Professional Documents
Culture Documents
Number 8 - August/2015
CLINICS
Editor
Edmund Chada Baracat
Area Editors
Ana Maria de Ulhoa Escobar
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Paulo Pgo-Fernandes
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Raul Coimbra
University of California, San Diego
La Jolla, CA, USA
Bruno Zilberstein
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Marcos Intaglietta
University of California, San Diego
San Diego, CA, USA
Rossana Francisco
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Carlos Serrano
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Mauricio Etchebehere
Universidade Estadual de Campinas
Campinas, SP, Brazil
Michele Correale
University of Foggia
Foggia, Italy
Ruth Guinsburg
Universidade Federal de So Paulo
So Paulo, SP, Brazil
Nelson Wolosker
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Sandro Esteves
ANDROFERT - Andrology & Human
Reproduction Clinic
Campinas, SP, Brazil
Newton Kara-Junior
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Geraldo Busatto
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Simone Appenzeller
Universidade Estadual de Campinas
Campinas, SP, Brazil
Valeria Aoki
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Paulo Hoff
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Editorial Board
Abhijit Chandra
King Georges Medical College
Lucknow, India
Artur Brum-Fernandes
Universit de Sherbrooke
Qubec, Canad
Adauto Castelo
Universidade Federal de So Paulo
So Paulo, SP, Brazil
Ademar Lopes
Fundao Antnio Prudente, Hospital
do Cncer
So Paulo, SP, Brazil
Edmund Neugebauer
Witten/Herdecke University
Witten, North Rhine - Westphalia,
Germany
Egberto Gaspar de Moura Jr.
Universidade do Estado do Rio de
Janeiro
Rio de Janeiro, RJ, Brazil
Ivan Cecconello
Faculdade de Medicina da Universidade
de So Paulo
So Paulo, SP, Brazil
Ke-Seng Zhao
Southern Medical University
Guangzhou, China
Francisco Laurindo
Faculdade de Medicina da Universidade
de So Paulo
So Paulo, SP, Brazil
Marcelo Zugaib
Faculdade de Medicina da Universidade
de So Paulo
So Paulo, SP, Brazil
Hiroyuki Hirasawa
Chiba University School of Medicine
Chiba, Japan
Pedro Puech-Leo
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Peter Libby
Brigham and Womens Hospital
Boston, Boston, MA, USA
Philip Cohen
University of Houston Health Center
Houston, Texas, USA
Rafael Andrade-Alegre
Santo Toms Hospital
Republic of Panamai, Panam
Ricardo Antonio Renetti
Faculdade de Medicina da Universidade Federal do
Rio de Janeiro Rio de Janeiro, RJ, Brazil
Roberto Chiesa
San Raffaele Hospital
Milan, Italy
Ronald A. Asherson
Netcare Rosebank Hospital
Rosebank, Johannesburg, South ifrica
Samir Rasslan
Faculdade de Medicina da Universidade de
So Paulo So Paulo,
SP, Brazil
Tarcisio Eloy Pessoa de Barros
Faculdade de Medicina da Universidade de
So Paulo
So Paulo, SP, Brazil
Valentim Gentil
Faculdade de Medicina da Universidade de
So Paulo So Paulo,
SP, Brazil
Wagner Farid Gattaz
Faculdade de Medicina da Universidade de
Scio Paulo So Paulo, SP, Brazil
Board of Governors
Alberto Jos da Silva Duarte
Aluisio Augusto Cotrim Segurado
Ana Claudia Latronico Xavier
Berenice Bilharinho de Mendona
Carlos Roberto Ribeiro de Carvalho
Clarice Tanaka
Claudia Regina Furquim de Andrade
Cyro Festa
Dalton de Alencar Fischer Chamone
Daniel Romero Muoz
Edmund Chada Baracat
Eduardo Massad
Eloisa Silva Dutra de Oliveira Bonf
Euripedes Constantino Miguel
Fbio Biscegli Jatene
Flair Jos Carrilho
Gerson Chadi
Gilberto Luis Camanho
Giovanni Guido Cerri
Irene de Lourdes Noronha
Irineu Tadeu Velasco
Ivan Cecconello
Editorial Director
Editorial Assistants
Nair Gomes
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Daniela Aquemi Higa
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Ariane Maris Gomes
Faculdade de Medicina da Universidade de So Paulo
So Paulo, SP, Brazil
Editorial Ofce: Rua Dr. Ovdio Pires de Campos, 225 - 6 Andar CEP 05403-010 So Paulo/SP Tel.: +55-11-2661-6235
Email: clinics@hc.fm.usp.br
Website: www.scielo.br/clinics
Submission: http://mc04.manuscriptcentral.com/clinics-scielo
Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of
Knowledge); Scopus; Ulrichs Periodical Directory; Qualis/Capes - Classied as an International Circulation Journal in Medicine.
Clinics. So Paulo: Scientic Journal of Hospital das Clnicas da Faculdade de Medicina da Universidade de So Paulo, 2005Monthly Periodical: January to December
ISSN 1807-5932 printed version
ISSN 1980-5322 online version
Formerly Revista do Hospital das Clnicas da FMUSP, 19462004.
1. Medicine-scientic production. 2.Medical Sciences I. Hospital das Clnicas da Faculdade deMedicina da Universidade de So Paulo.
CDD 610
Title page:
Title (up to 250 characters);
Running title (up to 40 characters, letters and spaces);
Full address of corresponding author only;
Authors names (without titles or degrees). Authors
Publication Fees
CLINICS uses a business model in which expenses are recoveredin
part by charging a publication fee to the authors or research
sponsors for each published article. Our 2015 prices are as
follows: original articles, review articles and rapid communications: US$ 1,500.00 or R$ 3.000,00. Invited reviews, editorials
and letters to the editors: no charge.
Manuscripts involving human subjects or the use of laboratory
animals must clearly state adherence to appropriate guidelines
and approval of protocols by their institutional review boards.
Photographs that may identify patients or other human
participants of studies shall be acceptable only when a legally
valid consent form is signed by the participating patient, other
human participant, or his/her legally constituted representative.
Manuscripts should be digitalized using aWord *.doc-compatible
software program and submitted online in English.
Authors are strongly advised to submit the manuscript in its
nal form to a spell check for English (US). Submissions with
excessive spelling or syntax mistakes as well as articles in which
the meaning is not sufciently clear shall be returned to authors
for correction. Authors are also strongly advised to use
abbreviations sparingly whenever possible to avoid jargon
and improve the readability of the manuscript. All abbreviations must be dened the rst time that they are used. Only
terms or expressions that are used at least 5 times through
out the text should be abbreviated. Never use abbreviations
that spell common English words, such as FUN, PIN, SCORE
and SUN.
Please make sure to submit your manuscript in the exact format
that is described below. Failure to do so will cause the submission
to be returned to you during the preliminary examination by the
Editorial Ofce.
RAPID COMMUNICATIONS:
Title page: As described in the Original Study section.
Manuscript: Rapid communications are limited to 1,500
ISSN-1807-5932
CLINICS
CONTENTS
Clinics 2015 70(8):535599
CLINICAL SCIENCES
Ocular risk management in patients undergoing general anesthesia: an analysis of 39,431 surgeries
Newton Kara-Junior, Rodrigo Franca de Espindola, Joao Valverde Filho, Christiane Pellegrino Rosa,
Andre Ottoboni, Enis Donizete Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Flow-through anastomosis using a T-shaped vascular pedicle for gracilis functioning free muscle
transplantation in brachial plexus injury
Yi Hou, Jiantao Yang, Yi Yang, Bengang Qin, Guo Fu, Xiangming Li, Liqiang Gu, Xiaolin Liu,
Qingtang Zhu, Jian Qi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544
Treatment with dasatinib or nilotinib in chronic myeloid leukemia patients who failed to respond to two
previously administered tyrosine kinase inhibitors a single center experience
Beatriz Felicio Ribeiro, Eliana C.M. Miranda, Dulcinia Martins de Albuquerque, Mrcia T. Delamain,
Gislaine Oliveira-Duarte, Maria Helena Almeida, Bruna Verglio, Rosana Antunes da Silveira,
Vagner Oliveira-Duarte, Irene Lorand-Metze, Carmino A. De Souza, Katia B.B. Pagnano . . . . . . . . . . . . . . . . . . . . 550
The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism
Kun Zou, Caigang Liu, Zhuo Zhang, Lijuan Zou . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556
BASIC RESEARCH
Effect of hypertonic saline treatment on the inammatory response after hydrochloric acid-induced
lung injury in pigs
Carla Augusto Holms, Denise Aya Otsuki, Marcia Kahvegian, Cristina Oliveira Massoco, Denise Tabacchi Fantoni,
Paulo Sampaio Gutierrez, Jose Otavio Costa Auler Junior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
REVIEW
Operative versus nonoperative treatment for displaced midshaft clavicle fractures: a meta-analysis
based on current evidence
Xin-Hua Wang, Wei-Jun Guo, A-Bing Li, Guang-Jun Cheng, Tao Lei, You-Ming Zhao. . . . . . . . . . . . . . . . . . . . . . . . . 584
The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes in
the Chinese Han population: a meta-analysis
Enting Ma, Huili Wang, Jing Guo, Ruirui Tian, Li Wei. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
CLINICAL SCIENCE
II
Department of Radiology
III
IV
OBJECTIVE: Typically, bone metastasis causes osteolytic and osteoblastic lesions resulting from the interactions
of tumor cells with osteoclasts and osteoblasts. In addition to these interactions, tumor tissues may grow inside
bones and cause mass lesions. In the present study, we aimed to demonstrate the negative impact of a tumor
mass in a large cohort of patients with bone metastatic cancer.
METHODS: Data from 335 patients with bone metastases were retrospectively reviewed. For the analysis, all
patients were divided into three subgroups with respect to the type of bone metastasis: osteolytic, osteoblastic,
or mixed. The patients were subsequently categorized as having bone metastasis with or without a tumor mass,
and statistically significant differences in median survival and 2-year overall survival were observed between
these patients (the median survival and 2-year overall survival were respectively 3 months and 16% in patients
with a tumor mass and 11 months and 26% in patients without a tumor mass; po0.001).
RESULTS: According to multivariate analysis, the presence of bone metastasis with a tumor mass was found to
be an independent prognostic factor (p=0.011, hazard ratio: 1.62, 95% confidence interval: 1.111.76). Bone
metastasis with a tumor mass was more strongly associated with osteolytic lesions, other primary diseases
(except for primary breast and prostate cancers), and spinal cord compression.
CONCLUSION: Bone metastasis with a tumor mass is a strong and independent negative prognostic factor for
survival in cancer patients.
KEYWORDS: Bone metastasis; Bone metastasis with a tumor mass; Prognostic factor; Survival.
ztoprak B, Hasbek Z, Bahar S, Kacan T, et al. The negative prognostic impact of bone metastasis with a tumor mass.
Yucel B, Celasun MG, O
Clinics. 2015;70(8):535-540
Received for publication on February 6, 2015; First review completed on March 23, 2015; Accepted for publication on March 31, 2015
E-mail: yucelbirsen@yahoo.com
*Corresponding author
INTRODUCTION
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/
4.0/) which permits unrestricted use, distribution, and reproduction in any
medium or format, provided the original work is properly cited.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)01
535
CLINICS 2015;70(8):535-540
Figure 1 - Types of bone metastasis (white arrows). A) Osteolytic metastasis. B) Osteoblastic metastasis. C) Bone metastasis with a tumor
mass.
RESULTS
The study group comprised 234 (70%) men and 101 (30%)
women. The median age at the time of cancer diagnosis was
59 years (range, 2182 years). The primary disease distribution was as follows: lung cancer in 107 (32%) patients, breast
cancer in 64 (19%), prostate cancer in 62 (19%), gastrointestinal system tumors in 40 (12%), genitourinary system
tumors in 20 (6%), head and neck tumors in 11 (3%), and
tumors in other organs in 31 (9%).
Osteolytic bone metastasis was observed in 99 (30%)
patients, whereas 155 (46%) had osteoblastic bone metastasis,
and 71 (21%) had mixed-type bone metastasis. Ten (3%)
patients had bone metastasis and only a tumor mass,
without any other lesions; these 10 patients were excluded
when categorizing the patients with respect to the type of
bone lesion (i.e., osteolytic, osteoblastic, or mixed). Bone
metastasis with a tumor mass was present in 73 (22%) cases.
Eleven (3%) patients had a single bone metastatic lesion, and
324 (97%) had two or more lesions. The 11 patients with
single bone lesions had no metastases in other organs. The
locations and frequencies of bone metastases were as follows:
vertebral column metastasis in 283 (84%) patients, pelvic
bone metastasis in 246 (73%), long bone metastasis in 189
(56%), costal metastasis in 189 (56%), and skull metastasis in
63 (19%).
536
CLINICS 2015;70(8):535-540
Table 1 - Comparison of features associated with bone metastases with or without tumor masses.
p-value
60 (61)
151 (97)
51 (72)
-
39 (39)
4 (3)
20 (28)
10 (100)
o0.001
(75)
(89)
(94)
(68)
(65)
(64)
(65)
27 (25)
7 (11)
4 (6)
13 (32)
7 (35)
4 (36)
11 (35)
o0.001
137 (75)
119 (84)
47 (25)
23 (16)
0.028
246 (79)
10 (63)
64 (21)
6 (37)
0.103
219 (83)
6 (30)
44 (17)
14 (70)
o0.001
228 (80)
34 (69)
58 (20)
15 (31)
0.079
249 (79)
13 (68)
67 (21)
6 (32)
0.213
12 (86)
25 (66)
57 (65)
2 (14)
13 (34)
30 (35)
0.312
23 (64)
71 (69)
13 (36)
32 (31)
0.306
80
57
58
27
13
7
20
537
CLINICS 2015;70(8):535-540
Survival Functions
Tumor mass
100
No
Ye
Yes
NoNo-censored
Yes-censore
Yes-censored
Cum survival
80
60
40
20
0
0
20
40
60
80
100
120
140
Months
DISCUSSION
The prevalence of bone metastasis is higher in advancedstage cancers. Patients diagnosed with bone metastasis
usually have incurable disease, though the survival duration
does vary based on the primary disease. Accordingly, it is
very important to determine prognostic factors once a
diagnosis of bone metastasis has been made. The present
study investigated the prognostic and clinical importance of
bone metastasis with a tumor mass and found that this
feature was an apparently strong negative prognostic factor
for survival. The higher incidence of these metastases in
association with osteolytic lesions might have contributed to
this result, as the presence of osteolytic lesions was found to
be a poor prognostic factor in a multivariate analysis. In
addition, growth of the tumor itself inside the bone might
indicate a larger tumor burden, which might also contribute
to a shorter survival duration. Given the soft tissue
component of bone metastasis with a tumor mass, spinal
cord compression was observed more frequently in these
patients; nonetheless, the presence of these lesions did not
538
CLINICS 2015;70(8):535-540
Table 2 - Prognostic factors affecting patient survival after the development of bone metastasis, as determined by univariate survival
analysis.
No. of patients
p-value
262
73
50
28
26
16
11
3
*o0.001
234
101
39
61
17
42
8
17
o0.001
248
87
53
24
27
12
12
5
o0.001
168
167
55
36
30
17
13
7
o0.001
184
142
50
39
29
16
12
9
0.004
310
16
46
-
24
-
10
3
0.027
107
64
62
40
20
11
27
72
69
24
20
9
10
47
31
6
10
-
5
18
15
5
5
3
o0.001
99
155
71
29
53
49
14
26
26
4
12
12
0.004
11
324
68
44
68
22
32
10
0.040
176
159
51
40
27
18
12
8
0.032
259
76
41
61
20
35
9
18
0.026
Abbreviations: 1ECOG PS, Eastern Cooperative Oncology Group performance status; 2ALP, alkaline phosphatase
539
CLINICS 2015;70(8):535-540
Table 3 - Independent prognostic factors affecting the duration of survival after the development of bone metastasis, as determined
by multivariate analysis.
Overall survival
95% CI2
p-value
1
1.62
1.111.76
*0.011
1
0.45
0.310.64
o0.001
1
1.39
1.021.90
0.034
0.200.57
o0.001
0.300.67
0.001
HR
Bone metastasis with a tumor mass
No
Yes
Gender
Male
Female
Weight loss
No
Yes
Primary disease
Lung
Breast
Lung
Prostate
Type of bone metastasis
Osteolytic
Osteoblastic
Osteolytic
Mixed
Serum ALP3 level
p129 U/L
4129 U/L
Serum calcium level
p10.6 mg/dL
410.6 mg/dL
1
0.32
1
0.45
1
0.56
1
0.56
0.390.81
0.002
0.380.83
0.004
1
1.34
1.032.00
0.030
1
2.22
1.034.81
0.042
Abbreviations: 1HR, hazard ratio; 2CI, confidence interval; 3ALP, alkaline phosphatase
AUTHOR CONTRIBUTIONS
Ycel B designed the research and analyzed the data. Ycel B, Celasun
MG, ztoprak B, Hasbek Z, Bahar S, Kacan T, Bahceci A, and Seker MM
performed the research. Kacan T and Seker MM contributed analytical
tools. Ycel B and ztoprak B wrote the paper. The authors have no
nancial disclosures to declare, no conicts of interest to report, and have
no commercial or proprietary interest.
