)

18 (21.7%)

Operative time (min)39 [25-63.8]

39 [25-61]

40 [24.5-69.5]

0.87

Anesthetic time (min)82 [64-118]

83 [66-118.5]

81 [62-116.0]

0.99

Blood loss (g)0 [0-1]

0 [0-75]

0 [0-70]

0.67

Urinary volume (mL)

0 [0-0]0 [0-0]0 [0-0]0.39

Infusion volume (mL)

110 [80-200]

100 [80-192.5]

122 [85-200]

0.43

Cases of exchanging tube

31 (14.8%)30 (23.6%)

1 (1.2%)＜0.001

ASA-PS

Insertion depth (cm)0.13

15.0 [14-15.5]

15.0 [14-16]

14.5 [13-15]＜0.001

Tube inner diameter (mm)430 (14.3%)

2 (1.6%)28 (33.7%)

4.5

47 (22.4%)

24 (18.9%)23 (27.7%)

5

77 (36.7%)

45 (35.4%)

32 (38.6%)

5.5

42 (20.0%)42 (33.1%)

0 (0%)

6

14 (6.7%)

14 (11.0%)

0 (0%)

20 [11.25-20]-

20 [11.25-20]

Cuff pressure (mm Hg) (n＝71)

0.01

＜0.01

Number of intubation trials
1

172 (81.9%)

91 (71.7%)

81 (97.6%)

2

34 (16.2%)

32 (25.2%)

2 (2.4%)3

4 (1.9%)4 (3.1%)0 (0%)

Cormack-Lehane grade (n＝201)

＊

＊
＊

＊

0.42

1

185 (92.0%)

114 (92.7%)

71 (91.0%)

2

14 (7.0%)

7 (5.7%)7 (9.0%)3

2 (1.0%)2 (1.6%)0 (0%)

Data are presented as median [interquartile range, IQR] or number (percentage). Mann-Whitney U test was
performed for continuous variables and Fisher s exact test or χ 2 analysis was used for categorical data. Asterisk ( ＊ )indicates statistical significance (p＜0.05).
Abbreviations: ASA-PS, American Society of Anesthesiologists physical status; UC, uncuffed tube (means Portex®);and MC, Microcuff ® PET.

strong. Additionally, the number of intubation trials was also not chosen as an explanatory variablebecause its relationship with tube exchange was too strong. As a result, we identified using
Microcuff ® PET as an independent negative risk factor for tube exchange. The odds ratio for therequirement of endotracheal tube exchange after the first intubation with Microcuff ® PET compared
to Portex® was 0.0408 (95% CI: 0.007-0.466; p ＝0.002). Sex and ASA-PS were not significantly
associated with the requirement of tube exchange.
Postoperative complications were also compared between both groups (Table 5). There were no- 113 -

Inada et al

statistically significant differences in the incidence of any perioperative complications (UC vs MC):
respiratory complications (3.1% vs 4.8%, p＝0.72), postoperative sore throat (3.9% vs 10.8%, p＝0.09),
postoperative nausea and vomiting (13.4% vs 16.1%, p＝0.49), and others (1.6% vs 2.4%, p＝0.65).
Respiratory complications included hoarseness (UC vs MC, 1 vs 1 case), atelectasis (1 vs 0),
laryngospasm after extubation (2 vs 1), and increased sputum (0 vs 2). Other complications included

Table 2. Details of tube exchange after the first intubation
UC group (n＝127)

MC group (n＝83)

Size up23 (18.1%)

0 (0%)

Size down

7 (5.5%)

1 (1.1%)Total

30 (23.6%)

1 (1.1%)Size up presents the number and percentage of patients who were reintubated using a tube witha larger inner diameter. Size down presents the number and percentage of patients who werereintubated using a tube with a smaller inner diameter.
Abbreviations: UC, uncuffed tube (means Portex®); and MC, Microcuff ® PET.

Table 3. ‌Surgical site in the study population, overall and by tube type
Surgical site

Overall (n＝210)

UC group (n＝127)

MC group (n＝83)

p value

Head and Neck51 (24.3%)

30 (23.6%)

21 (25.3%)

0.78-Brain2 (1.0%)1 (0.8%)1 (1.2%)1-Eye

6 (2.9%)3 (2.4%)3 (3.6%)0.68-Laryngopharyngeal

23 (11.0%)

13 (10.2%)

10 (12.0%)

0.68

-Ear

16 (7.6%)

10 (7.9%)

6 (7.2%)

0.86

-Nose

2 (1.0%)1 (0.8%)1 (1.2%)1-Neck

2 (1.0%)2 (1.6%)0 (0%)

0.52

Thorax7 (3.3%)4 (3.1%)3 (3.6%)1

Abdomen95 (45.2%)

55 (43.3%)

40 (48.2%)

0.49

Extremities

9 (4.3%)5 (3.9%)4 (4.8%)

0.74

Skin

48 (22.9%)

33 (26.0%)

15 (18.1%)

0.18

Data are presented as number (percentage). P values were generated by Fisher s exact test or χ analysis. Statisticalsignificance was set at p＜0.05.
2Abbreviations: UC, uncuffed tube (means Portex®); and MC, Microcuff ® PET.

Table 4. ‌Multivariate logistic regression analysis for detecting the risk factors for endotracheal tube
exchange
Odds ratio

95% CI

Tube type (UC＝0, MC＝1)

0.04080.007-0.466

Gender (Male＝1, Female＝2)

1.06

0.473-2.37

0.89

0.619

0.167-2.29

0.47

ASA-PSp value
＊

0.002

＊

Statistical significance was set at p＜0.05. Asterisk( ) indicates statistical significance.
Abbreviations: ASA-PS, American Society of Anesthesiologists physical status; UC, uncuffed tube (Portex®); MC,Microcuff ® PET; and CI, Confidence interval.

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The Usefulness of Microcuff ® Tube in Japanese.
Table 5. ‌Postoperative complications by tube type
UC group (n＝127)

MC group (n＝83)

p value

Respiratory complication

4 (3.1%)4 (4.8%)0.72

Sore throat

5 (3.9%)9 (10.8%)

0.09

PONV

17 (13.4%)

14 (16.1%)

0.49

Others2 (1.6%)2 (2.4%)

0.65

Total

27 (21.3%)

26 (31.3%)

0.10

Data are presented as number (percentage). P values were generated by Fisher s exact test or χ2 analysis. Statisticalsignificance was set at p＜0.05.
Abbreviations: UC, uncuffed tube (means Portex®); MC, Microcuff ® PET; and PONV, Postoperative nausea andvomiting.

agitation (1 vs 0), delayed awakening (1 vs 0), headache (0 vs 1), and nasal bleeding (0 vs 1). Becausesome patients experienced multiple complications, the total number of patients who experienced any
postoperative complications was 27 (21.3%) in the UC group and 26 (31.3%) in the MC group.
Although the total incidence of perioperative complications, especially sore throat, was higher in the
MC group, this result was not statistically significant (p＝0.10).