REFERENCES
1. Coleman RE, Rubens RD. The clinical course of bone metastases from
breast cancer. Br J Cancer. 1987;55(1):61-6, http://dx.doi.org/10.1038/
bjc.1987.13
2. Roodman GD. Mechanisms of bone metastasis. N Engl J Med. 2004;350
(16):1655-64, http://dx.doi.org/10.1056/NEJMra030831
3. Mundy GR. Metastasis to bone: causes, consequences and therapeutic
opportunities. Nat Rev Cancer. 2002;2(8):584-93, http://dx.doi.org/
10.1038/nrc867
540
CLINICAL SCIENCE
Srio-Libanes Hospital, Sao Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sao Paulo, Ophthalmology Department, Sao Paulo/SP, Brazil.
OBJECTIVE: This study sought to describe and analyze ocular findings associated with nonocular surgery in
patients who underwent general anesthesia.
METHODS: The authors retrospectively collected a series of 39,431 surgeries using standardized data forms.
RESULTS: Ocular findings were reported in 9 cases (2.3:10,000), which involved patients with a mean age of 58.9
19.5 years. These cases involved patients classified as ASA I (33%), ASA II (55%) or ASA III (11%). General
anesthesia with propofol and remifentanil was used in 4 cases, balanced general anesthesia was used in 4 cases,
and regional block was used in combination with balanced general anesthesia in one case. Five patients (55%)
underwent surgery in the supine position, one patient (11%) underwent surgery in the lithotomy position, two
patients (22%) underwent surgery in the prone position, and one patient (11%) underwent surgery in the
lateral position. Ocular hyperemia was detected in most (77%) of the 9 cases with ocular findings; pain/burning
of the eyes, visual impairment, eye discharge and photophobia were observed in 55%, 11%, 11% and 11%,
respectively, of these 9 cases. No cases involved permanent ocular injury or vision loss.
CONCLUSION: Ophthalmological findings after surgeries were uncommon, and most of the included patients
were relatively healthy. Minor complications, such as dehydration or superficial ocular trauma, should be
prevented by following systematic protocols that provide appropriate ocular occlusion with a lubricating
ointment and protect the eye with an acrylic occluder. These procedures will refine the quality of anesthesia
services and avoid discomfort among patients, surgeons and anesthesia staff.
KEYWORDS: Blindness; Anesthesia; Eye Injuries.
Kara-Junior N, Espindola RF, Valverde Filho J, Rosa CP, Andre Ottoboni, Silva ED. Ocular risk management in patients undergoing general
anesthesia: an analysis of 39,431 surgeries. Clinics. 2015;70(8):541-543
Received for publication on January 22, 2015; First review completed on April 7, 2015; Accepted for publication on May 12, 2015
E-mail: rodrigo166@uol.com.br
*Corresponding author
INTRODUCTION
Postoperative visual loss (POVL) following general surgery is a relatively uncommon but devastating complication
that is most frequently associated with cardiac, spine, head
and neck operations. Estimates have indicated that POVL
occurs in up to 0.2% (1) and 4.5% (2) of spine and cardiac
surgeries, respectively.
Although studies of 65,000 and 400,000 patients who
underwent anesthesia for all types of surgery at two large
academic institutions suggested a low prevalence of perioperative vision loss in surgeries other than cardiac and spinal
fusion procedures, the actual prevalences of perioperative
vision loss for the most common types of operations remain
unknown (3,4).
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/
4.0/) which permits unrestricted use, distribution, and reproduction in any
medium or format, provided the original work is properly cited.
DOI: 10.6061/clinics/2015(08)02
541
CLINICS 2015;70(8):541-543
All demographic variables were analyzed using descriptive statistics; in particular, means and SDs were determined
for continuous variables, and frequencies (in percentages)
were calculated for categorical variables.
RESULTS
This retrospective study included 39,431 nonocular surgeries.
Ocular findings were reported in 9 cases (2.3:10,000), which
involved patients with a mean age of 58.919.5 years. Table 1
presents the characteristics of all 9 cases. Examinations of
individual variables revealed that male patients (66%), ASA II
status (55%), elective surgery (88%) and the supine position
(66%) were each involved in the majority of these cases.
For patients with ocular findings who were subjected to
general anesthesia (9 cases), pain (55%) and photophobia
(22%) were the main symptoms, and hyperemia (77%) was
the main sign (Table 2). Only one patient presented with
blurred vision (11%).
The main diagnoses in these cases were direct trauma
(44%) and dry eye (33%) (Table 2). All 9 patients experienced
ocular occlusion during surgery, and 5 patients (55%) also
received lubricant. No patient exhibited permanent ocular
injury or significant visual loss.
DISCUSSION
Perioperative ischemic optic neuropathy (PION) has been
reported after spine (5-7), orthopedic (8), neck (9-13), heart,
and abdominal surgeries (14,15). Intraoperative variables
that reportedly play roles in the pathogenesis of PION
include hypotension, anemia, and elevated intraocular
pressure associated with the prone position during spinal
surgery (16). Vascular risk factors, such as diabetes, coronary
artery disease, and hypertension, are present in many
patients who experience PION (17,18), although vision loss
has been reported in children and healthy adults who exhibit
none of these factors (6).
Given that the mechanisms and risk factors for PION are
poorly understood, the risks of vision loss should be considered
in preoperative discussions with patients who expect to
undergo spine surgery or surgery requiring cardiopulmonary
bypass because such procedures are associated with the highest
incidences of this rare complication (19).
In the present study, the incidence of ocular findings was
2.3:10.000. No patient experienced permanent ocular injury
or significant visual loss. However, certain of the observed
symptoms/signs could significantly impact eye health.
All 9 of the patients with ocular findings experienced
ocular occlusion during their procedures, and 55% of these
Table 1 - Patient characteristics.
Patient
1
2
3
4
5
6
7
8
9
Sex
Age
Duration (min)
ASA
Surgery
male
male
female
male
female
male
female
male
male
69
21
69
77
34
62
60
58
80
105
450
345
150
255
275
345
135
135
I
I
I
II
II
II
II
II
III
elective
elective
elective
elective
emergency
elective
elective
elective
elective
542
Anesthesia
balanced general anesthesia
general anesthesia propofol
balanced general anesthesia
general anesthesia propofol
general anesthesia propofol
general anesthesia propofol
balanced general anesthesia
balanced general anesthesia
general anesthesia propofol
Position
and remifentanil
and remifentanil
and remifentanil
and remifentanil
and remifentanil
supine
supine
supine
prone
supine
lithotomy
prone
supine
supine
CLINICS 2015;70(8):541-543
Table 2 - Description of ocular findings (signs and symptoms), treatments and final diagnoses for patients subjected to general
anesthesia.
Patient
Use of ocular
lubricant
Type of
lubricant
yes
eye drops
yes
3
4
no
yes
serum
physiological
solution
gel
no
6
7
Sign(s)
Symptom(s)
Treatment(s)
Diagnosis
hyperemia,
edema
hyperemia
pain
antibiotics
direct trauma
pain
direct trauma/
dehydration by serum
discharge
hyperemia
pain
yes
no
gel
-
yes
ointment
hyperemia
hyperemia,
palpebral edema
hyperemia
photophobia
pain; photophobia;
blurred vision
pain
no
hyperemia
dry eye
corneal
deepithelialization
dry eye
injuries such as retinal ischemia and PION justify appropriate care and the active pursuit of high-quality anesthesia
services. Since 2010, a protocol involving ocular occlusion
with the instillation of lubricant eye drops during relatively
complex procedures has been systematically adopted by the
anesthesia services of Srio Libans Hospital. Beginning in
2014, guided by the results and insights of this study, which
was conducted and analyzed in collaboration with ophthalmologists, lubricating ointment and ocular occlusion with an
acrylic occluder for eye protection have been used for all
surgeries involving general anesthesia. It is recommended
that these procedures, which have been implemented to
achieve the objective of further improving patient safety
during surgery, should be followed from the induction of
anesthesia to the complete awakening of the patient in the
postanesthesia recovery room.
Minor complications, such as dehydration or superficial
ocular trauma, which can generally be rapidly resolved
during the postoperative period, should be prevented by
following systematic protocols that include appropriate
ocular occlusion with lubricating ointment and protection
of the eye with an acrylic occluder. These protocols will
refine the quality of anesthesia services and avoid discomfort
among patients, surgeons and anesthesia staff.
exposure keratopathy
direct trauma
direct trauma
dry eye
3. Roth S, Thisted RA, Erickson JP, Black S, Schreider BD. Eye injuries after
non ocular surgery: a study of 60,965 anesthetics from 1988 to 1992.
Anesthesiology 1996;85(5):10207.
4. Warner ME, Warner MA, Garrity JA, MacKenzie RA, Warner DO. The
frequency of perioperative vision loss. Anesth Analg. 2001;93(6):141721.
5. Katz DM, Trobe JD, Cornblath WT, Kline LB: Ischemic optic neuropathy
after lumbar spine surgery. Arch Ophthalmol. 1994;112(7):92531.
6. Alexandrakis G, Lam BL. Bilateral posterior ischemic optic neuropathy
after spinal surgery. Am J Ophthalmol. 1999;127(3):3545.
7. Cheng MA, Sigurdson W, Tempelhoff R, Lauryssen C. Visual loss after
spine surgery: A survey Neurosurgery. 2000;46(3):62531.
8. Roth S, Nunez R, Schreider BD. Unexplained visual loss after lumbar
spinal fusion. J Neurosurg Anesthesiol. 1997;9(4):3468.
9. Bhatti MT, Enneking FK. Visual loss and ophthalmoplegia after shoulder
surgery. Anesth Analg. 2003;96(3):899902.
10. Marks SC, Jaques DA, Hirata RM, Saunders JR Jr. Blindness following
bilateral radical neck dissection. Head Neck. 1990;12(4):3425.
11. Nawa Y, Jaques JD, Miller NR, Palermo RA, Green WR. Bilateral posterior
optic neuropathy after bilateral radical neck dissection and hypotension.
Graefes Arch Clin Exp Ophthalmol. 1992;230(4):3018.
12. Schobel GA, Schmidbauer M, Millesi W, Undt G. Posterior ischemic optic
neu- ropathy following bilateral radical neck dissection. Int J Oral
Maxillofac Surg. 1995;24(4):2837.
13. Worrell L, Rowe M, Petti G. Amaurosis: A complication of bilateral radical
neck dissection. Am J Otolaryngol. 2002;23(1):569.
14. Pazos GA, Leonard DW, Blice J, Thompson DH. Blindness after bilateral
neck dissection: Case report and review. Am J Otolaryngol. 1999;
20(5):3405.
15. Asensio JA, Forno W, Castillo GA, Gambaro E, Petrone P. Posterior
ischemic optic neuropathy related to profound shock after penetrating
thoracoabdominal trauma. South Med J. 2002;95(9):10537.
16. Johnson MW, Kincaid MC, Trobe JD. Bilateral retrobulbar optic nerve
infarctions after blood loss and hypotension. A clinicopathologic case
study. Ophthalmology. 1987; 94(12):157784.
17. Cheng MA, Todorov A, Tempelhoff R, McHugh T, Crowder CM,
Lauryssen C. The effect of prone positioning on intraocular pressure in
anesthetized patients. Anesthesiology. 2001;95(6):13515.
18. Kim JW, Hills WL, Rizzo JF, Egan RA, Lessell S. Ischemic optic neuropathy following spine surgery in a 16-year-old patient and a ten-year-old
patient. J Neuroophthalmol. 2006;26(1):303.
19. Holy SE, Tsai JH, McAllister RK, Smith KH. Perioperative Ischemic Optic
Neuropathy. A Case Control Analysis of 126,666 Surgical Procedures at a
Single Institution. Anesthesiology. 2009; 110(2):24653.
20. Carvalho RS, Kara-Jose N, Temporini ER, Kara-Junior N, Noma RK. Selfmedication: the first attempt in patients seen in an ophthalmologic
emergency room. Clinics. 2009;64(8):73541.
21. American Society of Anesthesiologists Task Force on Perioperative
Blindness: Practice advisory for perioperative visual loss associated with
spine surgery: A report by the American Society of Anesthesiologists Task
Force on Perioperative Blindness. Anesthesiology. 2006;104(6):131928.
AUTHOR CONTRIBUTIONS
Kara-Junior N: study conception and design; drafting of the manuscript;
and critical revision. Espindola RF: drafting of the manuscript; critical
revision; and analysis and interpretation of study data. Valverde Filho J,
Rosa CP, Ottoboni A, and Silva ED: study conception and design; data
acquisition; and analysis and interpretation of study data.
REFERENCES
1. Stevens WR, Glazer PA, Kelley SD, Lietman TM, Bradford DS. Ophthalmic complications after spinal surgery. Spine. 1997;22(12):131924.
2. Shaw PJ, Bates D, Cartlidge NE, Heaviside D, French JM, Julian DG, et al.
Neuro-ophthalmological complications of coronary artery bypass graft
surgery. Acta Neurol Scand. 1987;76(1):17.
543
CLINICAL SCIENCE
OBJECTIVE: In gracilis functioning free muscle transplantation, the limited caliber of the dominant vascular
pedicle increases the complexity of the anastomosis and the risk of vascular compromise. The purpose of this
study was to characterize the results of using a T-shaped vascular pedicle for flow-through anastomosis in
gracilis functioning free muscle transplantation for brachial plexus injury.
METHODS: The outcomes of patients with brachial plexus injury who received gracilis functioning free muscle
transplantation with either conventional end-to-end anastomosis or flow-through anastomosis from 2005 to 2013
were retrospectively compared. In the flow-through group, the pedicle comprised a segment of the profunda
femoris and the nutrient artery of the gracilis. The recipient artery was interposed by the T-shaped pedicle.
RESULTS: A total of 46 patients received flow-through anastomosis, and 25 patients received conventional endto-end anastomosis. The surgical time was similar between the groups. The diameter of the arterial anastomosis
in the flow-through group was significantly larger than that in the end-to-end group (3.87 mm vs. 2.06 mm,
respectively, po0.001), and there were significantly fewer cases of vascular compromise in the flow-through
group (2 [4.35%] vs. 6 [24%], respectively, p=0.019). All flaps in the flow-through group survived, whereas 2 in
the end-to-end group failed. Minimal donor-site morbidity was noted in both groups.
CONCLUSIONS: Flow-through anastomosis in gracilis functioning free muscle transplantation for brachial plexus
injury can decrease the complexity of anastomosis, reduce the risk of flap loss, and allow for more variation in
muscle placement.
KEYWORDS: Brachial plexus injury; Functioning free muscle transplantation; Flow-through anastomosis;
Gracilis muscle; T-shaped pedicle.
Hou Y, Yang J, Yang Y, Qin B, Fu G, Li X, et al. Flow-through anastomosis using a T-shaped vascular pedicle for gracilis functioning free muscle
transplantation in brachial plexus injury. Clinics. 2015;70(8):544-549
Received for publication on February 10, 2015; First review completed on March 30, 2015; Accepted for publication on May 12, 2015
E-mail: guliqiang1963@aliyun.com
*Corresponding author
INTRODUCTION
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/
4.0/) which permits unrestricted use, distribution, and reproduction in any
medium or format, provided the original work is properly cited.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)03
544
CLINICS 2015;70(8):544-549
Surgical technique
All of the FFMTs were performed by a single senior
professor and his team, including a resident and an attending
physician. Each patient was placed in the supine position
with hip joint flexion, abduction, and external rotation and
knee joint flexion. In this position, the adductor longus could
be palpated in the medial thigh. Additionally, a line was
drawn along the prominence from the pubic tubercle to the
medial knee to indicate the anterior boarder of the gracilis
muscle, and the skin flap overlying the gracilis muscle was
marked (Figure 1).
An incision was made along the anterior boarder of the
skin flap, and dissection between the adductor longus and
the gracilis was performed, preserving the fascia surrounding
the gracilis. The dominant pedicle and motor nerve were
identified beneath the adductor longus (Figure 2A); however,
the neurovascular pedicle was not dissected at this time.
Next, the incision was extended to the insertion of the gracilis,
and the posterior border of the skin flap was incised. The
gracilis was then isolated from adductor longus anterolaterally and from the adductor magus posterolaterally.
With the adductor longus retracted anteriorly, the vascular
pedicle of the gracilis was exposed and dissected, and the
branches to the adductor longus, brevis, and magnus were
ligated. The nerve branches of the muscle were also identified
(Figure 2B). The adductor longus was also retracted posteromedially so that the artery pedicle could be traced to its
origin at the profunda femoris. A 2 cm to 3 cm segment of
the profunda femoris was then isolated and cut (Figure 2C).
A T-shaped artery pedicle comprising the profunda femoris
and the nutrient artery of the gracilis was harvested, and the
venae comitantes of the nutrient artery were also harvested
from their origin. The profunda femoris arterial segment had
an obviously larger caliber (Figure 2D). After dividing the
motor nerve, the gracilis was harvested.
Incisions in the upper limb were performed based on the
aim of reconstruction. The recipient artery, which was usually
the brachial artery, axillary artery, or radial artery, was divided
and interposed with the T-shaped pedicle, with both ends
anastomosed (Figure 3). The venae comitantes of the gracilis
were also anastomosed with the matched recipient veins in an
end-to-end fashion. If there were 2 venae comitantes, they
were anastomosed with the superficial and deep venous
systems, respectively (Figure 3C). When only a single vena
comitans was present, only one anastomosis was performed.
In the end-to-end anastomosis group, a single arterial end-toend anastomosis was performed. The spinal accessory nerve
was the first choice for nerve innervation in both groups. For
patients whose spinal accessory nerves were unavailable,
intercostal nerves, the medial brachial cutaneous nerve, and a
bundle of ulnar nerves reinnervated by CC7 were utilized.
METHODS
Patients
The cases of consecutive patients with traumatic BPI
treated with gracilis FFMT at our center from 2005 to 2013
were retrospectively reviewed. The criteria for inclusion in
this study were patients with traumatic BPI who received
FFMT using the gracilis muscle as the donor muscle. Patients
with traumatic muscle loss and those who received FFMT
using muscles other than the gracilis as the donor muscle
were excluded. At the beginning of the study period, we
used traditional end-to-end vascular anastomosis when
performing FFMT, whereas in the later part of the study
period, we used the flow-through technique; thus, patients
were divided into a traditional anastomosis group and a
flow-through anastomosis group.
Data regarding patient age and gender, the etiology of the
injury, the harvest time, the total operation time, vascular
compromise and other postoperative complications, and
donor-site complications were collected from the medical
records. The data were specifically collected from the
medical records by a physician who was not involved in
the study and who was not aware of which type of
anastomosis was performed. As is routine in our department,
two groups of surgeons began preparation of the donor site
and recipient site simultaneously. The operation time was
defined as the time from incision to wound closure, and the
545
CLINICS 2015;70(8):544-549
Figure 2 - Intraoperative images - A) Exposure of the dominant vascular pedicle. To avoid injury during the operation, the pedicle was
not dissected at first. B) The neurovascular pedicle of the gracilis. Note that the sensory nerve branch ($) must be resected to ensure
enough motor nerve fiber regeneration (a, b). C) Exposure of the profunda femoris. A segment of the profunda femoris was prepared.
It is unnecessary to perform a long dissection. D) The T-shaped arterial pedicle of the gracilis musculocutaneous flap (flap placed with
the skin paddle downward).
546
CLINICS 2015;70(8):544-549
Figure 3 - Flow-through anastomosis of the T-shaped pedicle - A) The diameter of the profunda femoris segment is obviously larger
than that of the nutrient artery of the gracilis. B) The brachial artery was resected, and the diameters of the segment profunda femoris
and brachial artery were well matched. C) Interposed anastomosis to bridge the brachial artery. Two veins were anastomosed in direct
end-to-end fashion.
Statistical analysis
Continuous variables are presented as the meanstandard deviation (SD) and were compared using independentsample t-tests. Categorical variables are expressed as a
number and percentage and were compared using Fishers
exact test. A two-tailed po0.05 was considered statistically
significant. All analyses were performed using SPSS Version
20 (SPSS Statistics V20, IBM Corporation, Somers, New
York).
RESULTS
A total of 71 patients treated with FFMT for traumatic BPI
due to a motorcycle accident, a machine injury, or a crush
injury were included in the analysis. A total of 46 patients
received flow-through anastomosis, and 25 patients received
conventional end-to-end anastomosis; these 2 groups were
comparable with respect to age and sex (both p40.05;
Table 1). The purpose of reconstruction for the patients in the
flow-through group was to restore elbow flexion and finger
extension (n=33), to restore elbow flexion and finger flexion
(n=12), or to restore elbow extension (n=1). In the end-to-end
group, the goal of reconstruction was to restore elbow flexion
and finger extension (n=21) or to restore elbow flexion (n=4).
In the flow-through group, the recipient vessels included
547
CLINICS 2015;70(8):544-549
Age (y)
Gender, male
Harvest time (min)
Total operation time (min)
Diameter of arterial anastomosis* (mm)
Vascular compromise
Donor-site score
27.858.80
44 (95.6)
98.679.16
400.1173.14
3.870.42
2 (4.35)
1.090.69
26.447.94
24 (96)
101.526.02
408.2098.74
2.060.44
6 (24)
0.880.67
p-value
0.508
0.718
0.167
0.721
o0.001
0.019
0.228
* The diameter was measured once the vessels were mobilized during surgery.
The data are expressed as a number (percentage) or the meanstandard deviation.
DISCUSSION
548
CLINICS 2015;70(8):544-549
13. Lin SH, Chuang DC, Hattori Y, Chen HC. Traumatic major muscle loss in
the upper extremity: reconstruction using functioning free muscle transplantation. J Reconstr Microsurg. 2004;20(3):227-35, http://dx.doi.org/
10.1055/s-2004-823110
14. Chuang DC, Carver N, Wei FC. A new strategy to prevent the sequelae of
severe Volkmanns ischemia. Plast Reconstr Surg. 1996;98(6):1023-31,
http://dx.doi.org/10.1097/00006534-199611000-00015
15. Ihara K, Shigetomi M, Kawai S, Doi K, Yamamoto M. Functioning muscle
transplantation after wide excision of sarcomas in the extremity. Clin
Orthop Relat Res. 1999;(358):140-8.
16. Gardiner MD, Nanchahal J. Strategies to ensure success of microvascular
free tissue transfer. J Plast Reconstr Aesthet Surg. 2010;63(9):e665-73,
http://dx.doi.org/10.1016/j.bjps.2010.06.011
17. Macchi V, Vigato E, Porzionato A, Tiengo C, Stecco C, Parenti A, et al. The
gracilis muscle and its use in clinical reconstruction: an anatomical,
embryological, and radiological study. Clin Anat. 2008;21(7):696-704,
http://dx.doi.org/10.1002/ca.v21:7
18. Juricic M, Vaysse P, Guitard J, Moscovici J, Becue J, Juskiewenski S.
Anatomic basis for use of a gracilis muscle flap. Surg Radiol Anat. 1993;15
(3):163-8, http://dx.doi.org/10.1007/BF01627695
19. Giordano PA, Abbes M, Pequignot JP. Gracilis blood supply: anatomical
and clinical re-evaluation. Br J Plast Surg. 1990;43(3):266-72, http://dx.
doi.org/10.1016/0007-1226(90)90071-7
20. Foucher G, van Genechten F, Merle N, Michon J. A compound radial
artery forearm flap in hand surgery: an original modification of the
Chinese forearm flap. Br J Plast Surg. 1984;37(2):139-48, http://dx.doi.
org/10.1016/0007-1226(84)90001-8
21. Costa H, Guimares I, Cardoso A, Malta A, Amarante J, Guimares F.
One-staged coverage and revascularisation of traumatised limbs by a
flow-through radial mid-forearm free flap. Br J Plast Surg. 1991;44(7):
533-7, http://dx.doi.org/10.1016/0007-1226(91)90012-9
22. Kells AF, Broyles JM, Simoa AF, Lewis VO, Sacks JM. Anterolateral thigh
flow-through flap in hand salvage. Eplasty. 2013;13:e19.
23. Yokota K, Sunagawa T, Suzuki O, Nakanishi M, Ochi M. Short interposed
pedicle of flow-through anterolateral thigh flap for reliable reconstruction
of damaged upper extremity. J Reconstr Microsurg. 2011;27(2):109-14,
http://dx.doi.org/10.1055/s-0030-1268209
24. Kasten SJ, Chung KC, Tong L. Simultaneous revascularization and soft
tissue coverage in the traumatized upper extremity with a flow-through
radial forearm free flap. J Trauma. 1999;47(2):416-9, http://dx.doi.org/
10.1097/00005373-199908000-00042
25. Haffey TM, Lamarre ED, Fritz MA. Auto flow-through technique for
anterolateral thigh flaps. JAMA Facial Plast Surg. 2014;16(2):147-50,
http://dx.doi.org/10.1001/jamafacial.2013.2263
26. Parr JM, Adams BM, Wagels M. Flow-through flap for salvage of fibula
osseocutaneous vascular variations: a surgical approach and proposed
modification of its classification. J Oral Maxillofac Surg. 2014;72(6):
1197-202, http://dx.doi.org/10.1016/j.joms.2013.12.011
27. Kawamura K, Yajima H, Kobata Y, Shigematsu K, Takakura Y. Anatomy of
Y-shaped configurations in the subscapular arterial system and clinical
application to harvesting flow-through flaps. Plast Reconstr Surg. 2005;116
(4):1082-9, http://dx.doi.org/10.1097/01.prs.0000178791.85118.ca
28. Koshima I, Fujitsu M, Ushio S, Sugiyama N, Yamashita S. Flow-through
anterior thigh flaps with a short pedicle for reconstruction of lower leg
and foot defects. Plast Reconstr Surg. 2005;115(1):155-62.
29. Brooks D, Buntic RF, Nguyen N. Salvage of a radial forearm flap transferred onto a by-pass graft with conversion from a high-to-low-resistance
circulatory pattern: case report. J Reconstr Microsurg. 2005;21(6):355-7,
http://dx.doi.org/10.1055/s-2005-915201
30. Rozen WM, Leong J. Arterialized venous flow-through flaps with dual
discontiguous venous drainage: a new modification to improve flap
survival. Plast Reconstr Surg. 2012;130(1):229e-31e, http://dx.doi.org/
10.1097/PRS.0b013e3182550260
31. Carr MM, ManktelowRT, Zuker RM. Gracilis donor site morbidity. Microsurgery. 1995;16(9):598-600, http://dx.doi.org/10.1002/(ISSN)1098-2752
32. MathesSJ, Nahai F. Classification of the vascular anatomy of muscles:
experimental and clinical correlation. Plast Reconstr Surg. 1981;67(2):
177-87, http://dx.doi.org/10.1097/00006534-198167020-00007
33. Morris SF, Yang D. Gracilis muscle: arterial and neural basis for subdivision. Ann Plast Surg. 1999;42(6):630-3, http://dx.doi.org/10.1097/
00000637-199906000-00008
34. Kappler UA, Constantinescu MA, Buchler U, Vogelin E. Anatomy of the
proximal cutaneous perforator vessels of the gracilis muscle. Br J Plast
Surg. 2005;58(4):445-8, http://dx.doi.org/10.1016/j.bjps.2004.11.021
35. Miyamoto S, Okazaki M, Ohura N, Shiraishi T, Takushima A, Harii K.
Comparative study of different combinations of microvascular anastomoses in a rat model: end-to-end, end-to-side, and flow-through anastomosis. Plast Reconstr Surg. 2008;122(2):449-55, http://dx.doi.org/
10.1097/PRS.0b013e31817d62c5
36. Ichinose A, Terashi H, Nakahara M, Sugimoto I, Hashikawa K, Nomura T,
et al. Do multiple venous anastomoses reduce risk of thrombosis in free-flap
transfer? Efficacy of dual anastomoses of separate venous systems. Ann Plast
Surg. 2004;52(1):61-3, http://dx.doi.org/10.1097/01.sap.0000096425.18223.60
ACKNOWLEDGMENTS
This study was supported by the NHFPC Special Fund for Health Scientic
Research in the Public Welfare (Number 201402016).
AUTHOR CONTRIBUTIONS
We declare that all of the listed authors have participated actively in the
study and meet the requirements for authorship. Hou Y and Gu L designed
the study and wrote the protocol. Hou Y and Yang J performed the
research/study. Liu X and Zhu Q contributed constructive suggestions
about writing the article. Yang Y and Qin B managed the literature
searches and analyses. Fu G and Li X performed the statistical analysis.
Hou Y wrote the rst draft of the manuscript.
REFERENCES
1. Midha R. Epidemiology of brachial plexus injuries in a multitrauma
population. Neurosurgery. 1997;40(6):1182-9, http://dx.doi.org/10.1097/
00006123-199706000-00014
2. Gu YD, WU MM, Zhen YL, Zhao JA, Zhang GM, Chen DS, et al. Phrenic
nerve transferfor treatment of root avulsion of the brachial plexus. Chin
Med J (Engl). 1990;103(4):267-70.
3. Mcguiness CN, Kay SP. The prespinal route in contralateral C7 nerve root
transfer for brachial plexus avulsion injuries. J Hand Surg Br. 2002;27
(2):159-60, http://dx.doi.org/10.1054/jhsb.2001.0665
4. Lee SK, Wolfe SW. Nerve transfers for the upper extremity: new horizons
in nerve reconstruction. J Am AcadOrthop Surg. 2012;20(8):506-17,
http://dx.doi.org/10.5435/JAAOS-20-08-506
5. Terzis JK, Kostopoulos VK. The surgical treatment of brachial plexus
injuries in adults. Plast Reconstr Surg. 2007;119(4):73e-92e, http://dx.doi.
org/10.1097/01.prs.0000254859.51903.97
6. Terzis JK, Kostopoulos VK. Free muscle transfer in posttraumatic plexopathies: part 1: the shoulder. Ann Plast Surg. 2010;65(3):12-7, http://dx.
doi.org/10.1097/SAP.0b013e3181cbfe9d
7. Terzis JK, KostopoulosVK. Free muscle transfer in posttraumatic plexopathies part II: the elbow. Hand (N Y). 2010;5(2):160-70.
8. Terzis JK, KostopoulosVK. Free muscle transfer in posttraumatic plexopathies: part III. The hand. Plast Reconstr Surg. 2009;124(4):1225-36,
http://dx.doi.org/10.1097/PRS.0b013e3181b5a322
9. Chuang DC. Brachial plexus injury: nerve reconstruction and functioning
muscle transplantation. Semin Plast Surg. 2010;24(1):57-66, http://dx.doi.
org/10.1055/s-0030-1253242
10. Songcharoen P. Management of brachial plexus injury in adults. Scand J
Surg. 2008;97(4):317-23.
11. Chuang DC. Nerve transfer with functioning free muscle transplantation.
Hand Clin. 2008;24(4):377-88, http://dx.doi.org/10.1016/j.hcl.2008.03.012
12. Doi K, Kuwata N, Muramatsu K, Hottori Y, Kawai S. Double muscle
transfer for upper extremity reconstruction following complete avulsion
of the brachial plexus. Hand Clin. 1999;15(4):757-67.
549
CLINICAL SCIENCE
OBJECTIVE: To evaluate hematological, cytogenetic and molecular responses as well as the overall, progressionfree and event-free survivals of chronic myeloid leukemia patients treated with a third tyrosine kinase inhibitor
after failing to respond to imatinib and nilotinib/dasatinib.
METHODS: Bone marrow karyotyping and real-time quantitative polymerase chain reaction were performed at
baseline and at 3, 6, 12 and 18 months after the initiation of treatment with a third tyrosine kinase inhibitor.
Hematologic, cytogenetic and molecular responses were defined according to the European LeukemiaNet
recommendations. BCR-ABL1 mutations were analyzed by Sanger sequencing.
RESULTS: We evaluated 25 chronic myeloid leukemia patients who had been previously treated with imatinib and a
second tyrosine kinase inhibitor. Nine patients were switched to dasatinib, and 16 patients were switched to nilotinib
as a third-line therapy. Of the chronic phase patients (n=18), 89% achieved a complete hematologic response, 13%
achieved a complete cytogenetic response and 24% achieved a major molecular response. The following BCR-ABL1
mutations were detected in 6/14 (43%) chronic phase patients: E255V, Y253H, M244V, F317L (2) and F359V. M351T
mutation was found in one patient in the accelerated phase of the disease. The five-year overall, progression-free and
event-free survivals were 86, 54 and 22% ( po0.0001), respectively, for chronic phase patients and 66%, 66% and 0%
( po0.0001), respectively, for accelerated phase patients. All blast crisis patients died within 6 months of treatment.
Fifty-six percent of the chronic phase patients lost their hematologic response within a median of 23 months.
CONCLUSIONS: Although the responses achieved by the third tyrosine kinase inhibitor were not sustainable, a
third tyrosine kinase inhibitor may be an option for improving patient status until a donor becomes available
for transplant. Because the long-term outcome for these patients is poor, the development of new therapies for
resistant chronic myeloid leukemia patients is necessary.
KEYWORDS: CML; Dasatinib; Nilotinib; Third-line TKI treatment.
Ribeiro BF, Miranda EC, Martins de Albuquerque D, Delamain MT, Oliveira-Duarte G, Almeida MH, et al. Treatment with dasatinib or nilotinib in
chronic myeloid leukemia patients who failed to respond to two previously administered tyrosine kinase inhibitors a single
center experience. Clinics. 2015;70(8):550-555
Received for publication on February 25, 2015; First review completed on March 30, 2015; Accepted for publication on May 21, 2015
E-mail: kborgia@unicamp.br
*Corresponding author
INTRODUCTION
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0/)
which permits unrestricted use, distribution, and reproduction in any medium or
format, provided the original work is properly cited.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)04
550
CLINICS 2015;70(8):550-555
Statistical methods
Probabilities of overall survival (OS), progression-free
survival (PFS) and event-free survival (EFS) were calculated
using the Kaplan-Meier method. OS was calculated at the
initiation of therapy with the 3rd TKI until the final follow-up
or death for any reason. PFS was defined as survival without
transformation to the accelerated or blastic phase after
starting the 3rd TKI and was judged based on an event of
progression or death. EFS was defined as loss of complete
hematological response (CHR), CCyR, MMR, progression to
advanced phases, death or 3rd TKI discontinuation for any
reason (toxicity, resistance, transplant or patient lost to
follow-up). Po0.05 was considered statistically significant.