Discussion
In this study, we revealed that the use of Microcuff ® PET for Japanese children aged 2 to 8 years
old significantly decreased the risk of requiring endotracheal tube exchange after the first intubation(UC vs MC, 23.6% vs 1.2%). No patients suffered from severe air leakage in the MC group, whereas
18.1% of UC group cases needed an endotracheal tube exchange due to ventilation failure. Moreover,there were no significant differences in the frequency of perioperative complications between the UC
and MC groups.
There are several known benefits of cuffed endotracheal tubes over uncuffed endotracheal tubes.
The tight airway seal provided by the cuff may reduce the risk of aspiration15,21), may improve theaccuracy of capnogram and gas monitoring, and may reduce air contamination with volatile
anesthetics in the operation room5). Specifically, many studies demonstrated the superiority of theMicrocuff ® PET, which is a cuffed tube specially designed for children 9-18). Previous studies
demonstrated that the tube exchange ratio was 1.6%-3.3% when using Microcuff ® PET13,14,18). Our
study showed an even lower tube exchange ratio of 1.2% in the MC group compared to 23.6% in the
UC group, and there were no cases of air leakage in the MC group. As described in previous studies,
our results also reflect the excellent air sealing properties of Microcuff ® PET with high-volume lowpressure cuffs that work effectively, regardless of any individual differences in tracheal shape.
Considering the air sealing property, Microcuff ® PET seems to be especially useful for surgeries that
require high peak inspiratory pressure, such as laparoscopic surgeries or surgeries for obese patients.
Furthermore, Microcuff ® PET may be useful for nasopharyngeal surgeries including those with ahigh risk of aspiration, such as tonsillectomy. The tube sizes for each age described in the package
leaflet of Microcuff ® PET appeared to be well-adapted for Japanese children. The low incidence of
reintubation of Microcuff ® PET prevents multiple laryngoscopy and intubation procedures, which
may result in reduced postoperative laryngeal edema.
In our study, the median intubation depth was slightly but significantly shallower in the MC- 115 -

Inada et algroup than in the UC group (UC vs MC, 15 vs 14.5 cm), but there were no complications associatedwith the tube depth, such as endotracheal intubation or accidental extubation during surgery. This
result is attributable to each anesthesiologist s different definition of the tube depth between cuffed
and uncuffed tubes. In most cases wherein Portex ®, which has no depth mark, was used, theanesthesiologists adjusted the tube position by pulling the tube back from unilateral intubation depthwith an auscultation technique. Conversely, anesthesiologists may have decided the tube depth byadjusting the ring mark according to the vocal cord in most cases of Microcuff ® PET use. As Weiss etal demonstrated, the ring mark of Microcuff ® PET allowed adequate placement of the tube with a
cuff-free subglottic zone without the risk of unilateral intubation in children from birth to 14 years ofage19). The cuff length and ring mark of Microcuff ® PET is well-considered and specially designed forthe pediatric airway. Thus, we think the differences in the intubation depth between the UC and MC
groups is not so important in most clinical situations. However, we should take care to avoid
accidental extubation in surgeries requiring strong neck retroflection or handling the transesophageal
echo probe, especially when using Microcuff ® PET.
Several studies demonstrated that cuffed tubes did not increase morbidity among children under
8 years of age2,13,18). We also found no significant differences in postoperative complications between
the UC and MC groups; however, postoperative sore throat seemed to be more frequent in the MCgroup (10.8%) than in the UC group (3.9%). This may be attributed to the intraoperative
management of cuff pressure. In our patients, 56.6% (47 patients) of the MC group underwent
adjustment of the intraoperative cuff pressure to 20 cm H2O, and 6 of 9 patients who experienced
postoperative sore throat in the MC group were managed with a cuff pressure of 20 cm H2O. There isno clear standard regarding cuff pressure in children for now, but Seegobin et al reported that a cuff
pressure over 30 cm H2O impaired endotracheal mucosal blood flow in adults22). Because theacceptable cuff pressure seems to be lower in children, previous studies using Microcuff ® PET
reported an even lower mean sealing pressure of 9.6-11.7 cm H2O10,16,18). Al-Melwalli et al reported
that the incidence and severity of sore throat were significantly lower when the cuff pressure wasproperly maintained under 20 mm Hg compared to that with 57.55 mm Hg as determined by thefinger estimation technique 23). However, we did not find any significant statistical association
between cuff pressure and postoperative sore throat (p ＝1, Fisher s exact test). It is possible that we
could reduce the incidence of postoperative sore throat by managing with minimum cuff pressure toprevent air leakage. Further prospective studies with a strict protocol are needed to assess theassociation between cuff pressure and postoperative complications.
This study had several limitations. First, neonates and infants under 2 years of age were notincluded in this study. We did not use Microcuff ® PET under the size of ID 3.5 mm in our hospital
because the usage of cuffed endotracheal tubes for neonates and infants is still controversial 24).
Previous studies reported that cuffed tubes could be used without increasing complications forneonates and older children5,8,18), while other studies showed that cuffed tubes for premature and lowbirth-weight neonates increased the incidence of post-extubation strider25). Cuffed endotracheal tubes
might be too large in some cases, and it is possible that the tube exchange ratio and the incidence of
postoperative complications may increase in neonates and infants. Second, we retrospectively
obtained the information about postoperative complications from the electronic medical record
system; thus, the diagnostic criteria of each complication were not strictly defined. Moreover, there
may have been some omissions in the data in medical records; thus, it is possible that the data

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The Usefulness of Microcuff ® Tube in Japanese.

regarding the incidence of postoperative complications were less accurate in both groups. Third, thiswas a single center retrospective cohort study with a small number of patients, and thus, the evidence
level may be low to allow generalization. To address these issues, a well-structured, prospective
randomized study is needed in the future.
In conclusion, Microcuff ® PET showed a lower tube exchange ratio and exhibited superior airway
sealing properties compared to Portex® endotracheal tubes in Japanese children aged 2 to 8 years.
The incidence of postoperative complications, especially sore throat, was slightly higher in the MCgroup; however, no statistically significant difference was noted between the groups. Further
prospective randomized study is needed to validate our results.
Acknowledgements
All authors have no COI to declare regarding the present study.
References
1. Motoyama EK. Induction of Anesthesia and Maintenance of the Airway in Infants and Children. In: Motoyama
EK, Peter JD editor. Smith's Anesthesia for Infants and Children 7 th ed. St Louis: Mosby; 2006. pp. 319-358.
2. Deakers TW, Reynolds G, Stretton M, et al. Cuffed endotracheal tubes in pediatric intensive care. J Pediatr 1994;
125:57-62.
3. Eschertzhuber S, Salgo B, Schmitz A, et al. Cuffed endotracheal tubes in children reduce sevoflurane andmedical gas consumption and related costs. Acta Anaesthesiol Scand 2010;54:855-858.
4. Fine GF, Borland LM. The future of the cuffed endotracheal tube. Paediatr Anaesth 2004;14:38-42.
5. Khine HH, Corddry DH, Kettrick RG, et al. Comparison of cuffed and uncuffed endotracheal tubes in young
children during general anesthesia. Anesthesiology 1997;86:627-631; discussion 27A.
6. Newth CJ, Rachman B, Patel N, et al. The use of cuffed versus uncuffed endotracheal tubes in pediatricintensive care. J Pediatr 2004;144:333-337.
7. Pearson TE, Frizzola MA, Khine HH. Uncuffed endotracheal tubes: not appropriate for pediatric critical caretransport. Air Med J 2019;38:51-54.
8. Shi F, Xiao Y, Xiong W, et al. Cuffed versus uncuffed endotracheal tubes in children: a meta-analysis. J Anesth
2016;30:3-11.
9. Dullenkopf A, Gerber A, Weiss M. Fluid leakage past tracheal tube cuffs: evaluation of the new Microcuff
endotracheal tube. Intensive Care Med 2003;29:1849-1853.
10. Dullenkopf A, Schmitz A, Gerber AC, et al. Tracheal sealing characteristics of pediatric cuffed tracheal tubes.
Paediatr Anaesth 2004;14:825-830.
11. Dullenkopf A, Gerber A, Weiss M. The Microcuff tube allows a longer time interval until unsafe cuff pressures
are reached in children. Can J Anaesth 2004;51:997-1001.
12. Dullenkopf A, Schmitz A, Frei M, et al. Air leakage around endotracheal tube cuffs. Eur J Anaesthesiol 2004;
21:448-453.
13. Dullenkopf A, Gerber AC, Weiss M. Fit and seal characteristics of a new paediatric tracheal tube with highvolume-low pressure polyurethane cuff. Acta Anaesthesiol Scand 2005;49:232-237.
14. Funai Y. Miura Y, Mori Y, et al. The clinical assessment of Microcuff ® periatric endotracheal tubes in Japanesechildren. Nihon Syounimasui Gakkaishi 2016;22:215-220. (In Japanese)
15. Lau AC, Lam SM, Yan WW. Benchtop study of leakages across the Portex, TaperGuard, and Microcuff
endotracheal tubes under simulated clinical conditions. Hong Kong Med J 2014;20:7-15.
16. Mhamane R, Dave N, Garasia M. Use of Microcuff ® endotracheal tubes in paediatric laparoscopic surgeries.
Indian J Anaesth 2015;59:85-88.
17. Weiss M, Dullenkopf A, Gysin C, et al. Shortcomings of cuffed paediatric tracheal tubes. Br J Anaesth 2004;92:
78-88.
18. Weiss M, Dullenkopf A, Fischer JE, et al. Prospective randomized controlled multi-centre trial of cuffed or
uncuffed endotracheal tubes in small children. Br J Anaesth 2009;103:867-873.
19. Weiss M, Gerber AC, Dullenkopf A. Appropriate placement of intubation depth marks in a new cuffedpaediatric tracheal tube. Br J Anaesth 2005;94:80-87.
20. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow- 117 -