The cut-off for the data analysis was March 2015.
Ethics
The study protocol was approved and was conducted in
accordance with the ethical standards of the local Research
Ethics Committee on human experimentation and the
Helsinki Declaration of 1975, which was revised in 1983.
Patients provided written informed consent for their
participation.
RESULTS
Clinical and laboratory characteristics of the 25 CML
patients at the time of diagnosis and before the initiation of
the 3rd TKI are presented in Tables 1 and 2, respectively.
Chronic-phase CML patients (CP-CML) (n=18) were
analyzed separately. Thirteen CP-CML patients were resistant to imatinib (72%), and 5 were intolerant to imatinib
(28%). Five patients were treated with dasatinib (28%), and
13 patients were treated with nilotinib (72%). Sixteen patients
(89%) were resistant to the 2nd TKI, and 2 patients (11%) were
intolerant to the 2nd TKI. The resistant patients never
achieved a previous CCyR with imatinib or with the 2nd
TKI. The median follow-up duration was 52 (7-75) months,
and 16/18 patients (89%) achieved or maintained a complete
hematologic response during this period. Of 15 patients who
were subjected to cytogenetic analysis, 2 (13%) achieved
CCyR. Of 17 CP-CML patients with available molecular
analysis data, 4 (24%) achieved a major molecular response
(MMR), and 2 achieved a complete molecular response
(CMR). For CP-CML patients, the frequencies of the transcript
levels at baseline and at 3 and 6 months after the initiation of
the 3rd TKI are shown in Table 3.
551
CLINICS 2015;70(8):550-555
n.
45 (14-72)
13
52
5
1
9
10
01/09
11/16
06/11
137.10 (17.1 494.4)
352.0 (141.0 - 2,901.0)
10.2 (5.1 13.7)
3.5 (0 - 17)
4 (0 - 34)
20
4
36
40
11.1
68.7
54.4
Mutation analysis
Survival analysis
One patient in the CP stage died during 3rd TKI therapy.
CP-CML patients had 5-year OS, PFS and EFS values of 86,
54 and 22% (po0.0001), respectively, whereas AP-CML
patients had 5-year OS, PFS and EFS values of 66, 66 and
0%, respectively (po0.0001). BC-CML patients showed no
response in the first year after treatment (Figures 1, 2 and 3).
Long-term outcome
During treatment, 9/16 (56%) CP-CML patients lost CHR
within a median of 23 (3-37) months. Two patients lost CCyR
after 12 and 13 months. One patient lost MMR after
7 months. Six (34%) patients are currently taking their 3rd
TKI, although 3 of these patients lost their response (1 MMR,
1 CCyR and 1 CHR). Three CP-CML patients (17%)
progressed to the BC (blast crisis) stage, and 2 CP-CML
patients subsequently died. Discontinuation of the 3rd TKI
occurred in 16 (89%) cases due to resistance (8); intolerance
(3); loss to follow-up (3); and death (2) during the treatment.
Three AP-CML patients reached CHR, but one of these
patients lost their response. Only one patient achieved CCyR
and MMR, but those responses were lost. One patient
discontinued treatment due to intolerance in the 4th month.
tyrosine
Time
Variables
Median age (range) years
Median time of imatinib therapy (range) months
Achievement of CCyR with imatinib treatment n (%)
Interval diagnosis 3rd TKI (range) months
Treated with dasatinib 100-140 mg once daily n (%)
Treated with nilotinib 400 mg BID n (%)
Disease status before 3rd TKI n (%)
CP
AP
BC
Baseline
n= 25
56
30
3
98
16
09
(22-75)
(1-66)
(12%)
(12-404)
(64%)
(36%)
3 months
6 months
18 (72%)
03 (12%)
04 (16%)
552
RQ-PCR (IS)%
410
1 10
0.1 1o
p0.1
410
1 10
0.1 1o
p0.1
410
1 10
0.1 1o
p0.1
11/18
04/18
03/18
0
09/12
02/12
0
1/12
04/08
01/08
01/08
02/08
61
22
17
0
75
17
0
08
50
12.5
12.5
25
CLINICS 2015;70(8):550-555
Figure 1 - Kaplan-Meier survival analysis. Five-year OS of chronic myeloid leukemia patients treated with a 3rd tyrosine kinase inhibitor
according to disease phase.
Figure 2 - Kaplan-Meier survival analysis. Five-year PFS of chronic myeloid leukemia patients treated with a 3rd tyrosine kinase inhibitor
according to disease phase.
553
CLINICS 2015;70(8):550-555
Figure 3 - Kaplan-Meier survival analysis. Five-year event-free survival of chronic myeloid leukemia patients treated with a 3rd tyrosine
kinase inhibitor according to disease phase.
DISCUSSION
Our data show that only 22% of patients in the CP stage
showed long-term benefits from the administration of a 3rd
TKI after imatinib and a 2nd TKI failure. We found that 89%
of our patients in the CP stage achieved CHR, 13% achieved
CCyR, and 24% achieved MMR; however, 50% of those
patients lost CHR within a median of 23 months. All patients
with CCyR lost their response after 12 months, and 25% of
patients lost MMR after 7 months.
Our results are in agreement with prior reports. QuintasCardama et al. (15) performed a study on 23 CML patients
treated with dasatinib after imatinib and nilotinib failure and
found that 43% of these patients achieved CHR and 30%
achieved a cytogenetic response. Giles et al. (16), performed a
study analyzing 60 patients treated with nilotinib after
imatinib and dasatinib failure and found that 70% of CPCML patients achieved CHR and 43% of CP-CML patients
achieved a major cytogenetic response (MCyR). The authors
also found that after 18 months, 59% of CP-CML patients
were progression-free, and their estimated survival was 86%.
554
CLINICS 2015;70(8):550-555
ACKNOWLEDGEMENTS
Beatriz Felicio Ribeiro received a scholarship from FAPESP and Katia
Pagnano received nancial support from FAPESP. The authors thank the
Universidade de Campinas (UNICAMP), Centro de Hematologia e
Hemotherapia, Campinas/SP, Brazil for supporting this study.
AUTHOR CONTRIBUTIONS
Ribeiro BF and Pagnano KB conceived and designed the study. Ribeiro
BF, Duarte VO, Miranda EC, Almeida MH, and Pagnano KB performed
the data collection. Delamain MT, Oliveira-Duarte G, and Pagnano KB
treated the patients. Lorand-Metze I, Souza CA, Pagnano KB, Ribeiro BF,
Verglio B, Silveira RA, and Albuquerque DM performed the BCR-ABL1
mutation analysis and quantitative PCR experiments. Miranda ECM
managed, analyzed, and interpreted the data. Ribeiro BF, Miranda ECM,
Albuquerque DM, Delamain MT, Oliveira- Duarte G, Almeida MH,
Verglio B, Silveira RA, Oliveira-Duarte V, Lorand-Metze I, Souza CA,
and Pagnano KB approved the nal manuscript. All authors contributed to
the collection, analysis and interpretation of the data and contributed to the
critical revision of the article for intellectual content.
REFERENCES
1. Giles FJ, Le Coutre PD, Pinilla-Ibarz J, Larson RA, Gattermann N,
Ottmann OG, et al. Nilotinib in imatinib-resistant or imatinib-intolerant
patients with chronic myeloid leukemia in chronic phase: 48-month
follow-up results of a phase II study. Leukemia. 2013;27(1):10712,
http://dx.doi.org/10.1038/leu.2012.181.
2. Hochhaus A, Baccarani M, Deininger M, Apperley JF, Lipton JH,
Goldberg SL, et al. Dasatinib induces durable cytogenetic responses in
patients with chronic myelogenous leukemia in chronic phase with
resistance or intolerance to imatinib. Leukemia. 2008;22(6):12006, http://
dx.doi.org/10.1038/leu.2008.84.
3. Milojkovic D, Apperley JF, Gerrard G, Ibrahim AR, Szydlo R, Bua M,
et al. Responses to second-line tyrosine kinase inhibitors are durable: an
intention-to-treat analysis in chronic myeloid leukemia patients. Blood.
2012;119(8):183843, http://dx.doi.org/10.1182/blood-2011-10-383000.
4. Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley
JF, et al. European LeukemiaNet recommendations for the management of
chronic myeloid leukemia: 2013. Blood. 2013;122(6):87284, http://dx.
doi.org/10.1182/blood-2013-05-501569.
5. Hughes T, Saglio G, Branford S, Soverini S, Kim D-W, Mller MC, et al.
Impact of baseline BCR-ABL mutations on response to nilotinib in
patients with chronic myeloid leukemia in chronic phase. J Clin Oncol.
2009;27(25):420410, http://dx.doi.org/10.1200/JCO.2009.21.8230.
6. Cortes J, Jabbour E, Kantarjian H, Yin CC, Shan J, Brien SO, et al. Dynamics
of BCR-ABL kinase domain mutations in chronic myeloid leukemia after
sequential treatment with multiple tyrosine kinase inhibitors. Blood.
2007;110(12):400511, http://dx.doi.org/10.1182/blood-2007-03-080838.
7. Baccarani M, Saglio G, Goldman J, Hochhaus A, Simonsson B,
Appelbaum F, et al. Evolving concepts in the management of chronic
myeloid leukemia: recommendations from an expert panel on behalf of
the European LeukemiaNet. Blood. 2006;108(6):180920, http://dx.doi.
org/10.1182/blood-2006-02-005686.
8. Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J, et al.
Chronic myeloid leukemia: an update of concepts and management
recommendations of European LeukemiaNet. J Clin Oncol. 2009;27
(35):604151, http://dx.doi.org/10.1200/JCO.2009.25.0779.
555
CLINICAL SCIENCE
OBJECTIVE: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular
mechanism.
METHODS: Apoptosis of A549 cells was detected by flow cytometry and fluorescence microscopy. The effect of
double-strand break (DSB) damage repair in A549 cells was evaluated using the neutral comet assay. Protein
expression levels were detected using western blotting, and the correlation between protein levels was
analyzed.
RESULTS: Elemene exhibited a radiosensitizing effect on A549 cells. The level of apoptosis induced by elemene
combined with radiation was significantly greater (po0.01) than that elicited by either radiation or elemene
alone. Following radiation and subsequent repair for 24 h, the tail intensity of A549 cells treated with a
combination of elemene and radiation was greater than that of cells treated with either elemene or radiation
alone (po0.01). This result indicates that elemene inhibits cellular DSB repair. Both elemene combined with
radiation and radiation alone decreased the protein expression of DNA-PKcs and Bcl-2 compared to elemene
alone (po0.01), while p53 protein expression was increased (po0.01). A negative correlation was observed
between DNA-PKcs and p53 expression (r=-0.569, p=0.040), while a positive correlation was found between
DNA-PKcs and Bcl-2 expression (r=0.755, p=0.012).
CONCLUSIONS: Elemene exhibits a radiosensitizing effect on A549 cells, and its underlying molecular
mechanism of action may be related to the downregulation of DNA-PKcs gene expression.
KEYWORDS: Elemene; Radiosensitivity; A549 cells; DNA-PKcs; Bcl-2; p53.
Zou K, Liu C, Zhang Z, Zou L. The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism.
Clinics. 2015;70(8):556-562
Received for publication on January 6, 2015; First review completed on February 27, 2015; Accepted for publication June 1, 2015
E-mail: 799832582@qq.com, zoulijuanfl@163.com
*Corresponding authors
INTRODUCTION
damage repair-related proteins (5). Thus, inhibition of DNAPKcs gene expression can block DNA double-strand break
(DSB) repair and improve cellular radiosensitivity.
Cellular apoptosis is the core characteristic of radiotherapy, and regulation of this process thus plays an
important role in cellular radiosensitivity (6,7). Previous
studies have shown that apoptosis-related genes, such as
phosphoprotein (p53), p16, B-cell lymphoma-2 (Bcl-2), and
erythroblastic leukemia viral oncogene homolog 2 (erbB-2),
are associated with tumor radiosensitivity (8,9), especially
p53 and Bcl-2. It has also been reported that elemene
interacts with the frontier orbitals of DNA bases to form
complexes between DNA molecules. Specifically, Jiang et al.
(10) showed that elemene increases the radiosensitivity of
A549 cells, the mechanism for which may be related to the
upregulation of p53, downregulation of Bcl-2, and induction
of cellular apoptosis.
Elemene, which is extracted from Zingiberaceae plants
(Curcuma aromatica Salisb.), is a non-cytotoxic antitumor
compound that can improve the radiosensitivity of tumor
cells (11). Results of an in vitro study showed that elemene
increased the radiosensitivity of renal carcinoma cells,
tongue squamous cancer cells, and non-small cell lung
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/
4.0/) which permits unrestricted use, distribution, and reproduction in any
medium or format, provided the original work is properly cited.
No potential conflict of interest was reported.
DOI: 10.6061/clinics/2015(08)05
556
CLINICS 2015;70(8):556-562
Dq)/experimental
group (D0,
Dq).
Apoptosis standard
Normal cells demonstrated uniform dispersion of lowdensity fluorescence, while apoptotic cells showed highdensity fluorescence, characterized by a bright blue hue.
Assessment of apoptosis
The cells used were grouped and treated as specified
above. Then, samples of 3 105 cells were collected from
each group, treated with pancreatic enzyme digesting cells,
rinsed twice with PBS, and centrifuged at 1,000 rpm for
5 min. The cells were treated with 100 mL 2% Triton X-100 for
20 min, rinsed twice with PBS, and centrifuged at 1,000 rpm
for 5 min. Next, 200 mL of DNA-Prep LPR reagent (BeckmanCoulter Ltd) was added for 20 min, and the cells were rinsed
twice with PBS, followed by centrifugation at 1,000 rpm for
5 min. The cells were resuspended in PBS and 50 mg/mL
propidium iodide (PI) reagent containing 480 mL of PBS, 5 mL
of PI (5 mg /mL), and 5 mL of RNase (10 mg /mL), and
10 mL of Triton X-100 (10%) was added 30 s later. Single-cell
suspensions were analyzed by flow cytometry to determine
the cellular apoptosis rate.
Irradiation conditions
Cell irradiation was performed using the Varian 2300C/D
medical linear accelerator (Varian Companies, USA) with a
coverage field of 20 cm 20 cm. The culture dish was
placed in the radiation field above 1.5 cm of organic glass.
Cells with irradiated with 6 MV X-ray irradiation at a dosage
rate of 300 cGy/min, a rack angle of 180 , and source-tosurface distance (SSD) of 100 cm.
Clonogenic assay
Neutral comet assay
557
CLINICS 2015;70(8):556-562
0 Gy
2 Gy
4 Gy
6 Gy
8 Gy
10 Gy
100
97.720.2
95.418.8
84.113.2
76.210.4
63.67.5
70.210.5
49.56.4
30.43.0
66.31.9
22.52.3
5.71.1
47.32.5
7.51.8
2.70.6
26.01.3
3.40.2
0.70.1
Cells were incubated with 10 or 20 mg/mL concentrations of elemene for 24 h and were then irradiated with 0, 2, 4, 6, 8, and 10 Gy and cultured for
another 14 days. The number of cells forming more than 50 clones was counted under the inverted microscope to calculate the cloning efficiency (CE):
CE (%)=(Clone formation average of treatment group/inoculated cell number) 100%. Survival fraction (SF)=(irritated group CE/non-irradiated group
PE) 100%. The experiment was repeated 3 times to calculate the average.
Statistical analysis
RESULTS
Effects of elemene on cell radiosensitivity
The survival fraction of A549 cells decreased following
treatment with different doses of radiation and the same
concentration of elemene. Conversely, the A549 cell
survival fraction decreased in the groups treated with the
same dose of radiation in combination with increasing
concentrations of elemene (Table 1). Following treatment
with 10 or 20 mg/mL elemene, the A549 cell survival curve
shifted to the left, the shoulder area was diminished, and
the steepness of the curve increased (Figure 1). Based on
the cell survival curve, the radiobiological parameters and
radiosensitization ratio were obtained and are listed in
Table 1. These data show that, compared with the control
group, the SERD0 and SERDq values for the 10 and 20 mg/mL
elemene treatment groups were greater than 1. Furthermore,
the ratio gradually increased with an increasing drug
concentration (Table 2).
D0 (Gy)
Dq (Gy)
SF2 (%)
SERD0
SERDq
Control
2.540.24 2.680.25 84.620.9
10 mg/mL 1.640.15 1.870.22 56.314.9 1.540.20 1.430.15
20 mg/mL 1.550.13 1.530.11 43.210.7 1.630.32 1.750.19
SPSS17.0 was used to calculate survival parameters including SF2
(surviving fraction of 2 Gy), D0 (mean lethal dose or final slope), and
Dq (quasi-thres-hold dose). SER (sensitization enhancement ratio). SER D0
(Dq)=control group D0 (Dq)/drug group D0 (Dq) . Three parallel samples
were set at each radiation dosage.
558
CLINICS 2015;70(8):556-562
Figure 2 - Elemene induces apoptosis in A549 cells. Cells were incubated with 10 or 20 mg/mL elemene for 24 h, followed by irradiation with
4 Gy X-rays and washing with PBS. (A) Cells were imaged with an Olympus BX-51 fluorescent microscope using appropriate filter cubes. (B)
Chromatin condensation was analyzed by fluorescence microscopy after DNA staining with Hoechst 3334. The results are expressed as the
mean SD of three independent experiments, n=3, **po0.01 compared to control, ##po0.01 compared to radiation alone.
559
CLINICS 2015;70(8):556-562
Figure 3 - Influence of elemene on radiation-induced DSB. A549 cells were irradiated with 4 Gy X-rays in the absence or presence of
elemene (10 or 20 mg/mL) and assayed immediately after radiation or returned to the incubator for 24 h to permit repair. Comet assays
were performed immediately after incubation with elemene treatment, irradiation, or combination treatment. Columns represent the
means of the tail moments from three independent experiments, and the bars represent the SD, **po0.01 vs. control, ##po0.01
compared to radiation alone.
DISCUSSION
Basic research in radiation biology has shown that
radiation therapy works mainly by damaging tumor cell
DNA and altering the expression of apoptosis-related genes
and proteins. The radiosensitivity of tumor cells relates to
their capacity to repair DSB via the related genes DNAPKcs, Ku70/80, and ataxia telangiectasia mutated (ATM).
Other genes known to be involved in radiosensitivity and
responsible for apoptosis regulation include p53, Bcl-2,
c-myc proto-oncogene (c-myc), and survivin (9). Beta
elemene, which is the active component of elemene, has
recently been demonstrated to enhance the radiosensitivity
of human cancer cell lines in vitro and in an animal tumor
model in vivo (16,20). In particular, beta elemene was found
to enhance radiosensitivity by influencing the cell cycle
distribution of gastric cancer MKN28 cells, and the
mechanisms responsible for this effect include the induction
of G2/M phase arrest, inhibition of sublethal damage repair,
and induction of cell apoptosis, which enhances the killing
effects of radioactive rays (21). The results of the current
study show that the SERD0 and SERDq values of A549 cells
exposed to a low concentration of cytotoxic elemene were
greater than 1. In addition, elemene enhanced the sensitivity
560
CLINICS 2015;70(8):556-562
of A549 cells to radiotherapy. Cellular apoptosis is fundamental to radiotherapy, and its regulatory mechanism plays
an important role in cellular radiosensitivity. Apoptosisrelated genes such as p53 and Bcl-2 have important
regulatory functions in the progression of rapid apoptosis
induced by radiation therapy. For instance, a previous study
showed that the levels of the antiapoptotic genes Bcl-2 and
Bcl-xl in A549 cells decreased, while p53 expression and the
production of exosomes increased, following elemene
treatment (22). This result demonstrates that both p53 and
Bcl-2 have important regulatory actions in cervical cancer
cell apoptosis induced by radiation. A number of experimental studies have further shown that elemene is involved
in regulating the expression of Bax, c-myc, p53, poly (ADPribose) polymerase (PARP), survivin, and livin as well as
inducing tumor cell apoptosis (23-26). Our results showed
that, compared with the exposure alone group, the group
that received elemene combined with irradiation exhibited
increased p53 gene expression and significantly decreased
Bcl-2 gene expression, and the expression of both genes was
significantly correlated. Furthermore, elemene was shown to
regulate expression of the apoptosis-related genes Bcl-2 and
561
CLINICS 2015;70(8):556-562
15. She JJ, Wang ZM, Che XM, Pan CE. Radiosensitization of beta-elemene on
VX2 carcinoma transplanted on kidney in rabbits in vivo. Zhong Xi Yi Jie
He Xue Bao. 2006;4(4):392-6, http://dx.doi.org/10.3736/jcim20060415.
16. Li LJ, Zhong LF, Jiang LP, Geng CY, Zou LJ. b-Elemene radiosensitizes lung
cancer A549 cells by enhancing DNA damage and inhibiting DNA repair.
Phytother Res. 2011; 25(7):1095-7, http://dx.doi.org/10.1002/ptr.v25.7.
17. Lindblom E, Dasu A, Lax I, Toma-Dasu I. Survival and tumour control
probability in tumours withheterogeneous oxygenation: A comparison
between the linearquadratic and the universal survival curve models for
high doses. Acta Oncol. 2014;53(8):1035-40, http://dx.doi.org/10.3109/
0284186X.2014.925582.
18. Forchhammer L, Johansson C, Loft S, Moller L, Godschalk RW, Langie
SA, et al. Variation in the measurement of DNA damage by comet assay
measured by the ECVAG inter-laboratory validation trial. Mutagenesis.
2010;25(2):113-23, http://dx.doi.org/10.1093/mutage/gep048.
19. Li Z, Berk M, McIntyre TM, Gores GJ, Feldstein AE. The lysosomalmitochondrial axis in free fatty acid-induced hepatic lipotoxicity. Hepatology. 2008;47(5):1495-503, http://dx.doi.org/10.1002/(ISSN)1527-3350.
20. Li G, Xie B, Li X, Chen Y, Wang Q, Xu Y, et al. Down-regulation of
survivin and hypoxia-inducible factor-1 alpha by beta-elemene enhances
the radiosensitivity of lung adenocarcinoma xenograft. Cancer Biother
Radiopharm. 2012;27(1):56-64, http://dx.doi.org/10.1089/cbr.2011.1003.
21. He S, Liu J, Zhang Z, Che X, Fan L, Chang S, et al. Enhancement of gastric
cancer MKN28 cell line radiosensitivity induced by beta-elemene. Zhonghua Wai Ke Za Zhi. 2014;52(6):442-5.
22. Li J, JunYu, Liu A, Wang Y. b-Elemene against human lung cancer via upregulation of P53 protein expression to promote the release of exosome. Lung
Cancer. 2014;86(2):144-50, http://dx.doi.org/10.1016/j.lungcan.2014.08.015.
23. Zhang Y, Zhao MF, Liu YP. The effects of b-elemene on the activation of
Akt and the expressions of apoptosis-related proteins in gastric cancer.
Shanxi Oncology Medicine. 2012;3:451-4.
24. Zhou XQ, Qiu XJ, Zhao HY. Influence on apoptosis relevant protein
expression of human lung adenocarcinoma A549 cell line managed by
Elemene. Journal of Modern Oncology. 2012; 20(10):2027-30.
25. Yang WZ, Chen CM, Shi SS. The Study on inhibition of the expression of
c-myc gene of human glioma U251cell line and the effect mechanism of
effect on cellular apoptosis by elemene. Chin J Clin Oncol. 2005;
32(13):763-6.
26. Zhao QT, Yang Y, Sun CB. Effect of b-elemene combined with radiotherapy on the expression of Livin mRNA and apoptosis of lung adenocarcinoma cell line A549. Journal of Modern Oncology. 2013; 21(2):257-60.
27. Calsou P, Delteil C, Frit P, Drouet J, Salles B. Coordinated assembly of Ku
and p460 subunits of the DNA-dependent protein kinase on DNA ends is
necessary for XRCC4-ligase IV recruitment. J Mol Biol. 2003;326(1):93-103,
http://dx.doi.org/10.1016/S0022-2836(02)01328-1.
28. Pan Y, Li WX, Li JM, Zhu JQ, Liang YQ, Guo AL. Correlation of DNAdependent protein kinase catalytic subunit expression to radiosensitivity
of non-small cell lung cancer cell lines. Ai Zheng. 2009;28(7):714-7.
29. Zou W, Che J, Wang CJ. DNA-PKcs silencing inhibit the DNA repair
induced by low dose radiation on human breast epithelial cells. Chin J
Biotech. 2009;25(5):727-32.
30. An J, Sui JL, Xu QZ. Inhibition of DNA-PKcs by siRNA and its effect on
the growth of HeLa cells. Carcinogenesis, Teratogenesis & Mutagenesis.
2005;17(6):327-31.
31. Yan YQ, Zhou PK. Mechanism of non-homologous end joing and its
bilolgical implications. Journal of Medical Molecular Biology. 2006;
3(1):69-72.
32. Yu S, Xiong Y, Tian S. The expression of DNA-PKcs in non-small cell lung
cancer and its relationship with apoptosis associated proteins. Zhongguo
Fei Ai Za Zhi. 2003;6(5):356-9.
33. Daido S, Yamamoto A, Fujiwara K, Sawaya R, Kondo S, Kondo Y. Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy. Cancer Res.
2005;65(10):4368-75, http://dx.doi.org/10.1158/0008-5472.CAN-04-4202.
34. Lee JH, Kang Y, Khare V, Jin ZY, Kang MY, Yoon Y, et al. The p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA damage.
Oncogene. 2010;29(10):1431-50, http://dx.doi.org/10.1038/onc.2009.438.
ACKNOWLEDGEMENT
This study was supported by grants from the National Natural Science
Foundation of China (No. 81473452).
AUTHOR CONTRIBUTIONS
Zou K, Zhang Z and Zou L designed the research study and wrote the
paper. Zou K and Liu C performed the research. Zou K and Zhang Z
analyzed the data.
REFERENCES
1. Maciejczyk A, Skrzypczynska I, Janiszewska M. Lung cancer. Radiotherapy in lung cancer: Actual methods and future trends. Rep Pract Oncol
Radiother. 2014;19(6):353-60, http://dx.doi.org/10.1016/j.rpor.2014.04.012.
2. Collis SJ, Swartz MJ, Nelson WG, DeWeese TL. Enhanced radiation and
chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res. 2003; 63(7):1550-4.
3. Marples B, Cann NE, Mitchell CR, Johnston PJ, Joiner MC. Evidence for
the involvement of DNA-dependent protein kinase in the phenomena of
low dose hyper-radiosensitivity and increased radioresistance. Int J Radiat
Biol. 2002;78(12):1139-47, http://dx.doi.org/10.1080/09553000210166606.
4. Sak A, Stuschke M, Wurm R, Schroeder G, Sinn B, Wolf G, et al. Selective
inactivation of DNA-dependent protein kinase with antisense oligodeoxynucleotides: consequences for the rejoining of radiation-induced
DNA double-strand breaks and radiosensitivity of human cancer cell
lines. Cancer Res. 2002;62(22):6621-4.
5. Jackson SP. Sensing and repairing DNA double-strand breaks. Carcinogenesis. 2002;23(5):687-96, http://dx.doi.org/10.1093/carcin/23.5.687.
6. Kurdoglu B, Cheong N, Guan J, Corn BW, Curran WJ, Jr, Iliakis G.
Apoptosis as a predictor of paclitaxel-induced radiosensitization in
human tumor cell lines. Clin Cancer Res. 1999;5(9):2580-7.
7. Wang LP, Liang K. Apotosis and Radiosensitivity of Cells. Foreign Medical Science. 2000;24(2):75-8.
8. Rosen EM, Fan S, Rockwell S, Goldberg ID. The molecular and cellular
basis of radiosensitivity: implications for understanding how normal tissues and tumors respond to therapeutic radiation. Cancer Invest. 1999;
17(1):56-72, http://dx.doi.org/10.3109/07357909909011718.
9. Rosen EM, Fan S, Goldberg ID, Rockwell S. Biological basis of radiation
sensitivity. Part 2: Cellular and molecular determinants of radiosensitivity.
Oncology (Williston Park). 2000;14(5):741-57; discussion 757-8, 761-6.
10. Jiang H, Ma S, Feng J. In vitro study of radiosensitization of b-elemene in
A549 cell line from denocarcinoma of lung. Chinese-German J of Clin
Oncol. 2009;8(1):12-5, http://dx.doi.org/10.1007/s10330-008-0139-3.
11. Zou K, Tong E, Xu Y, Deng X, Zou L. Down regulation of mammalian
target of rapamycin decreases HIF-1a and survivin expression in anoxic
lung adenocarcinoma A549 cell to elemene and/or irradiation. Tumor
Biol. 2014;35(10):9735-41, http://dx.doi.org/10.1007/s13277-014-2226-0.
12. Cheng W, Li JP, Wang ZM, Song W, Huang C. The variation of gene
expression profile in radiosensitivity to kidney cancer cells induced by
elemenen. Chin J Urol. 2007;28(2):87-90.
13. Wu DP, Li XM, Zhao JF, Wang HB, Zhao DQ. A Study of Radiosensitivity
of b-Elemene to Squamous Cell Carcinoma of Tongue Tca-8113 Cell Line
in Vitro. Journal of Basic and Clinical Oncology. 2006;19(2):116-7.
14. Zou LJ, Sun XH, Xu XY. The study of the b-elemene effect on the augment
of radiation treatment in mouse inoculated U14 strain. Chin J Radiological
Medicine and Protection. 2004;24(3):254-5.
562
CLINICAL SCIENCE
OBJECTIVE: Little is known about metabolic factors in cirrhotic patients in China. Therefore, we aimed to quantify
the prevalence of both metabolic factors and non-alcoholic steatohepatitis-related liver cirrhosis in China.
METHODS: The medical records of 1,582 patients diagnosed with liver cirrhosis from June 2003 to July 2013 at
Daping Hospital (Chongqing, China) were retrospectively reviewed through a computer-generated search.
RESULTS: Serum hepatitis B virus surface antigen was present in 1,083 (68.5%) patients, and hepatitis B was found
to be the only etiological factor in 938 (59.3%) of all patients. Obesity, diabetes mellitus, and arterial hypertension
were observed in 229 (14.5%), 159 (10.1%), and 129 (8.2%) patients, respectively. From 2012-2013, the proportion
of non-alcoholic steatohepatitis-related liver cirrhosis increased to 3.2%, whereas the average proportion of nonalcoholic steatohepatitis-related liver cirrhosis in the previous ten years was 1.9%. The incidence of hepatocellular
carcinoma was much higher in males than in females (6.3% vs. 3.7%, respectively, p=0.036). Obesity and diabetes
mellitus did not significantly increase the incidence of hepatocellular carcinoma in the whole cirrhotic group. The
presence of hepatitis B virus was the only risk factor for hepatocellular carcinoma in cirrhotic patients (po0.001).
CONCLUSIONS: Although hepatitis B virus remains the main etiology of liver cirrhosis in China, steatohepatitisrelated liver cirrhosis is increasing in frequency. Hepatitis B virus was the sole significant risk factor for
hepatocellular carcinoma in the whole cirrhotic group in the present study, in contrast to obesity and diabetes
mellitus, for which only a trend of increased hepatocellular carcinoma was found.
KEYWORDS: Non-Alcoholic Steatohepatitis; Non-Alcoholic Fatty Liver Disease; Liver Cirrhosis; Obesity; Metabolism.
Xiong J, Wang J, Huang J, Sun W, Wang J, Chen D. Non-alcoholic steatohepatitis-related liver cirrhosis is increasing in China: A ten-year
retrospective study. Clinics. 2015;70(8):563-568
Received for publication on January 29, 2015; First review completed on March 20, 2015; Accepted for publication on June 1, 2015
E-mail: chendongfeng1981@163.com
*Corresponding author
INTRODUCTION
DOI: 10.6061/clinics/2015(08)06
563
CLINICS 2015;70(8):563-568
Category
HBV alone
HCV alone
HBV/HCV
HBV/HEV
Alcohol
HBV/Alcohol
HCV/Alcohol
AIH
PBC
AIH/PBC
Wilsons disease
NASH-LC
Cryptogenic
Other
Total
(59.3)
(1.0)
(0.5)
(0.1)
(9.9)
(8.5)
(0.3)
(2.0)
(4.4)
(0.3)
(0.4)
(1.9)
(10.6)
(0.8)
(100)
HBV alone: hepatitis B virus was the only etiological factor for cirrhosis;
HCV alone: hepatitis C virus was the only etiological factor for cirrhosis;
HBV/HCV: hepatitis B virus combined with hepatitis C virus; HBV/HEV: hepatitis B virus combined with hepatitis E virus; HBV/Alcohol: hepatitis B virus
combined with alcohol abuse; HCV/Alcohol: hepatitis C virus combined
with alcohol abuse; AIH: autoimmune hepatitis; PBC: primary biliary cirrhosis; AIH/PBC: autoimmune hepatitis combined with primary biliary
cirrhosis.
Statistical analysis
The dichotomous data and continuous data were analyzed
by the chi-square test and Students t test, respectively, using
Statistical Package for the Social Sciences (SPSS, version 15.0;
Chicago, IL, USA). Logistic regression was used to analyze
the selected risk factors for HCC. When examining the
differences between males and females, Fishers exact test
and the Mann-Whitney U test were also used. A p-value of
o0.05 was considered statistically significant.
RESULTS
Etiology of LC
A total of 1,582 patients diagnosed with LC from 20032013 were included in this retrospective study. A total of
1,097 (69.4%) patients were male, 485 (30.6%) were female,
and 87 (5.5%) were diagnosed with HCC during
hospitalization.
In our study, 14 etiological categories were set for LC
(Table 1). The most common etiology was HBV; hepatitis B
surface antigen (HBs Ag) was present in the sera of 1,083
(68.5%) patients. HBV was found to be the only etiological
factor in 938 (59.3%) patients. Hepatitis C virus (HCV)
accounted only for 1.8% of cases (1.0% of patients had HCV
alone, 0.5% had HCV combined with HBV, and 0.3% had
HCV combined with alcohol abuse). Hepatitis E virus
combined with HBV was found in only two patients. In
total, 298 (18.7%) patients had a history of alcohol abuse (135
of them also had HBV infection, and 5 patients also had HCV
infection; alcohol was the dominant factor in 158 patients).