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Transplant 2013;48:452-458.
21. Browning DH, Graves SA. Incidence of aspiration with endotracheal tubes in children. J Pediatr 1983;102:582584.
22. Seegobin RD, van Hasselt GL. Endotracheal cuff pressure and tracheal mucosal blood flow: endoscopic studyof effects of four large volume cuffs. Br Med J (Clin Res Ed) 1984;288:965-968.
23. Al-Metwalli RR, Sadek S. Safety and reliability of the sealing cuff pressure of the Microcuff pediatric tracheal
tube for prevention of post-extubation morbidity in children: a comparative study. Saudi J Anaesth 2014;8:484488.
24. De Orange FA, Andrade RG, Lemos A, et al. Cuffed versus uncuffed endotracheal tubes for generalanaesthesia in children aged eight years and under. Cochrane Database Syst Rev 2017;11:CD011954.
25. Sathyamoorthy M, Lerman J, Asariparampil R, et al. Stridor in neonates after using the Microcuff ® anduncuffed tracheal tubes: a retrospective review. Anesth Analg 2015;121:1321-1324.

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Osaka City Med. J. Vol. 65, 119-133, 2019

Time-lapse Observation of Stabilized Mitochondrial Membrane
Potential in Chemotherapy-induced Granulosa Cell Damage
Treated with L-carnitine
AKI TAKASE1), DAISUKE TACHIBANA1), YUKIMI KIRA2),
AKIHIRO HAMURO1), TAKUYA MISUGI1), and MASAYASU KOYAMA1)
Departments of Obstetrics and Gynecology1) and Research Support Platform2), Osaka City
University Graduate School of Medicine

Abstract
Background
Chemotherapy-induced infertility has become problems faced by reproductive-age women
receiving chemotherapy and many anticancer drugs mainly affect proliferating cells such as
granulosa cells, which support the oocyte. The aims of this study were to investigate mitochondrial
membrane potential and to test the protective effect of L-carnitine (LC) in the granulosa cells treatedwith anticancer drugs.
Methods
Female ICR mice were divided into 5 groups (control, cisplatin, cisplatin＋LC, paclitaxel,
paclitaxel＋LC) and the number of follicles in three sections of ovaries was counted every 100 μm ateach development stage. Immunohistochemical analysis of cleaved caspase-3 was performed. Thechanges due to administration of anticancer drugs with/without LC treatment were analyzed by cell
viability and western blotting for active caspase-3, using COV434 cells. The mitochondrial membranepotential was analyzed with the time-lapse function.
Results
Low-dose chemotherapy, which affected the number of primordial cells, caused morphologicalchanges in murine granulosa cells and significant prolongation of mitochondrial membrane potential
was observed in pretreatment with LC in chemotherapy-induced cell damage in the COV434 cell line.
In addition, cleaved caspase-3 levels were strikingly decreased in LC-treated groups.
Conclusions
Our study is the first to investigate the mitochondrial membrane potential in granulosa cell line
COV434 using time-lapse methods, and we found that LC pretreatment had a protective effect tostabilize the mitochondrial membrane potential and postpone apoptosis. Our findings may provide
new insight into protection against chemotherapy-induced ovarian failure for women who wish to
Received August 20, 2019; accepted October 8, 2019.
Correspondence to: Daisuke Tachibana MD. PhD.
Department of Obstetrics and Gynecology, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3862; Fax: +81-6-6646-5800
E-mail: dtachibana@med.osaka-cu.ac.jp- 119 -

Takase et alpreserve fertility.
Key Words: ‌L-carnitine; Granulosa cell; Mitochondrial membrane potential; Timelapse; Chemotherapy

Introduction
The survival period and quality of life for patients with various cancers have improved due todevelopment of chemotherapy. However, as the number of reproductive-age women receiving
chemotherapy is increasing, chemotherapy-induced infertility and premature ovarian failure havebecome problems faced by an increasing number of patients1). At birth, the ovary contains a finite
number of oocytes that are surrounded by a single layer of pregranulosa cells to form primordial
follicles, and the female ovary establishes a fixed number of primordial follicles by 5 months ofgestational age. Therefore the number of primordial follicles is a direct indicator of fertility reserve,
and the prevention of chemotherapy-induced follicle loss is a critical problem.
Anticancer drugs act on the ovary by different mechanisms, and both oocytes and granulosa cells
are vulnerable to the chemotherapic agents. Direct toxicity to the ovary results in diminishedprimordial follicle pool, ovarian atrophy, and irreversible change of blood vasculature 2). A recent
study has proposed a new hypothesis, called the burn-out theory. The burn-out theory is amechanism in which the anticancer drugs induce that development from the primordial follicles tothe antral follicles continues to be accelerated wastefully, and these follicles become follicle atresia,
and to cause the depletion of ovarian reserve and consequently ovarian failure. On the other hand,
granulosa cells are active to proliferate and highly sensitive to the anticancer drugs, so chemothetapy
causes the granulosa cell apoptosis3,4).
Mitochondrial membranes are important sites for steroidogenesis in granulosa cells. Cholesterol,a substrate of steroid hormones, is transferred from the outer to the inner mitochondrial membraneby steroidogenic acute regulatiry (StAR) protein, which is in turn metabolized to pregnenolone bycytochrome P450 cholesterol side-chain cleavage enzyme (P450scc). Damage to mitochondrial
membranes by oxidative stress impairs steroidogenesis in granulosa cells. In fact, oxidative stresshas been reported to inhibit steroidogenic enzymes and a mitochondrial carrier protein (StAR protein)
involved in cholesterol transport into mitochondria of luteal cells 5). Therefore, stabilized themitochondrial membrane potential of granulosa cells may have protective effect against chemotherapyinduced follicle apoptosis. However, the exact mechanism of granulosa cell apoptosis induced bychemotherapy remains unclear.
L-carnitine (LC) is a natural nutrient and essential for β-oxidation of fatty acids in mitochondria
to generate ATP. Because hydrophobic anions exhibit detergent-like action and perturb membrane/lipid bilayers, abnormally elevated long-chain fatty acids might impair mitochondrial membranes
and induce permeability transition, a prerequisite reaction to cytochrome c release. In addition, LC
effectively inhibits mitochondrial dysfunction induced by oxidative stress and mitochondriadependent apoptosis of various cell types6), and to prove these phenomena, several reports have usedtechniques that can capture changes in mitochondrial membrane potential at the very early stage ofapoptosis temporally and quantitatively7,8). Therefore, we hypothesized that chemotherapy induces
mitochondrial membrane impairment of granulosa cells and treatment with LC can inhibit
chemotherapy-induced follicle apoptosis.