There were 32 patients with autoimmune hepatitis (AIH), 69
patients with primary biliary cirrhosis (PBC), 4 patients
with AIH combined with PBC, and 6 patients with
Wilsons disease. For NASH-related LC, 30 patients (1.9%)
met our criteria. Thirteen patients had other identified etiologies, including intra-hepatic bile-duct stones
(9 patients), Budd-Chiari syndrome (2 patients), parasitic
infection (1 patient), and veno-occlusive disease (1 patient).
NASH-related LC
Among the total of 197 patients with cryptogenic LC, 30
patients met our criteria for NASH-related LC. As shown in
Figure 2, the proportion of NASH-related LC increased in
the last two years of the study to 3.2%, compared with 1.9%
for the whole study period.
564
CLINICS 2015;70(8):563-568
DISCUSSION
Age (years)
Female
Male
Male/female
BMI (Mean SD)
Obesity (%)
DM (%)
Hypertension (%)
HBs Ag+ (%)
HCC (%)
Number of patients
Total
20032008
20082013
p-value#
52.812.8
485
1,097
2.26
22.13.3
229 (14.5)
159 (10.1)
129 (8.2)
1,083 (68.5)
87 (5.5)
1,582
52.312.8
147
344
2.34
22.13.5
71 (14.5)
44 (8.9)
33 (6.7)
325 (66.2)
36 (7.3)
491
52.712.7
338
753
2.23
22.13.2
158 (14.5)
115 (10.5)
96 (8.8)
758 (69.5)
51 (4.7)
1,091
0.519
0.678
0.894
0.991
0.334
0.162
0.193
0.033
BMI: body mass index; DM: diabetes mellitus; HBS Ag+: hepatitis B virus
surface antigen positive; HCC: hepatocellular carcinoma.
#
p-value comparing 20032008 with 20082013.
565
CLINICS 2015;70(8):563-568
prior to the start of the vaccination campaign; the development of cirrhosis usually requires a long period of time (11).
Nevertheless, the efficacy of the Chinese HBV immunization
program is indicated by the decreased prevalence of HBV
carrier status to 2.1% among all children and to 1.0% among
those born after 1999, together with the presence of anti-HBs
antibodies (1,12). Therefore, it is likely that HBV-related
etiology of Chinese LC will be significantly reduced in the
future.
In Western countries, NAFLD is now considered to be the
most common cause of chronic liver disease, with 20-30% of
the total population presenting with NAFLD (13,14). In
China, NAFLD affects approximately 15% of adults and is
present in 68.5% of obese children (15, 16). The proportion of
NASH-related LC (1.9%) in our study was similar to that in
Japan (2.1%) (4). However, our study was retrospective and
may not represent the incidence of LC in the general
population. In our study, an increasing frequency was noted
over the ten years covered by the study; this trend is likely to
result in a significant increase in patients with NASH-related
LC in the future.
A limitation of the current study, especially in the context
of a retrospective study, is that diagnosing LC related to
NASH requires a BMI higher than 25. However, our criteria
were based on those adopted by the Japanese nationwide
Table 3 - Clinical characteristics of NASH-related LC.
Number of patients
Age (years)
BMI (Kg/m2)
Arterial hypertension (%)
DM (%)
Complications
Ascites (%)
HCC (%)
UGB (%)
HRS (%)
HE (%)
Child-Pugh grading
A (%)
B (%)
C (%)
Total
Female (F)
Male (M)
p-value (F vs. M)
30
57.714.1
27.22.1
11 (36.7)
8 (26.7)
11
60.612.4
27.22.2
6 (54.6)
1 (9.1)
19
55.915.0
27.22.0
5 (26.3)
7 (36.8)
0.388a
0.965a
0.238b
0.199b
(40.0)
(0)
(20.0)
(0)
(0)
5 (45.5)
0 (0)
3 (27.3)
0 (0)
0 (0)
7 (36.8)
0 (0)
3 (15.8)
0 (0)
0 (0)
16 (53.3)
12 (40.0)
2 (6.7)
5 (45.5)
4 (36.4)
2 (18.2)
11 (57.9)
8 (42.1)
0 (0)
12
0
6
0
0
0.712b
0.641b
0.308c
BMI: body mass index; DM: diabetes mellitus; HCC: hepatocellular carcinoma; UGB: upper gastrointestinal bleeding; HRS: hepatorenal syndrome; HE: hepatic
encephalopathy.
p-value determined by a: Students t test; b: Fishers exact test; c: the Mann-Whitney U test.
566
CLINICS 2015;70(8):563-568
Age
o65
X65
Sex
Female
Male
Obesity
Obese
Non-obese
Hypertension
Hypertensive
Non-hypertensive
DM
Diabetic
Non-diabetic
HBs Ag
HBs Ag positive
HBs Ag negative
Hepatitis B*
HBV+obesity
HBV+non-obesity
HBV+DM
HBV+non-DM
Alcohol#
HBV+alcohol abuse
HBV+no alcohol abuse
Control
HCC
N=1,495 (%)
N=87 (%)
OR (95% CI)
p-value
1,227 (94.6)
268 (94.0)
70 (5.4)
17 (5.9)
0.93 (0.52-1.64)
0.796
468 (96.3)
1,027 (93.7)
18 (3.7)
69 (6.3)
1.81 (1.04-3.14)
0.036
216 (94.3)
1,279 (94.5)
13 (5.7)
74 (5.5)
1.06 (0.58-1.96)
0.862
123 (95.3)
1,362 (94.4)
6 (4.6)
81 (5.6)
0.90 (0.37-2.21)
0.825
151 (95.0)
1,344 (94.5)
8 (5.0)
79 (5.6)
0.93 (0.43-1.99)
0.849
1,008 (93.1)
487 (98.6)
75 (6.9)
7 (1.4)
2.64 (1.01-6.92)
0.049
11
52
5
58
(8.4)
(6.5)
(7.1)
(6.7)
1.31 (0.66-2.58)
0.436
1.27 (0.53-3.07)
0.589
12 (8.9)
63 (6.7)
0.74 (0.39-1.42)
0.364
121
754
65
810
(91.7)
(93.6)
(92.8)
(93.3)
123 (91.1)
885 (93.4)
Univariate analysis
Multivariate analysis
OR (95% CI)
p-value
3.35 (1.76-6.36)
0.000
* Cirrhotic patients with chronic liver disease caused only by HBV infection (938 patients).
Alcohol abuse in cirrhotic patients with HBs Ag positivity (1,083 patients).
567
CLINICS 2015;70(8):563-568
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of
China (Grant No. 81170382 and 81200297).
AUTHOR CONTRIBUTIONS
Xiong J designed the questionnaire for data collection from the database,
was responsible for the data entry and statistical analysis, and wrote the rst
version of the manuscript. Wang J collected data from the database and
participated in data entry. Huang J collected data from the database and
participated in data entry. Sun W checked the data and collected references
for this manuscript. Wang J revised the manuscript. Chen D participated in
the overall process of writing and revising the manuscript and served as the
corresponding author.
REFERENCES
1. Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, et al. Epidemiological
serosurvey of hepatitis B in China--declining HBV prevalence due to
hepatitis B vaccination. Vaccine. 2009;27(47):6550-7, http://dx.doi.org/
10.1016/j.vaccine.2009.08.048.
2. Schutte K, Kipper M, Kahl S, Bornschein J, Gotze T, Adolf D, et al. Clinical
characteristics and time trends in etiology of hepatocellular cancer in Germany. Digestion. 2013;87(3):147-59, http://dx.doi.org/10.1159/000346743.
3. Nayak NC, Vasdev N, Saigal S, Soin AS. End-stage nonalcoholic fatty
liver disease: evaluation of pathomorphologic features and relationship to
cryptogenic cirrhosis from study of explant livers in a living donor liver
transplant program. Hum Pathol. 2010;41(3):425-30, http://dx.doi.org/
10.1016/j.humpath.2009.06.021.
4. Michitaka K, Nishiguchi S, Aoyagi Y, Hiasa Y, Tokumoto Y, Onji M.
Etiology of liver cirrhosis in Japan: a nationwide survey. J Gastroenterol.
2010;45(1):86-94, http://dx.doi.org/10.1007/s00535-009-0128-5.
5. Schuppan. D, Afdhal. NH. Liver cirrhosis. Lancet. 2008;371(9615):838-51,
http://dx.doi.org/10.1016/S0140-6736(08)60383-9.
6. Fleming KM, Aithal GP, Solaymani-Dodaran M, Card TR, West J. Incidence and prevalence of cirrhosis in the United Kingdom, 19922001: A
general population-based study. J Hepatol. 2008;49(5):732-8, http://dx.
doi.org/10.1016/j.jhep.2008.05.023.
7. Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis
and primary liver cancer worldwide. J Hepatol. 2006;45(4):529-38,
http://dx.doi.org/10.1016/j.jhep.2006.05.013.
8. Huang P, Zhu LG, Zhai XJ, Zhu YF, Yue M, Su J, et al. Hepatitis C
virus infection and risk factors in the general population: a large
community-based study in eastern China, 2011-2012. Epidemiol Infect.
2015;Jan 20:1-10.
9. Qing Wang, Lianyou Hu, Daihong Zhao, Sun M. Seroepidemiological
Survey and Analysis of Virus Hepatitis C in Chongqing. J Prev Med Inf.
2007;23(2):152-4.
568
CLINICAL SCIENCE
University of Malaya, Faculty of Medicine, Department of Medicine, Kuala Lumpur, Malaysia. II University of Malaya, Faculty of Medicine, Department of
Anesthesiology, Kuala Lumpur, Malaysia. III University of Malaya, Faculty of Medicine, Department of Social and Preventive Medicine, Kuala Lumpur,
Malaysia. IV University of Malaya, Faculty of Medicine, Department of Pharmacy, Kuala Lumpur, Malaysia. V University of Malaya, Faculty of Science,
Department of Geology, Kuala Lumpur, Malaysia.
OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis,
mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical
vein endothelial cells.
MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine
and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the
production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity
assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay.
RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and
cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was
significantly (po0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed
significantly reduced apoptosis (po0.005) in the glutamine-treated cells. Moreover, glutamine alone or in
combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular
endothelial growth factor were up-regulated while tumor necrosis factor-a was down-regulated after
treatment with glutamine.
CONCLUSION: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial
stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of
inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner.
KEYWORDS: Hyperglycemia; Sepsis; Apoptosis; Cytokine; Glutamine.
Safi SZ, Batumalaie K, Mansor M, Chinna K, Mohan S, Karimian H, et al. Glutamine treatment attenuates hyperglycemia-induced mitochondrial
stress and apoptosis in umbilical vein endothelial cells. Clinics. 2015;70(8):569-576
Received for publication on April 29, 2015; First review completed on June 9, 2015; Accepted for publication on June 9, 2015
E-mail: safi.nust@yahoo.com
*Corresponding author
INTRODUCTION
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0/)
which permits unrestricted use, distribution, and reproduction in any medium or
format, provided the original work is properly cited.
DOI: 10.6061/clinics/2015(08)07
569
CLINICS 2015;70(8):569-576
divided into three groups. In the first group, 40 mM of glutamine was added. In the second group, 1.0 x 10-6 units/ml of
insulin was added. In the third group, glutamine (40 mM)
and insulin (1.0 x 10-6 units/ml) were added. The cells were
then incubated for the required length of time (24 hours). For
the cytokine and TUNEL analyses, 0.7x106 cells were grown
in T25 flasks using the same treatment groups. The cells were
harvested and frozen until required for analysis.
Western blotting
The endothelial cells were first lysed in cold lysis buffer
containing 20 mmol/l of TRIS HCl, 140 mmol/l of NaCl, 1
mmol/l of EDTA and complete miniprotease inhibitor cocktail, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1
mmol/l NaF, and 1 mmol orthovanadate. The proteins (30 mg)
were then loaded on 10% SDS polyacrylamide gels and
transferred to activated nitrocellulose membranes. The membranes were blocked with Tris-buffered saline (TBS) containing 5% nonfat milk and incubated overnight with the primary
antibodies to IL-10 and TNF-a, obtained from Santa Cruz, at
4 C. Beta-actin was used as a loading control. After extensive
washes in TBS, the membranes were incubated for one hour at
room temperature with the appropriate horseradish peroxidase-conjugated secondary antibodies, and the proteins were
visualized using a chemiluminescence substrate according to
the manufacturers instructions (Amersham Life Sciences).
Cell treatment
The cells were seeded at 1x104 cells in each well and
incubated for 24 hours. Various concentrations of glucose,
ranging from a normal value (5 mM) to a hyperglycemic
level (20 mM), were added to the individual wells. The
hyperglycemic cells (glucose concentration 20 mM) were
570
CLINICS 2015;70(8):569-576
Bio-Plex Cytokine Assay (Bio-Rad Laboratories) was conducted according to the manufacturers protocol. The
calculated concentrations for each cytokine were averaged,
and the standard deviations were determined. Statistical
significance was determined using the t-test, where po0.05
designated increased/decreased cytokine production in the
presence of glutamine or glutamine in combination with
insulin.
DNA damage was investigated using a 96well colorimetric apoptosis detection kit (R&D System) according to the
manufacturers instructions. Umbilical vein endothelial cells
were cultured and transferred to a 96-well plate (1x105 cells/
well). The cells were then fixed with 3.7% buffered
formaldehyde for 5 minutes, followed by washing with
PBS. The washing was followed by permeabilization of the
cells with 100% methanol for 20 minutes and another wash
with PBS. Following the manufacturers protocol, the cells
were then subjected to the labeling procedure, and the
reaction was stopped with 0.2 N HCl after 30 minutes. The
cells were treated with NucleaseTM to generate DNA breaks
and to confirm the permeabilization and labeling reactions.
An unlabeled control was included to indicate the level of
background labeling associated with non-specific binding of
the Strep-HRP. The absorbance at 450 nm was measured
using a microplate reader.
Statistical analysis
Each experiment was performed at least two times.
Statistical analysis was performed using one-way analysis
of variance (ANOVA).
RESULTS
Glutamine reduces the cytochrome C levels and
apoptosis in hyperglycemic human umbilical vein
endothelial cells
Umbilical vein endothelial cells were stained with Hoechst
in the presence of glucose (20 mM) alone, 20 mM glucose +
40 mM glutamine, 20 mM glucose + 10-6 M insulin, or 20
mM glucose + 40 mM glutamine + 10-6 M insulin, and the
staining intensity was determined. As shown in Figures 1
and 2, glucose alone (20 mM) reduced the number of cells,
possibly by apoptosis, as well as reduced the level of
Cytokine measurements
The cytokines TNF-a, IL-6, and IL-10 were measured in
triplicate using the Protein Bio-Plex Cytokine Assay (Bio-Rad
Laboratories). T25 flasks containing 0.7x106 cells were
cultured, and the lysate was filter-sterilized (0.22-mm pore
size). The protein concentrations were determined, and the
Figure 1 - Representative images of endothelial cells treated with medium alone (control), glucose alone (20mM), glucose (20mM) +
insulin (10-6 M), glucose (20mm) + glutamine (40mM) and glucose (20mM) + glutamine (40mM) + insulin (10-6 M). Cells were stained
with Hoechst for nuclear, cell permeability dye, mitochondrial membrane potential dye and cytochrome c. The image from each row
was obtained from the same field of the same treatment sample (magnification 20 xs).
571
CLINICS 2015;70(8):569-576
Figure 2 - Shows (A) TUNEL assay which revealed a significantly reduced apoptosis when hyperglycemic cells were treated with insulin
(po0.05) glutamine (po0.005) and glutamine + insulin (po0.005). (B) Cytochrome c intensity for the endothelial cells treated with
glucose (20 mM) + glutamine (40 mM) significantly changes when compare to glucose (20 mM) alone. Cytochrome c intensity for the
cells treated with glucose (20 mM) + insulin (10-6 M) and glucose (20 mM) + insulin (10-6 M) + glutamine (40 mM) changes
insignificantly as compare to glucose (20 mM) alone. (C) and (D) show mitochondrial membrane potential and cell permeability
respectively.
572
CLINICS 2015;70(8):569-576
Figure 3 - (A) shows that insulin did not alter the expression of IL10 when treated alone but insulin in combination with glutamine
increased IL10 in endothelial cells (po0.05). (B) Shows a significantly reduced TNFa when cells were treated with the same
concentration of glutamine in combination with insulin. (C) IL6 expression which is significantly increased when treated with
glutamine in combination with insulin (po0.005). (D) A mild increase in the expression of VEGF was noted when treated with
glutamine or glutamine in combination with insulin.
573
CLINICS 2015;70(8):569-576
carried out Western blotting for IL-10 and TNF-a (Figure 4).
The levels of both IL-10 and TNF-a changed in the same
manner, as shown in Figure 3.
Hu IL-10
Hu TNF-a
Hu VEGF (45)
Group
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
mM
mM+Ins
mM+Gln
mM+Ins+Gln
mM
mM+Ins
mM+Gln
mM+Ins+Gln
mM
mM+Ins
mM+Gln
mM+Ins+Gln
mM
mM+Ins
mM+Gln
mM+Ins+Gln
Mean
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
55.5
56.2
73.7
91.0
26.3
27.5
32.2
34.8
13.3
12.8
10.7
9.7
22.0
20.7
22.5
24.7
p-value
DISCUSSION
NS
0.035
0.004
NS
NS
0.011
NS
NS
0.006
NS
NS
NS
Figure 4 - (A) shows the effect of insulin and glutamine on the expression of IL10 and TNFa in endothelial cell. (B) shows significantly
reduced IL10 and (C) shows TNFa in graphical form.