- 120 -

Prevention of Chemotherapy-induced Ovarian Damage

Methods
Animal experiments
Experimental protocols used in this study conformed to the Institutional Committee for Animal
Care and Experiments in Osaka City University, Graduate School of Medicine, and were approved by
the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in
Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports,
Science, and Technology. Seven-week-old female ICR mice weighing 30g were purchased from SLC(Japan SLC). The animals were kept in a cage under controlled light (12-h light/12-h dark cycle),temperature, and humidity conditions.
Fifty mice were divided evenly into 5 groups. Group 1 was a control group in which the ovaries
were removed at 10 weeks of age. Group 2 was the cisplatin group in which cisplatin (10 mg/kg)9) was
intraperitoneally injected. Group 3 was the paclitaxel group in which paclitaxel (5 mg/kg)10) was
intraperitoneally injected. Groups 4 and 5 were LC pretreatment groups, and mice consumed LC in
their drinking water (LC concentration 1 mg/mL)11) for 2 weeks (LC intake: 200 mg/kg/day). Group 4
received the same amount of cisplatin as group 2, and Group 5 received the same amount of paclitaxel
as in group 3. Mice in Groups 2-5 were euthanized 1 week after administration of anticancer drugs,
and ovaries were removed at 10 weeks of age. For histology, one ovary from each mouse was fixedwith formalin and embedded in paraffin.
Change in follicle number by follicular development stageOvaries were fixed with formalin, embedded in paraffin, and sectioned at 4 μm. Sections were
stained with hematoxylin and eosin, and the number of follicles in three sections was counted every
100 μm. Follicles were categorized as primordial follicles (oocyte surrounded by a single layer of flat
follicular epithelium cells), primary follicles (oocyte surrounded by a single layer of cubic granulosa
cells), secondary follicles (oocyte surrounded by at least two cell layers of cubic granulosa cells), andantral follicles (consisting of oocytes, many layers of granulosa cells, and a cavity filled with follicular
fluid)12).
Immunohistochemistry
Paraffin-embedded sections were deparaffinized by rinsing with 100% xylene three times, 100%
ethanol twice, 70% ethanol once, and 50% ethanol once. Citrate buffer (pH 6.0) was used for antigen
activation. Endogenous peroxidases were quenched with 3% H2O2, and blocking was performed with
5% goat serum. Then, sections were incubated with primary antibody (active caspase-3, #9661, 1:200,
Cell Signaling Technology) overnight at 4℃. After two washes in Tris Buffered Saline (TBS), sections
were incubated with secondary antibody (Histofine Simple Stain Mouse MAX-PO ®, Nichirei
Biosciences). A diaminobenzidine solution was dropped onto sections and incubated until the desired
staining was achieved. Finally, sections were stained with hematoxylin and eosin (HE).
Cell lines
COV434 cells13), which are a human granulosa cell tumor cell line, were used. COV434 cells arederived from a 27-year-old female with a metastatic granulosa cell tumor but possess many
characteristics of normal granulosa cells. COV434 cells were cultured in Dulbecco s Modified Eagle
Medium F12 (Wako Pure Chemical Industries) supplemented with 5% fetal bovine serum in a 5%
CO2 incubator at 37℃.
Cell viability
Cell viability was analyzed using a Cell Counting Kit-8 (CCK-8) kit (Dojindo Laboratories).

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COV434 cells were plated at 2.5×104/well in a 96-well microplate and allowed to attach overnight.
COV434 cells were cultured at different concentrations of cisplatin and paclitaxel for 24 h, andanticancer drug concentrations at which 50%-60% cell viability was observed were applied in thisstudy. After cells were treated with or without 0.5 mM LC14) for 12 h, culture medium was changed tomedium containing anticancer drugs at the indicated concentration (without LC), and then 12 h later
a CCK-8 solution was added at 10 µL/well. Plates were incubated at 37℃ for 2 h and the absorbancewas measured at 450 nm using a microplate reader (Varioskan™ LUX, Thermo Fisher Scientific).
Cell viability was calculated based on the absorbance of control cells and changes due toadministration of anticancer drugs with/without LC treatment were analyzed.
Detection of mitochondrial membrane potential
COV434 cells were cultured with/without LC treatment for 6 h in 3-cm glass-bottom dishes andmitochondrial membrane potential was assessed by fluorescence microscopy after applying the JC-1
Mitochondrial Membrane Potential Assay Kit (BD™ MitoScreen Kit, Biosciences)15). The fluorescentcarbocyanine dye, JC-1, labels mitochondria with high membrane potential as red and mitochondria
with low membrane potential as green. After incubating COV434 cells with/without LC treatment,
cells were incubated in 5 µM JC-1 fluorescence dye in a 5% CO2 incubator for 30 min at 37℃. Cells
were slowly washed with JC-1 working solution three times. The mitochondrial membrane potential
was analyzed by imaging every 1 min after administration of anticancer drugs with the time-lapse
function of the LSM 700 confocal laser scanning microscope system. The green signal of JC-1 wasmeasured at 485/555 nm, whereas that of the red signal was measured at 590/610 nm16).
Western blotting
Cells were pretreated with LC for 12 h, incubated with anticancer drugs for 24 h, and thencollected. The supernatant was also collected. After centrifugation for 5 min, the supernatant was
discarded, washed with Phosphate Buffered Saline (PBS), centrifuged again for 5 min, and the pellet
was recovered. Proteins were extracted with radioimmunoprecipitation assay buffer. Total protein
was quantified using the Bicinchoninic acid (BCA) protein assay. Protein samples with loading buffer
were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred topolyvinylidene difluoride membranes. Membranes were blocked with blocking buffer (5% skim milk
in TBST) for 1 h and then incubated with the following primary antibodies diluted in blocking buffer
at 4℃ overnight: anti-caspase-3, anti-active caspase-3, and anti-β-actin (1:1000, 1:1000, 1/10000; Cell
Signaling Technology). Bound antibodies were detected after incubation with horseradish peroxidaseconjugated rabbit anti-mouse immunoglobulin G using ImmunoStar LD (Wako Pure Chemical
Industries, Ltd.) and Fusion SOLO.7S (Vilber Lourmat). Chemiluminescent bands were quantified
with Image Quant TL (GE Healthcare Life Sciences).
Statistical analysis
Results are expressed as the mean±standard deviation. All statistical analyses were performed
using the Mann-Whitney U-test. A value of p＜0.05 was considered significant.

Results
L-carnitine affects the number of follicles at each development stage
In the primordial follicle stage, the number of follicles in the chemotherapy-only group was
significantly lower than that in the control group (cisplatin, p＝0.003; paclitaxel, p＝0.030) and thenumber of follicles in the paclitaxel＋LC group was significantly higher than that in the non-

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treatment group (p ＝0.0001). In the primary follicle stage, the number of follicles in the
chemotherapy-only group was significantly lower than that in the control group (cisplatin, p＝0.015;
paclitaxel, p＝0.028). In the secondary follicle stage, the number of follicles in the LC pretreatmentgroup was significantly higher than in the non-treatment group (cisplatin, p＝0.033; paclitaxel, p＝0.044). In the antral follicle stage, there was no significant difference between groups. (Fig. 1).

Figure 1. The number of follicles at each development stage (primordial, primary, secondary, and antral follicles).
Fifty mice were divided evenly into 5 groups. Sections of ovary were stained with hematoxylin and eosin, and thenumber of follicles in three sections was counted every 100 μm. In the primordial follicle stage, the number offollicles in the chemotherapy-only group was significantly lower than that in the control group (cisplatin, p＝0.003;
paclitaxel, p＝0.030) and the number of follicles in the paclitaxel＋LC was significantly higher than that in the nontreatment group (p＝0.0001). In the primary follicle stage, the number of follicles in the chemotherapy-only group was
significantly lower than that in the control group (cisplatin, p＝0.015; paclitaxel, p＝0.028). In the secondary follicle
stage, the number of follicles in the LC pretreatment group was significantly higher than in the non-treatment group(cisplatin, p＝0.033; paclitaxel, p＝0.044). In the antral follicle stage, there was no significant difference between
groups. Data represent the mean±SD. Asterisks denote significant differences ( ＊ p＜0.05). LC, L-carnitine.

Immunohistochemical analysis of cleaved caspase-3 in mouse ovaries
In antral follicles, the structure of granulosa cell layers around the oocyte was disordered in the
anticancer drugs treatment group (Figs. 2C and 2G), whereas cell layers were maintained in the LC
pretreatment group (Figs. 2E and 2I). As a result of immunohistochemical analysis of active
caspase-3, which is a mitochondrial-dependent apoptosis marker, granulosa cells were stained only inthe anticancer drugs treatment groups (Figs. 2D and 2H). However, in the LC pretreatment group,
almost no staining was seen (Figs. 2F and 2J). In primordial follicles, the structure was relatively
maintained even in the anticancer drugs treatment group (Figs. 3C and 3G). Active caspase-3 wasdetected in granulosa cells of the anticancer drugs treatment groups (Figs. 3D and 3H). However,active caspase-3 was not detected in granulosa cells of the LC pretreatment groups (Figs. 3F and 3J).

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Figure 2. Representative histological sections of mouse ovaries stained with hematoxylin and eosin (A, C, E, G, andI), and immunohistochemical analysis of cleaved caspase-3 (B, D, F, H, and J). Antral follicles from (A and B) control,(C and D) cisplatin-only, (E and F) cisplatin＋LC, (G and H) paclitaxel-only, and (I and J) paclitaxel＋LC groups areshown. Active caspase-3 was detected in granulosa cells of cisplatin-only (D) and paclitaxel-only (H) groups. However,active caspase-3 was not detected in granulosa cells of cisplatin＋LC (F) and paclitaxel＋LC (J) groups. Scale bars＝
100 μm. LC, L-carnitine.

Figure 3. Representative histological sections of mouse ovaries stained with hematoxylin and eosin (A, C, E, G, and I),
and immunohistochemical analysis of cleaved caspase-3 (B, D, F, H, and J). Primordial follicles from (A and B) control,(C and D) cisplatin-only, (E and F) cisplatin＋LC, (G and H) paclitaxel-only, and (I and J) paclitaxel＋LC groups areshown. Active caspase-3 was detected in granulosa cells of cisplatin-only (D) and paclitaxel-only (H) groups. However,active caspase-3 was not detected in granulosa cells of cisplatin＋LC (F) and paclitaxel＋LC group (J) groups. Scalebars＝50 μm. LC, L-carnitine.

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This result demonstrates that mitochondria-dependent apoptosis caused by anticancer drugs was
suppressed by LC pretreatment.
Effect of LC in cell viability
The effect of different concentrations of cisplatin (1-100 µM) or paclitaxel (10-500 nM) for 24 h on
the viability of COV434 cells was measured by CCK-8. Anticancer drug concentrations at which 50%60% cell viability was observed were determined as 25 µM for cisplatin (Fig. 4A) and 250 nM forpaclitaxel (Fig. 4B). These concentrations were applied in the following experiment.
COV434 cells were treated with each anticancer drug for 12 h after LC pretreatment, and thenthe CCK-8 assay was performed. Cell viability of the cisplatin＋LC group was approximately 15%
higher than that of the cisplatin-only group. Furthermore, the viability of the paclitaxel＋LC group
was approximately 20% higher than that of the paclitaxel-only group (Fig. 5). These results suggestthat LC has a protective effect against anticancer drug-induced cell death.

Figure 4. Cell viability of each group using Cell Counting Kit-8 (CCK-8). The effect of treatment with different
concentrations of cisplatin (1-100 µM) or paclitaxel (10-500 nM) for 24 h on the viability of COV434 cells was measured
by CCK-8. Anticancer drug concentrations at which 50%-60% cell viability was observed were applied in this study.

Effect of LC pretreatment in mitochondrial membrane potential
COV434 cells had high mitochondrial membrane potential before chemotherapy (Fig. 6A), which
disappeared following chemotherapy (Fig. 6B). COV434 cells were incubated with or without LC (0.5mM) for 6 h before the addition of cisplatin or paclitaxel. Time-lapse images of mitochondrial
membrane potential were detected by JC-1 and captured every 1 min (Figs. 7A, 7C, 9A, and 9C). The
shift in mitochondrial membrane potential (crossing point between red and green signals) occurred 3
min after administration of cisplatin (Fig. 7B). When LC pretreatment was performed, the crossing
point was prolonged to 7 min (Fig. 7D). The fluorescence ratio (red/green) indicates the mitochondrial
membrane potential. The mitochondrial membrane potential of the LC pretreatment group was
significantly increased as compared with the cisplatin-only group (0 min, p＝0.046) (Fig. 8).

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The crossing point occurred 3-4 min after administration of paclitaxel (Fig. 9B). When LC
pretreatment was performed, the crossing point was prolonged to 10-11 min (Fig. 9D). The
mitochondrial membrane potential of the LC pretreatment group was significantly increased ascompared with the paclitaxel-only group (0 min, p ＝0.009; 5 min, p＝0.016; 10 min, p＝0.016) (Fig. 10).
LC pretreatment decreased active caspase-3
In cisplatin- and paclitaxel-treated COV434 cells, active caspase-3 was significantly increased

Figure 5. COV434 cells were incubated in the presence or absence of LC (0.5 mM) for 12 h before the addition of
cisplatin or paclitaxel. Cell viability of the cisplatin＋LC group was significantly higher than that of the cisplatin-onlygroup (p＝0.024). Cell viability of the paclitaxel＋LC group was significantly higher than that of the paclitaxel-onlygroup (p＝0.035). Asterisks denote significant differences ( ＊ p＜0.05). LC, L-carnitine.

Figure 6. Representative images of mitochondrial membrane potential using JC-1 are shown. (A) COV434 cells
before chemotherapy and (B) COV434 cells after chemotherapy. JC-1 labels mitochondria with high membranepotential as red and mitochondria with low membrane potential as green.