574
CLINICS 2015;70(8):569-576
575
CLINICS 2015;70(8):569-576
ACKNOWLEDGEMENTS
This work was supported in part by grants (No. RG074/09AFR, and RG52813HTM (UMRG)) from the University of Malaya. We thank Arokiasamy
Vinsent Rayappan (Department of Medicine, Faculty of Medicine, UM) for
helping in the cell culture work. We declare there is no conict of interest.
AUTHOR CONTRIBUTIONS
Sa SZ performed the basic work and wrote the manuscript. Batumalaie K
helped with the lab work. Karimian H helped with the reagents. Mansor M,
Mohan S, Qvist R, and Yan GOS designed the study and reviewed
the manuscript several times. Chinna K and Ahraf MA helped with the
statistical analysis.
REFERENCES
1. The Diabetes Control and Complications Trial Research Group. The effect of
intensive treatment of diabetes on the development and progression of longterm complications in insulin-dependent diabetes mellitus. N Engl J Med.
1993;329(14):977-86, http://dx.doi.org/10.1056/NEJM199309303291401.
2. Safi SZ, Qvist R, Kumar S, Batumalaie K, Ismail IS. Molecular mechanisms of diabetic retinopathy, general preventive strategies, and novel
therapeutic targets. Biomed Res Int. 2014;2014:801269, http://dx.doi.org/
10.1155/2014/801269.
3. Safi SZ, Qvist R, Yan GO, Ismail IS. Differential expression and role of
hyperglycemia induced oxidative stress in epigenetic regulation of b1, b2
and b3-adrenergic receptors in retinal endothelial cells. BMC Med
Genomics. 2014;7:29, http://dx.doi.org/10.1186/1755-8794-7-29.
4. Ruderman NB, Williamson JR, Brownlee M. Glucose and diabetic vascular disease. FASEB J. 1992;6(11):2905-14.
5. van den Berghe G, Wouters P, Weekers F, Verwaest C, Bruyninckx F,
Schetz M, et al. Intensive insulin therapy in critically ill patients. N Engl J
Med. 2001;345(19):1359-67, http://dx.doi.org/10.1056/NEJMoa011300.
6. Oritz A, Ziyadeh FN, Neilson EG. Expression of apoptosis regulatory
gene in renal proximal epithelial cells exposed to high ambient glucose
and diabetic kidneys. J Investig Med. 1997;45(2):50-6.
7. Safi SZ, Shah H, Siok Yan GO, Qvist R. Insulin resistance provides the
connection between hepatitis C virus and diabetes. Hepat Mon. 2014;15
(1):e23941, http://dx.doi.org/10.5812/hepatmon.
8. Li W, Yanoff M, Liu X, Ye X. Retinal capillary pericyte apoptosis in early
human diabetic retinopathy. Chin Med J (Engl). 1997;110(9):659-63.
9. Kern TS, Tang J, Mizutani M, Kowluru RA, Nagaraj RH, Romeo G et al.
Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia. Invest
Ophthalmol Vis Sci. 2000;41(12):3972-8.
10. Fiordaliso F, Li B, Latini R, Sonnenblick EH, Anversa P, Leri A et al.
Myocyte death in streptozotocin-induced diabetes in rats is angiotensin IIdependent. Lab Invest. 2000;80(4):513-27, http://dx.doi.org/10.1038/
labinvest.3780057.
11. Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A, Maseri A et al.
Myocardial cell death in human diabetes. Circ Res. 2000;87(12):1123-32,
http://dx.doi.org/10.1161/01.RES.87.12.1123.
12. Marik PE, Varon J. Sepsis: State of the art. Dis Mon. 2001; 47:463-532,
http://dx.doi.org/10.1067/mda.2001.119745.
13. Szabo G, Mandrekar P, Dolganiuc A. Innate immune response and hepatic
inflammation. Semin Liver Dis. 2007;27(4):339-50, http://dx.doi.org/
10.1055/s-2007-991511.
14. Barnes PJ, Karin M. Nuclear FactorKb-A pivotal transcription Factor in
chronic inflammatory diseases. N Engl J Med. 1997;336(15):1066-71,
http://dx.doi.org/10.1056/NEJM199704103361506.
15. Peters K, Unger RE, Brunner J, Kirkpatrick CJ. Molecular basis
of endothelial dysfunction in sepsis. Cardiovasc Res. 2003;60(1):49-57,
http://dx.doi.org/10.1016/S0008-6363(03)00397-3.
16. Frey EA, Finlay BB. Lipopolysaccharide induces apoptosis in a bovine
endothelial cell line via a soluble CD14 dependent pathway. Microb
Pathog. 1998;24(2):101-9, http://dx.doi.org/10.1006/mpat.1997.0178.
17. Robaye B, Mosselmans R, Fiers W, Dumont JE, Galand P. Tumour necrosis
factor induces apoptosis (Programmed cell death) in normal endothelial
cells in vitro. Am J. Pathol. 1991; 138(2):447-53.
18. Heike B and Klaus SO. Cell death in sepsis: a matter of how, when, and
where? Critical Care. 2009;13(4):173, http://dx.doi.org/10.1186/cc7966.
19. Bone RC, Grodzin CJ, Balk RA. Sepsis: a new hypothesis for pathogenesis
of the disease process. Chest. 1997;112(1):235-43, http://dx.doi.org/
10.1378/chest.112.1.235.
20. Garrabou G, Morn C, Lpez S, Tobas E, Cardellach F, Mir O, et al. The
Effects of Sepsis on Mitochondria. J Infect Dis. 2012;205(3):392-400,
http://dx.doi.org/10.1093/infdis/jir764.
576
BASIC RESEARCH
Faculdade de Medicina da Universidade de Sao Paulo, Laboratory of Anesthesiology (LIM-08), Sao Paulo/SP, Brazil. II Faculdade de Medicina Veterinaria e
Zootecnia da Universidade de Sao Paulo, Department of Pathology. III Department of Surgery, Sao Paulo/SP, Brazil. IV Instituto do Coracao do Hospital das
Clnicas da Faculdade de Medicina da Universidade de Sao Paulo, Laboratory of Pathology, Sao Paulo/SP, Brazil.
OBJECTIVES: Hypertonic saline has been proposed to modulate the inflammatory cascade in certain
experimental conditions, including pulmonary inflammation caused by inhaled gastric contents. The present
study aimed to assess the potential anti-inflammatory effects of administering a single intravenous dose of
7.5% hypertonic saline in an experimental model of acute lung injury induced by hydrochloric acid.
METHODS: Thirty-two pigs were anesthetized and randomly allocated into the following four groups: Sham,
which received anesthesia and were observed; HS, which received intravenous 7.5% hypertonic saline solution
(4 ml/kg); acute lung injury, which were subjected to acute lung injury with intratracheal hydrochloric acid; and
acute lung injury + hypertonic saline, which were subjected to acute lung injury with hydrochloric acid and
treated with hypertonic saline. Hemodynamic and ventilatory parameters were recorded over four hours.
Subsequently, bronchoalveolar lavage samples were collected at the end of the observation period to measure
cytokine levels using an oxidative burst analysis, and lung tissue was collected for a histological analysis.
RESULTS: Hydrochloric acid instillation caused marked changes in respiratory mechanics as well as blood gas and
lung parenchyma parameters. Despite the absence of a significant difference between the acute lung injury and
acute lung injury + hypertonic saline groups, the acute lung injury animals presented higher neutrophil and
tumor necrosis factor alpha (TNF-a), interleukin (IL)-6 and IL-8 levels in the bronchoalveolar lavage analysis. The
histopathological analysis revealed pulmonary edema, congestion and alveolar collapse in both groups;
however, the differences between groups were not significant. Despite the lower cytokine and neutrophil levels
observed in the acute lung injury + hypertonic saline group, significant differences were not observed among
the treated and non-treated groups.
CONCLUSIONS: Hypertonic saline infusion after intratracheal hydrochloric acid instillation does not have an
effect on inflammatory biomarkers or respiratory gas exchange.
KEYWORDS: Acute lung injury; Hypertonic saline; Pigs; Hydrochloric acid; Inflammation.
Holms CA, Otsuki DA, Kahvegian M, Massoco CO, Fantoni DT, Gutierrez OS, et al. Effect of hypertonic saline treatment on the inflammatory
response after hydrochloric acid-induced lung injury in pigs. Clinics. 2015;70(8):577-583
Received for publication on May 5, 2015; First review completed on May 19, 2015; Accepted for publication on May 19, 2015
E-mail: lim08@usp.br
*Corresponding author
INTRODUCTION
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/
4.0/) which permits unrestricted use, distribution, and reproduction in any
medium or format, provided the original work is properly cited.
DOI: 10.6061/clinics/2015(08)08
577
CLINICS 2015;70(8):577-583
METHODS
Study design
After preparation, all of the animals were submitted to
three series of recruitment maneuvers consisting of 30 s of
sustained inflation with 20 cmH2O pressure followed by 30 s
of regular ventilation. The animals were allowed to stabilize
for 30 min and then randomly assigned to four groups: Sham
(n=8), hypertonic saline (HS; n=8), acute lung injury (ALI;
n=8) and acute lung injury + hypertonic saline (ALI+HS;
n=8). ALI was induced in the ALI and ALI+HS groups via
intratracheal instillation of HCl through the distal port of a
bronchoscope. The ALI+HS animals were treated with 7.5%
hypertonic saline (4 mg/kg) 15 min after HCl instillation.
The Sham and HS groups served as controls. The animals
from the HS group were administered 7.5% hypertonic saline
(4 ml/kg) 15 min after the baseline measurement.
The experimental protocol is outlined in Figure 1.
Collecting points
Following a 30-min stabilization period, baseline (BL)
measurements were performed. ALI was induced immediately after BL measurements in the ALI and ALI+HS
groups. A new series of measurements was collected 30 min
after the administration of HCl (T30) and hourly thereafter
(T90, T150, T210, T270).
Blood samples were collected at the above time points, and
bronchoalveolar lavage (BAL) sampling was performed with
3 x 20 ml phosphate buffered saline (PBS) at T270 through
bronchoscopy of the upper right lobe of the lung.
Monitoring
An arterial catheter was inserted into the right femoral
artery, and a 7.5 French pulmonary artery catheter that
measured continuous cardiac output (Edwards Lifesciences
Corp., Irvine, CA) was inserted into the right jugular vein.
The heart rate (HR), mean arterial pressure (MAP), mean
pulmonary artery pressure (MPAP), pulmonary artery
wedge pressure (PAWP) and central venous pressure (CVP)
were obtained directly using a multiparametric monitor
(IntelliVue MP40, Phillips, Bblinger, Germany). Using
578
CLINICS 2015;70(8):577-583
Table 1 - Hemodynamics and respiratory variables in the control (SHAM and hypertonic saline) and acid lesion (acute lung injury and
acute lung injury + hypertonic saline) groups.
Variable
2
CI (L/min/m )
HR (bpm)
MAP (mmHg)
MPAP (mmHg)
CVP (mmHg)
PVRI (dyne.sec.cm
SVRI (dyne.sec.cm
PPlat (cmH2O)
Compl (cmH2O)
.m2)
.m2)
Group
BL
T30
T90
T150
T210
T270
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
SHAM
HS
ALI
ALI+HS
3.60.6
3.70.3
3.90.6
3.50.3
948
10415
10613
9212
547
673
708
663
142
162
162
143
93
102
101
91
13120
12815
15231
13735
1317188
1185124
1228152
1266175
131
141
141
141
314.1
294.5
284.2
284.2
3.70.5
4.10.4
4.20.9
4.30.4*
9711
10511
12415
11413
596*
724*
8016*
8317*
162
161
253*w#
265*w#
112
111
9 1
111
12918
11930
331114*w#
364127*w#
1249138
1178140
1343335
1349291
141*
141,8
231.3*w#
231.6*w#
304.5
284.6
141.4*w#
132*w#
4.00.5
4.90.4*w
4.40.9
4.80.5*
10119
11515
11219
12021
6910*
8410*
808*
8311*
153
191*
193*w#
213*w#
103
111
101
91
12116
13040
22551*w#
20757*w#
1348158
1157202
1317354
1222254
141.7*
141.8*
211.5*w#
211.7*w#
294
263.8*
162*w#
151.6*w#
4.30.5
4.90.6*
4.40.8
4.80.4*
10415
12918
12231
12112
6612*
8112*
849*
8511*
153
166
223*w#
243*w#
103*
101*
101*
101*
13130
13027
26677*w#
26653*w#
1302236
1115200
1372320
1247280
141.6*
151.6*
211.7*w#
211.6*w#
294.3*
263.8*
161.9*w#
151.7*w#
4.30.7
4.60.7*
4.20.7
4.80.4*
10721
12218
11930
12417
619*
769*
828*
8512*
163
182**
213*w#
244*w#
102*
102*
101*
101*
13228
13231
23058*w#
26666*w#
1362202
1116228
1419352
1251274
141.6*
151.9*
211.8*w#
211.2*w#
284*
254.4*
161.5*w#
151.7*w#
4.30.7
4.50.7*
4.20.5
4.60.5*
10828
11710
11921
12119
618*
777*
815*
828*
152
192*
224*w#
233*w#
102
102
101
101
13130
14928*
29051*w#
26358*w#
1274111
1172272
1375187
1241269
141.6*
152*
211.7*w#
211.2*w#
283.6*
244.3*
162.5*w#
152*w#
Data are expressed as the meanstandard deviation. CI: cardiac index; HR: heart rate; MAP: mean arterial pressure; MPAP: mean pulmonary arterial
pressure; CVP: central venous pressure; PVRI: pulmonary vascular resistance index; SVRI: systemic vascular resistance index; PPlat: plateau pressure; Compl:
pulmonary compliance; * po0.05 compared with the baseline; w po0.01 compared with the Sham group; # po0.05 compared with the HS group.
Lung histology
At the end of the study, the trachea was clamped at the end of
the inspiratory cycle, and the animals thorax was opened. Four
samples were collected from the middle area of the left apical
(Wests zone 2) and diaphragmatic lung lobes as well as from
the middle of the right apical and diaphragmatic lung lobes. The
samples were fixed in a 10% formaldehyde solution for
subsequent histologic analysis. The tissue samples were
embedded in paraffin, and 5 mm histological sections were
stained with hematoxylin and eosin. Two pathologists who
were blinded to the study groups performed the histological
analyses simultaneously. Examinations included testing for the
presence of edema, intra-alveolar and interstitial hemorrhages
and polymorphonuclear and mononuclear cell infiltration.
Each assessed histological characteristic was attributed a
score from 0 to 3 according to the level observed in the tissue
(absent (0), mild (1), moderate (2) or severe (3)). The final score
for the animal was determined according to the sum of the
scores from each lobe (maximum score 12).
Cytokine measurements
BAL samples (10 ml) were centrifuged (2,000 rpm, 10 min,
4 C), and the supernatant was stored at -80 C for subsequent
analysis. BAL cytokine levels (interleukin (IL)-1, IL-8, IL-10, and
tumor necrosis factor alpha (TNF-a)) were assessed using
commercially available immunoenzymatic assay (ELISA) kits
579
CLINICS 2015;70(8):577-583
Statistical analysis
RESULTS
The body weights of the animals did not differ between
groups. In addition, the administered amount of infused
7.5% hypertonic saline (HS: 17116 ml; ALI+ HS: 11211 ml)
and HCl (ALI: 11912 ml; ALI + HS: 11212 ml) were also
similar among the groups.
Histological analysis
The score for histological injury was significantly higher in
the ALI and ALI+HS groups compared with the Sham and
HS groups (Figure 4).
The pattern of lung injury observed in the ALI and ALI+
HS groups was heterogeneous and more evident in the
diaphragmatic lobes. Examination of these lung tissues
revealed large areas of alveolar architecture destruction,
hemorrhage, edema and inflammatory polymorphonuclear
and mononuclear cell infiltration (Figure 5). However,
significant differences were not observed between the scores
exhibited by animals in the ALI and ALI+HS groups.
DISCUSSION
In the present study, we demonstrated that hypertonic
saline infused after intratracheal HCl instillation attenuated
increases in BAL neutrophil counts and inflammatory
cytokine concentrations. HCl instillation alone induced a
severe direct lung injury as evidenced by an intense
inflammatory reaction observed in the lung histology, BAL
cytokine levels and oxidative burst. Lung function was also
adversely affected, which was indicated by decreased gas
exchange and reduced lung compliance.
Previous studies have attributed beneficial effects to hypertonic saline in a number of ALI models, such as oleic acid and
ischemia/reperfusion-induced lung injury (7,18,19). The use of
hypertonic saline solution has also demonstrated potential antiinflammatory effects related to neutrophil activation (20) in cell
cultures as well as in experimental models of sepsis and
hemorrhagic shock (21-23). Hypertonic saline solution acts on
polymorphonuclear A2 adenosine receptors and causes a
feedback mechanism that stimulates cAMP and PKA release,
thus blocking neutrophil activation (21-23).
It is believed that hyperosmolar solutions can also
decrease pulmonary vascular permeability and leukocyte
adhesion molecule expression, especially P-selectin and
L-selectin. This expression hinders neutrophil adhesion to the
endothelium and may result in reduced lung injury (24,25).