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Figure 7. COV434 cells were incubated with (A and B) or without (C and D) LC (0.5 mM) for 6 h before the additionof cisplatin. Parts A and C shows time-lapse images of mitochondrial membrane potential every 1 min, and parts B
and D shows the time-lapse of mitochondrial membrane potential intensity. Note the crossing point between red andgreen signals. The crossing point in the without LC group was ＜4 min after cisplatin administration, whereas the
crossing point in the LC treatment group was postponed by ＞10 min. LC, L-carnitine.

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Takase et al

Figure 8. Representative images taken at 0, 5, and 10 min after cisplatin administration were selected from a setof time-lapse dual images for comparison. The fluorescence ratio (red/green) indicates the mitochondrial membranepotential. The mitochondrial membrane potential of the LC pretreatment group was significantly increased ascompared with that of the cisplatin-only group. Asterisks denote significant differences ( ＊ p＜0.05). LC, L-carnitine.

compared with that of the control. Conversely, in the LC pretreatment group, active caspase-3 wassignificantly decreased compared with treatment with anticancer drugs only (cisplatin, p＝0.025;
paclitaxel, p＝0.025) (Fig. 11A). Pro-caspase-3 was not significantly changed in the groups (Fig. 11B).
These results suggest that cell death of COV434 cells occurred due to mitochondria-dependent
apoptosis by anticancer drug treatment, and LC can rescue this cell death through mitochondrial
protective effects.

Discussion
The present results showed that low-dose chemotherapy, which affected the number of primordial
cells, caused morphological changes in murine granulosa cells. Furthermore, we first observed that
pretreatment with LC showed significant prolongation of mitochondrial membrane potential inchemotherapy-induced cell damage in the COV434 cell line. In addition, active caspase-3 levels were
strikingly decreased in LC-treated groups. These phenomena might be due to stabilization of themitochondrial membrane potential, which is thought to play pivotal roles in cell apoptosis17).
Cancer treatment can have devastating consequences for young females, resulting in ovarian
damage accompanied by diminished fertility potential. The extent of damage is related tochemotherapeutic regimen and dosage. Mitochondrial damage caused by cisplatin has been reported- 128 -

Prevention of Chemotherapy-induced Ovarian Damage

Figure 9. COV434 cells were incubated with (A and B) or without (C and D) LC (0.5 mM) for 6 h before the additionof paclitaxel. Parts A and C shows time-lapse images of mitochondrial membrane potential every 1 min, and parts
B and D shows time-lapse of mitochondrial membrane potential intensity. Note the crossing point between red andgreen signals. The crossing point in the without LC group was ＜3 min after paclitaxel administration, whereas the
crossing point in the LC treatment group was postponed by ＞7 min. LC, L-carnitine.

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Takase et al

Figure 10. Representative images taken at 0, 5, and 10 min after paclitaxel administration were selected from a setof time-lapse dual images for comparison. The fluorescence ratio (red/green) indicates the mitochondrial membranepotential. The mitochondrial membrane potential of the LC pretreatment group was significantly increased ascompared with that of the paclitaxel-only group. Asterisks denote significant differences ( ＊ p＜0.05). LC, L-carnitine.

Figure 11. Western blot analysis was performed with COV434 cells for (A) cleaved caspase-3 and (B) pro-caspase-3.
Data represent the mean±SD. Asterisks denote significant differences ( ＊ p＜0.05). In the anticancer drug-only
treatment group, active caspase-3 was significantly increased compared with that of the control group (p＝0.025). In
the LC pretreatment group, active caspase-3 was significantly decreased compared with that of the anticancer drugonly treatment group. Pro-caspase-3 was not significantly changed in any group. LC, L-carnitine.

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Prevention of Chemotherapy-induced Ovarian Damage

in several models of cisplatin-induced nephrotoxicity, neurotoxicity, and auditory toxicity18-20). Cellularexposure to cisplatin causes direct damage to mitochondrial DNA (mtDNA) resulting in a reduction of
mitochondrial protein synthesis, impairment of electron transport chain function, and subsequently,
increases in intracellular reactive oxygen species (ROS) levels. ROS promote mitochondriadependent apoptosis and enhance cytotoxic effects of cisplatin through nuclear DNA damage 21).
Paclitaxel activates Raf-1 and causes phosphorylation of Bcl-2, which leads to an increase in the levelof free intracellular Bax. Free intracellular Bax activates caspase proteins through Bax-induced
release of cytochrome c, which finally promotes mitochondrial apoptosis22). Cisplatin causes direct
damage to mitochondrial DNA and induces apoptosis by promoting ROS production. On the other
hand, paclitaxel increases the permeability of the mitochondrial membrane by increasing BAX(apoptosis inducing factor) and induces apoptosis. Regarding the different effects of LC for two
anticancer drugs, one possible mechanism might be explained that paclitaxel gradually damages the
mitochondria, and it may take time to reach the mitochondrial membrane through some steps
mentioned above, and the protective effect of pretreated LC on the mitochondrial membrane waspromptly and profoundly observed.
The apoptosis pathway, which starts with mitochondrial damage, has been recently studied bymeasuring the magnitude and time courses of mitochondrial membrane potential in pancreatic betacells and astrocytes 7,8). Furthermore, using isolated mitochondria purified by Percoll gradientcentrifugation to exclude any indirect mechanism of the effect of paclitaxel on permeability transition,
Varbiro et al22) showed that paclitaxel induces mitochondrial permeability transition, including large
amplitude swelling of mitochondria, dissipation of mitochondrial membrane potential, opening of thepermeability transition pore by an enzymatic mechanism, and cytochrome c release. Jou et al7)
reported that cell stress caused by ROS showed unstabilized mitochondrial permeability transition
and mitochondrial oxidation. They also reported that melatonin provided multiple levels of
mitochondria-targeted protection. Antioxidant agents such as melatonin are suggested to havesubstantial roles for protective stabilization of mitochondrial function.
The present results showed that the number of the antral follicles tended to decrease with theadministration of anticancer drugs, and to protect with LC pretreatment, but there was no significant
difference. However, in the secondary follicles, the number of follicles was significantly increased by
LC pretreatment. The primordial follicle develops step by step into the primordial follicle, theprimary follicle, the secondary follicle, and finally the antral follicle. By the damage of multilayered
granulosa cells, the number of secondary follicles decreases and the developmental damage from thesecondary follicles to the antral follicles occurs. The protective effect of LC against the damage to
granulosa cells in the antral follicles shown in Figure 3 was not effective to increase the number of
the antral follicles, but at least in the level of secondary follicles, LC was contributed to stabilize themitochondrial membrane potential of granulosa cells.
LC is a water-soluble nutrient with a choline-like structure and is distributed mainly in the liver,kidney, heart, brain, and skeletal muscles in humans. LC is biosynthesized from lysine andmethionine in the liver and kidney and plays an essential role for β-oxidation of free fatty acids on
the inner mitochondrial membrane for ATP production6). Accumulated data of LC in female infertility
have been recently documented and a beneficial effect of antioxidant properties and amelioration ofenergy supply to reproductive organs have been shown in vitro as well as in vivo23). One potential
mechanism of LC action within the oocyte is that LC is converted to acetyl L-carnitine and balances- 131 -

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the acetyl CoA/CoA ratio to maintain glucose metabolism through the TCA cycle, yielding higher
energy production24). This further reduces oxidative stress and lipotoxicity by scavenging free radicals
and removing excess palmitate 25). As such, while the roles of LC in oocytes have been studied
extensively, apoptosis in granulosa cells, especially that induced by chemotherapy, lacks experimental
observation.
To the best of our knowledge, our study is the first to investigate the mitochondrial membranepotential in granulosa cell line COV434 using time-lapse methods. In addition, we found that LC
pretreatment had a protective effect to stabilize the mitochondrial membrane potential and postpone
apoptosis. Our findings may provide new insight into protection against chemotherapy-induced
ovarian failure for women who wish to preserve fertility. Further studies are needed to elucidate
mechanisms underlying the interaction between inhibition of granulosa cell apoptosis andpreservation of oocyte quality.
Acknowledgements
All authors have no COI to declare regarding the present study.
The authors would like to acknowledge Keisuke Inoue, Emi Donoue and Yoriko Yabunaka(Research support platform of Osaka City University Graduate School of Medicine) for special
technical assistance.
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2. Nelson SM, Telfer EE, Anderson RA. The ageing ovary and uterus: new biological insights. Hum Reprod
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3. Bedoschi G, Navarro PA, Oktay K. Chemotherapy-induced damage to ovary: mechanisms and clinical impact.
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4. Meirow D, Biederman H, Anderson RA, et al. Toxicity of chemotherapy and radiation on female reproduction.
Clin Obstet Gynecol 2010;53:727-739.
5. Tanabe M, Tamura H, Taketani T, et al. Melatonin protects the integrity of granulosa cells by reducing
oxidative stress in nuclei, mitochondria, and plasma membranes in mice. J Reprod Dev 2015;61:35-41.
6. Vaz FM, Wanders RJ. Carnitine biosynthesis in mammals. Biochem J 2002;361:417-429.
7. Jou MJ, Peng TI, Reiter RJ. Protective stabilization of mitochondrial permeability transition andmitochondrial oxidation during mitochondrial Ca2＋ stress by melatonin s cascade metabolites C3-OHM and
AFMK in RBA1 astrocytes. J Pineal Res 2019;66:e12538.
8. Gerencser AA, Mookerjee SA, Jastroch M, et al. Measurement of the absolute magnitude and time courses of
mitochondrial membrane potential in primary and clonal pancreatic beta-cells. PLoS One 2016;11:e0159199.
9. Chang EM, Lim E, Yoon S, et al. Cisplatin induces overactivation of the dormant primordial follicle throughPTEN/AKT/FOXO3a pathway which leads to loss of ovarian reserve in mice. PLoS One 2015;10:e0144245.
10. Gücer F, Balkanli-Kaplan P, Doganay L, et al. Effect of paclitaxel on primordial follicular reserve in mice.
Fertil Steril 2001;76:628-629.
11. Makowski L, Noland RC, Koves TR, et al. Metabolic profiling of PPARalpha-/- mice reveals defects in carnitine
and amino acid homeostasis that are partially reversed by oral carnitine supplementation. FASEB J 2009;23:
586-604.
12. Ozcelik B, Turkyilmaz C, Ozgun MT, et al. Prevention of paclitaxel and cisplatin induced ovarian damage inrats by a gonadotropin-releasing hormone agonist. Fertil Steril 2010;93:1609-1614.
13. Havelock JC, Rainey WE, Carr BR. Ovarian granulosa cell lines. Mol Cell Endocrinol 2004;228:67-78.
14. Wang Q, Ju X, Chen Y, et al. Effects of L-carnitine against H2O2-induced oxidative stress in grass carp ovary
cells (Ctenopharyngodon idellus). Fish Physiol Biochem 2016;42:845-857.
15. Chang S, Ren G, Steiner RD, et al. Elevated autophagy and mitochondrial dysfunction in the Smith-LemliOpitz syndrome. Mol Genet Metab Rep 2014;1:431-442.
16. Chen W, Xu X, Wang L, et al. Low expression of Mfn2 is associated with mitochondrial damage and apoptosis- 132 -

Prevention of Chemotherapy-induced Ovarian Damageof ovarian tissues in the premature ovarian failure model. PLoS One 2015;10:e0136421.
17. Kumar S, Yedjou CG, Tchounwou PB. Arsenic trioxide induces oxidative stress, DNA damage, andmitochondrial pathway of apoptosis in human leukemia (HL-60) cells. J Exp Clin Cancer Res 2014;33:42.
18. De Koning P, Neijt JP, Jennekens FG, et al. Evaluation of cis-diamminedichloroplatinum (II) (cisplatin)neurotoxicity in rats. Toxicol Appl Pharmacol 1987;89:81-87.
19. Stadnicki SW, Fleischman RW, Schaeppi U, et al. Cis-dichlorodiammineplatinum (II) (NSC-119875): hearing
loss and other toxic effects in rhesus monkeys. Cancer Chemother Rep 1975;59:467-480.
20. Ward JM, Fauvie KA. The nephrotoxic effects of cis-diammine-dichloroplatinum (II) (NSC-119875) in male
F344 rats. Toxicol Appl Pharmacol 1976;38:535-547.
21. Marullo R, Werner E, Degtyareva N, et al. Cisplatin induces a mitochondrial-ROS response that contributes
to cytotoxicity depending on mitochondrial redox status and bioenergetic functions. PLoS One 2013;8:e81162.
22. Varbiro G, Veres B, Gallyas F Jr, et al. Direct effect of Taxol on free radical formation and mitochondrial
permeability transition. Free Radic Biol Med 2001;31:548-558.
23. Agarwal A, Sengupta P, Durairajanayagam D. Role of L-carnitine in female infertility. Reprod Biol Endocrinol
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24. Infante JP, Tschanz CL, Shaw N, et al. Straight-chain acyl-CoA oxidase knockout mouse accumulates
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Osaka City Med. J. Vol. 65, 135-140, 2019

Bilateral Ovarian Fibromatosis Diagnosed with Magnetic
Resonance Imaging: A Case Report
NORIKO TAKESHITA1), TAKUHITO TADA1), MASAHIRO TOKUNAGA1), AKIRA OKIMURA2),
MASARU MAKIHARA1), and TOHRU TAKESHITA3)
Departments of Radiology1) and Pathology2), Izumi City General Hospital; andDepartment of Radiology3), PL Hospital

Abstract
This report describes a case of ovarian fibromatosis, which is a rare non-neoplastic disease. In
ovarian fibromatosis, one or both ovaries are enlarged owing to fibroblastic proliferation associatedwith collagen deposition within the ovarian stroma. Ovarian fibromatosis is not a well-recognizedentity. It is often misdiagnosed as a neoplastic disease, and treated with excessive surgery.
In our case of ovarian fibromatosis, preoperative magnetic resonance imaging revealed normal
ovarian tissue surrounded by thick fibrous tissue not infiltrated, but preserved with a black garlandlike appearance. This reflects the pathognomonic finding of the disease. Under the clinicaldiagnosis of ovarian fibromatosis, the outer portions of both ovarian masses were resected andovarian function was preserved.
The pathogenesis of ovarian fibromatosis appears to be related to massive ovarian edema. The
patient in the present case had a history of ovarian hyperstimulation syndrome, which may havecaused the massive ovarian edema and may be suggestive of the hypothesis that massive ovarianedema and ovarian fibromatosis are a series of pathological conditions.
Key Words: ‌Ovarian fibromatosis; Non-neoplastic lesion; Black garland-likeappearance; Magnetic resonance imaging

Introduction
Ovarian fibromatosis is a miscellaneous non-neoplastic lesion that was defined by Young andScully in 19841). It presents with a tumor-like enlargement of one or both ovaries. When surgery is
performed on the basis of a clinical diagnosis of ovarian neoplasm, ovarian function is lost. This
report describes a case of ovarian fibromatosis that was correctly diagnosed using magnetic resonanceimaging (MRI) before surgery.

Received November 19, 2018; accepted April 19, 2019.
Correspondence to: Noriko Takeshita, MD.
Department of Radiology, Izumi City General Hospital,
Wake-cho 4-5-1, Izumi, Osaka, 594-0073, Japan
Tel: +81-725-41-1331; Fax: +81-725-43-3660
E-mail: mqfwk775@ybb.ne.jp- 135 -

Takeshita et al

Case Report
A 39-year-old woman was referred to our institution for menstrual abnormalities. She had neither
abdominal pain nor masculinization and hirsutism. She was nulliparous and had a history of ovarian
hyperstimulation syndrome at the age of 34 years. She had no malignant medical history.
Her physical examination was normal. Routine laboratory test results were within the normal
limits, with the exception of increased levels of CA 19-9 (68 U/mL) and CA 125 (77.7 U/mL).
Transvaginal ultrasonography revealed bilateral solid ovarian masses and neoplasm was suspected.
To obtain further information, non-enhanced MRI was performed. It revealed bilateral enlarged
ovaries, with lobulated surfaces. T1-weighted images showed a homogeneous, low signal intensity(Fig. 1). In both ovarian masses, T2-weighted images demonstrated a high signal intensity at the
inner portion, which indicated a normal stroma, and a low signal intensity similar to that of skeletal
muscles at the outer portion (Fig. 2). Several small high-intensity areas were also observed at theouter portion, which indicated spared normal stroma (Fig. 2). Diffusion-weighted images showed ahigh signal intensity at the inner portion, which indicated a normal ovarian stroma, and a low signal
intensity at the outer portion (Fig. 3). In addition to the bilateral ovarian masses, several uterine
myomas and ascites were observed. When the MRI findings were summarized, normal ovarian tissue
was surrounded by thick fibrous tissue. Therefore, a clinical diagnosis of ovarian fibromatosis wasmade in spite of elevated tumor markers.
In accordance with the diagnosis, the outer portion of both ovarian masses was resected andmyomectomy was also performed. On gross examination of the specimen, the surface of both ovarian
masses was coarsely lobulated; the sectioned specimen was mostly light gray and partly yellowish

Figure 1. T1-weighted magnetic resonance image, of both ovarian masses, showing homogeneous low signal intensity(white arrows).

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Diagnosis of Ovarian Fibromatosis Using MRI

Figure 2. T2-weighted magnetic resonance image showing high signal intensity (white arrow-heads) in the inner
portion of each ovarian masses, which is surrounded by a low signal intensity (white arrows) similar to that of skeletal
muscles.

Figure 3. High b-value, diffusion-weighted magnetic resonance image of the inner portion of the ovarian masses,
showing a high signal intensity, which was consistent with normal ovarian stroma (white arrow- heads).

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Takeshita et al-white (Fig. 4). A cystic lesion was also identified. The histopathological findings demonstrated
proliferative, collagen-producing, spindle-shaped cells in incomplete clusters with a storiform patternsurrounding the expanded follicles (Fig. 5). Malignant findings were not present. On the basis of thepathological findings, a final diagnosis of bilateral ovarian fibromatosis was made.
The postoperative course was uneventful. The patient was discharged 6 days after the surgery,and her menstrual cycle normalized.

Figure 4. A sectioned specimen with mostly light gray and partly yellowish -white color.

Figure 5. Histopathological finding demonstrating atypical, proliferative, collagen-producing, spindle-shaped
cells in incomplete clusters, with a storiform pattern surrounding the expanded follicles in both ovaries (original
magnification, ×10; Hematoxylin Eosin stain).

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Discussion
Ovarian fibromatosis is a rare clinicopathological entity, with only approximately 30 cases havebeen described in the literature1-8). In ovarian fibromatosis, one or both ovaries are enlarged owing tofibroblastic proliferation associated with collagen deposition within the ovarian stroma9). It is mostcommonly reported in women aged 13-39 years (mean, 25 years). Preservation of the preexistingovarian structures is typical and unrelated to soft- tissue fibromatosis, which rarely involves theovary. In approximately 20% of cases, ovarian fibromatosis involves both ovaries2). The predominant
symptoms are menstrual abnormalities and abdominal pain, and sometimes, masculinization andhirsutism occur due to hormonal activities1,3-5). Tumor markers are within their normal ranges orslightly elevated3-7). The patient s slightly elevated tumor markers might be also caused by myoma
uteri10,11). The pathogenesis of ovarian fibromatosis is unclear; however, local tissue disorders, surgery,and ovarian torsion are possible contributors1).
The pathogenesis of ovarian fibromatosis appears to be related to massive ovarian edema.
Massive ovarian edema is characterized by ovarian enlargement and stromal edema. Russell andFarnsworth suggested that ovarian fibromatosis presented the burned out stage of reactive
fibroblastic proliferation that result from massive edema at one end of the spectrum. Foci of stromal
edema similar to those of massive ovarian edema have been reported in roughly 50% of cases ofovarian fibromatosis 12). The presented patient had a history of ovarian hyperstimulation syndrome
due to infertility treatment. This syndrome is characterized by cystic enlargement of the ovaries anda fluid shift from the intravascular to the third space due to increased capillary permeability andovarian neoangiogenesis13). The syndrome may have caused massive ovarian edema13,14). The patient s
history of ovarian hyperstimulation syndrome may be suggestive of the hypothesis that massive
ovarian edema and ovarian fibromatosis are a series of pathological conditions.
The differential diagnoses of bilateral ovarian masses with very low signal intensity on T2weighted image include ovarian stromal hyperplasia, metastatic ovarian tumors, and bilateralovarian fibrothecomas15). Although ovarian stromal hyperplasia is generally associated withhyperandrogenism, obesity, hypertension, hyperinsulinemia, and glucose intolerance, without sparednormal ovarian follicles16), these coexisting conditions were absent and normal ovarian follicles wereobserved in the present case. Metastatic ovarian tumors usually infiltrate and replace normal
ovarian follicles, and fibrothecomas replace normal follicles. In the presented case, this phenomenon
was not observed. Furthermore, most fibrothecomas are unilateral, but they may also be bilateral(8%) 17), especially in patients with Gorlin or basal cell nervous syndrome (bilateral ovarian
fibrothecomas, multiple basal cell carcinomas of the skin, odontogenic keratocysts, and otherabnormalities)18). The fibrothecoma in Gorlin syndrome usually shows coarse calcification. In thiscase, no coarse calcification in both ovaries was observed.
Takeuchi et al described that the low intensity surrounding the ovary like a garland on T2weighted MRI was a characteristic finding of ovarian fibromatosis and named it a black garlandlike appearance5). In this case, a preexisting ovarian stroma with follicles was surrounded by thickfibrous tissue like a garland. This case can be added to cases with ovarian fibromatosis showing a
black garland-like appearance.
On the basis of the characteristic MRI findings, a clinical diagnosis of ovarian fibromatosis was
made. As MRI was useful in differentiating this disease from neoplastic diseases, ovarian function
was preserved. Recognition of ovarian fibromatosis by surgeons and pathologists has important- 139 -

Takeshita et al

therapeutic implications.
The magnetic resonance imaging finding, such as black garland-like appearance on T2 weighted
image, may be a diagnostic clue to this rare disease. The patient s history of ovarian hyperstimulation
syndrome may be suggestive of the hypothesis that massive ovarian edema and ovarian fibromatosis
are a series of pathological conditions.
Acknowledgements
All authors have no COI to declare regarding the present study.
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