Contrary to the results obtained in studies performed in a
HCl-induced lung injury model (8) and an experimental oleic
acid-induced lung injury model (7), which demonstrated that
pulmonary edema decreased in rats treated with 7.5%
hypertonic saline, our histopathological results did not show
a significant differences between the ALI and ALI+HS
groups with regard to the investigated parameters. However,
the ALI+HS group tended to show lower histopathological
scores relative to the ALI group, although this difference was
not significant. Regarding the control groups, significant
differences were not observed between the HS and Sham
groups, which demonstrates that the administration of 7.5%
580
CLINICS 2015;70(8):577-583
Figure 3 - Bronchoalveolar lavage cytokines. The values are expressed in pg/ml. Data are presented as the group meanSD. w: po0.05
compared with the Sham group; #: po0.05 compared with the hypertonic saline group; y: po0.05 compared with the acute lung injury group.
Figure 4 - Scores for histological injury of the lungs. w: po0.05 compared with the Sham group; #: po0.05 compared with the
hypertonic saline group.
581
CLINICS 2015;70(8):577-583
Figure 5 - Representative photomicrographs with hematoxylin and eosin (H&E) staining (x200) of the lungs of pigs submitted to acute
lung injury. A) Sham group. B) hypertonic saline group. C) acute lung injury group. D) acute lung injury + hypertonic saline group.
Extensive alveolar and interstitial inflammatory infiltration was seen in both injury groups.
582
CLINICS 2015;70(8):577-583
ACKNOWLEDGMENTS
AUTHOR CONTRIBUTIONS
Holms CA conducted the study and data analysis. Otsuki DA helped design
the study, conduct the study, collect and analyze data and prepare the
manuscript. Kahvegian M helped conduct the study. Massoco CO helped
conduct the study and data analysis. Fantoni DT designed the study and
helped analyze the data. Gutierrez PS performed the histological analysis.
Auler Jr JO helped design the study and prepare the manuscript. All of the
authors read and approved the nal manuscript.
*Presented in part at the 30th International Symposium on Intensive Care
and Emergency Medicine, 2010, Brussels.
REFERENCES
1. Wheeler AP, Bernard GR. Acute lung injury and the acute respiratory
distress syndrome: a clinical review. Lancet. 2007;369(9572):1553-64,
http://dx.doi.org/10.1016/S0140-6736(07)60604-7.
2. Estenssoro E, Dubin A, Laffaire E, Canales H, Saenz G, Moseinco M, et al.
Incidence, clinical course, and outcome in 217 patients with acute
respiratory distress syndrome. Crit Care Med. 2002;30(11):2450-6,
http://dx.doi.org/10.1097/00003246-200211000-00008.
3. Ng A, Smith G. Gastroesophageal reflux and aspiration of gastric contents
in anesthetic practice. Anesth Analg. 2001;93(2):494-513.
4. Marik PE. Aspiration pneumonitis and aspiration pneumonia. N Engl J Med.
2001;344(9):665-71, http://dx.doi.org/10.1056/NEJM200103013440908.
5. Barbas CS, Matos GF, Amato MB, Carvalho CR. Goal-oriented respiratory
management for critically ill patients with acute respiratory distress
syndrome. Crit Care Res Pract. 2012;2012:952168.
6. Meers CM, De Wever W, Verbeken E, Mertens V, Wauters S, De Vleeschauwer SI, et al. A porcine model of acute lung injury by instillation of
gastric fluid. J Surg Res. 2011;166(2):e195-204, http://dx.doi.org/10.1016/
j.jss.2010.10.015.
7. Kennedy MT, Higgins BD, Costello JF, Curtin WA, Laffey JG. Hypertonic
saline reduces inflammation and enhances the resolution of oleic acid
induced acute lung injury. BMC Pulm Med. 2008;8:9, http://dx.doi.org/
10.1186/1471-2466-8-9.
8. Rabinovici R, Vernick J, Hillegas L, Neville LF. Hypertonic saline treatment of acid aspiration-induced lung injury. J Surg Res. 1996;60(1):176-80,
http://dx.doi.org/10.1006/jsre.1996.0028.
9. Safdar Z, Yiming M, Grunig G, Bhattacharya J. Inhibition of acid-induced
lung injury by hyperosmolar sucrose in rats. Am J Respir Crit Care Med.
2005;172(8):1002-7, http://dx.doi.org/10.1164/rccm.200501-005OC.
10. Inoue Y, Chen Y, Pauzenberger R, Hirsh MI, Junger WG. Hypertonic saline
up-regulates A3 adenosine receptor expression of activated neutrophils and
increases acute lung injury after sepsis. Crit Care Med. 2008;36(9):2569-75,
http://dx.doi.org/10.1097/CCM.0b013e3181841a91.
11. Chen Y, Hashiguchi N, Yip L, Junger WG. Hypertonic saline enhances
neutrophil elastase release through activation of P2 and A3 receptors. Am
J Physiol Cell Physiol. 2006;290(4):C1051-9, http://dx.doi.org/10.1152/
ajpcell.00216.2005.
12. Martin TR, Matute-Bello G. Experimental models and emerging
hypotheses for acute lung injury. Crit Care Clin. 2011;27(3):735-52,
http://dx.doi.org/10.1016/j.ccc.2011.05.013.
13. Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung
injury. Am J Physiol Lung Cell Mol Physiol. 2008;295(3):L379-99,
http://dx.doi.org/10.1152/ajplung.00010.2008.
583
REVIEW
Literature searches of the Cochrane Library, PubMed, EMBASE, Web of Science, LILACS, China National Knowledge
Infrastructure, and Wanfang Data databases were performed from 1966 to September 2014. Only randomized
and quasi-randomized controlled clinical trials comparing operative and nonoperative treatments for displaced
midshaft clavicle fractures were included. Data collection and extraction, quality assessment, and data analyses
were performed according to the Cochrane standards. Thirteen studies were considered in the meta-analysis.
Constant scores and the Disabilities of the Arm, Shoulder and Hand scores were improved in the operative fixation
group at a follow up of one year or more. The nonunion and symptomatic malunion rates were significantly
lower in the operative group. Additionally, the nonoperative group had a higher likelihood of neurological
symptoms compared with the operative group. A significantly higher risk of complications was found in patients
treated conservatively than in those who underwent operative fixation. However, when patients with nonunion
and symptomatic malunion were excluded from the analysis, no significant differences in the complication rate
were found. We concluded that based on the current clinical reports, operative treatment is superior to
nonoperative treatment in the management of displaced midshaft clavicle fractures. However, we do not support
the routine use of primary operative fixation for all displaced midshaft clavicle fractures in adults.
KEYWORDS: Clavicle; Midshaft clavicles; Operative treatment; Nonoperative treatment; Meta-analysis.
Wang XH, Guo WJ, Li AB, Cheng GJ, Lei T, Zhao YM Operative versus nonoperative treatment for displaced midshaft clavicle fractures: a meta-analysis
based on current evidence. Clinics. 2015;70(8):584-592
Received for publication on February 04, 2015; First review completed on March 23, 2015; Accepted for publication on April 30, 2015
E-mail: wzmuorthopaedic@sina.com
*Corresponding author
INTRODUCTION
A few meta-analyses comparing operative versus nonoperative approaches for the treatment of midshaft clavicle
fractures have been published in recent years (11,12), but the
results were inconclusive due to the relatively small sample
size in each published study. However, because several
relevant studies have been published on this topic in recent
years, the present meta-analysis is more precise.
The purpose of the present systematic review and metaanalysis was to determine the effectiveness of operative
versus nonoperative treatment for displaced midshaft
clavicle fractures by comparing the clinical results reported
in all of the available related evidence.
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0/)
which permits unrestricted use, distribution, and reproduction in any medium or
format, provided the original work is properly cited.
DOI: 10.6061/clinics/2015(08)09
584
CLINICS 2015;70(8):584-592
Critical Appraisal
RESULTS
Characteristics of the eligible studies
Details of the literature search are presented in a flow
diagram (Figure 1). Thirteen studies with relatively low
quality were included in the final analysis. Among them, the
report by Smith et al. (17) was an abstract that met the
inclusion criteria, and the sample sizes of the studies ranged
from 40 to 178 patients. Information on the general
characteristics, participants, and methodological quality of
the 13 studies is summarized in Table 1. Of a total of 959
included patients, 507 were treated with operative
approaches, and the others were treated with conservative
approaches. Allocation concealment was reported in 8 trials
(18-25) and was not stated in the other trials. Blinding was
rarely used in the included studies; only one study by
Robinson et al. (23) was blinded in the functional assessment.
Data extraction
Statistical analysis
Estimates of the treatment effect were expressed as risk
ratios (RRs) for dichotomous outcomes and weighted mean
differences (WMDs) for continuous outcomes, both with 95%
confidence intervals (CIs). For studies that did not present
standard deviations, the standard deviations were calculated
from the P-value or CI following the guidance of the
Cochrane Handbook for Systematic Reviews of Interventions
(13). Homogeneity across the studies was assessed with a
chi-square analysis, considering po0.10 significant. A fixed
effects model was used when the heterogeneity was not
significant, and a random effects model was adopted if
significant heterogeneity was present. A sensitivity analysis
was performed by omitting one study each time to explore
potential sources of heterogeneity and to test the stability of
pooled results. Publication bias was observed with the funnel
plot. Review Manager (RevMan) software (Version 5.3.5. The
Nordic Cochrane Centre, Copenhagen, Denmark) (15),
provided by The Cochrane Collaboration, was used for
graphical representation of the pooled data.
Functional outcomes
Nine studies (18,10-23,25,27-29) reported Constant scores
(eight at a follow up of one year or more and one at a follow
up of 6 months); the Constant scores of the operative group
were higher than those of the nonoperative group. Three
(27-29) of the nine studies were excluded from the analysis
due to a lack of data on the standard deviation or to
insufficient follow-up. The test for heterogeneity was
significant (Chi2=14.13, df=5, I2=65%, p=0.01). Using the
random effects model, the aggregated results suggested that
the Constant score was significantly higher in the operative
group compared with the nonoperative group (WMD 4.74,
95%CI, 2.457.03, po0.0001) (Figure 3A). Subsequently, we
performed a sensitivity analysis to explore potential sources
of heterogeneity. Exclusion of the trial conducted by
585
CLINICS 2015;70(8):584-592
Complications
Because the definition of complications varied in all of the
studies, we defined complications as all adverse events that
were reported in those trials: nonunion (usually defined as no
evidence of healing at fifty-two weeks after injury), delayed
union (no evidence of healing at twenty-four weeks after
injury), symptomatic malunion, infection, hardware removal,
neurological symptoms, and refracture, among others.
In an overall analysis of the 13 selected studies, significant heterogeneity (Chi2=22.50, df=12, I2=47%, p=0.03) was
detected among these studies. Sensitivity analysis found that
the study reported by Judd et al. (19) was the source of
heterogeneity, probably owing to a high rate of hardwarerelated complications associated with the use of Hagie pins
in this study. Thus, the random effects model was applied. A
significantly higher risk of complications was found in
patients treated conservatively than in those who underwent
Smith (2001)
Jubel (2005)
COTS (2007)
Figueiredo (2008)
Judd (2009)
Smekal (2009)
Bohme (2011)
Chen (2011)
Mirzatolooei (2011)
Kulshrestha (2011)
Virtanen (2012)
Robinson (2013)
Mohsen (2014)
Design
Range of Ages
(years)
Follow-up
(months)
Internal
Fixation
Nonoperative
Treatment
Jadad
Score
RCT
QRCT
RCT
RCT
RCT
RCT
QRCT
RCT
RCT
QRCT
RCT
RCT
QRCT
30/35
26/27
62/49
24/16
29/28
30/30
58/38
30/30
26/24
45/28
26/25
86/92
35/30
Adults
Adults
1660
18-58
1740
1865
18-70
18-63
1865
20-50
1870
1660
18-60
12
6
12
12
12
24
8
15
12
18
12
12
6
Plate
Nail
Plate
Plate
Nail
Nail
Plate/Nail
Nail
Plate
Plate
Plate
Plate
Plate
Sling
Bandage
Sling
Sling
Sling
Sling
Bandage
Sling
Sling
Sling
Sling
CollarCuff
Bandage
3
1
3
3
3
3
1
3
3
1
3
4
1
O/N: operative group/nonoperative group, RCT: randomized controlled trial, QRCT: quasi-randomized controlled trial.
586
CLINICS 2015;70(8):584-592
Figure 2 - Forest plot showing comparison of nonunion rate (A) and symptomatic malunion rate (B) between operative (experimental)
and nonoperative (control) groups.
Neurologic symptoms
Nine studies reported neurological symptoms (17-22,25,
27,28). Pooled data showed that the operative group had a
significantly lower likelihood of developing neurological
symptoms compared with the nonoperative group (RR 0.40,
95%CI 0.230.70, p=0.001). No significant heterogeneity was
587
CLINICS 2015;70(8):584-592
Figure 3 - Forest plot showing comparison of Constant scores (A) and DASH scores (B) between operative (experimental) and
nonoperative (control) groups.
Publication bias
Publication bias was assessed by comparing the WMDs of
nonunion; no evidence of publication bias was detected
(Figure 5).
DISCUSSION
Our meta-analysis revealed that primary operative fixation
could effectively reduce the rates of nonunion, symptomatic
malunion, neurological symptoms and overall complications. In addition, DASH and Constant scores were
significantly improved after operative fixation compared
with nonoperative treatment after a follow up of one year or
more. Based on current clinical reports, we conclude that
operative treatment is superior to nonoperative treatment in
the management of displaced midshaft clavicle fractures.
Pooled data showed that 14% of 452 patients in the
nonoperative group developed a nonunion, which is
significantly higher (p=0.00001) than the 1.7% rate of
nonunion in the 507 patients of the operative group.
Symptomatic malunion was also significantly more common
in the nonoperative group (20% in the nonoperative group
versus 1.8% in the operative group, po0.00001). However,
with the data available, we were not able to draw any
588
CLINICS 2015;70(8):584-592
Figure 4 - Forest plot showing comparison of complications rates (A and B) and neurologic symptoms rates (C) between operative
(experimental) and nonoperative (control) groups.
implants or improved surgical techniques. The predominant complications in the nonoperative group were nonunion, neurological symptoms (including brachial plexus
irritation and compression) and symptomatic malunion;
however, most of those complications require operative
intervention.
589
CLINICS 2015;70(8):584-592
Although modern plate fixation techniques provide reliable healing rates, the optimal plate position and type remain
controversial. The clavicle contour and anatomy are curved
in multiple planes. The reconstruction plate is easier to
contour in all planes than the stiffer dynamic compression
plates (DCP), which allow bending only along the length of
the plate. For superior plating, a reconstruction plate or
precontoured plate can more precisely fit the S-shaped
anatomy. For anteroinferior plating, DCPs can be bent to
conform to the anatomy very well (39). Regarding stability,
two biomechanical studies have found greater stability with
compression plates compared with reconstruction plates
(31,32). In addition, a finite element study showed that
anteroinferior plating best resists the effects of most daily
living forces that act on the clavicle and can be considered
more mechanically physiological (33).
Will et al. (34) suggested that locked compression plates
(LCPs) provided more stiffness and less deflection than lowcontact dynamic compression plates (LC-DCPs). Using a
simulated segmental clavicle fracture model, another biomechanical study by Iannotti et al. (35) reported that LCDCPs offer significantly greater biomechanical stability than
reconstruction plates and DCPs and that clavicles plated at
the superior aspect exhibited significantly greater biomechanical stability than those plated at the anterior aspect.
However, most of the biomechanical studies must be
interpreted with caution because such testing can offer clean
comparisons of instrumentation and technique without
the confounding factors of patient and surgeon variations.
Nine of the 13 studies included in this review used plate
fixation; among them, three studies used reconstruction
590
CLINICS 2015;70(8):584-592
AUTHOR CONTRIBUTIONS
Wang XH conceived the study, collected the data, participated in the analysis
of samples, drafted the manuscript and performed the statistical analysis. Guo
WJ conceived the study and participated in its design, coordination and
drafting. Li AB participated in the analysis and interpretation of samples and
in the language translation of non-English studies. Cheng GJ participated in
the language translation of non-English studies. Lei T participated in the
revision of the manuscript. Zhao YM participated in the review, revision,
coordination and drafting of the manuscript and performed the analysis with
constructive discussions. The aim of this article was to identify the effects of
operative versus nonoperative treatment in the management of displaced
midshaft clavicle fractures in adults.
REFERENCES
1. Neer C. Fractures of the clavicle. In: Rockwood CA Jr, Green DP, editors.
Fractures in adults. 2nd ed. Philadelphia: Lippincottp. 1984;707-13.
2. Neer CS II. Fractures of the distal third of the clavicle. Clin Orthop Relat
Res. 1968;58:43-50.
3. Van der Meijden OA, Gaskill TR, Millett PJ. Treatment of clavicle fractures: current concepts review. J Shoulder Elbow Surg. 2012;21(3):423-9,
http://dx.doi.org/10.1016/j.jse.2011.08.053.
4. Sankarankutty M, Turner BW. Fractures of the clavicle. Injury. 1975;
7(2):101-6, http://dx.doi.org/10.1016/0020-1383(75)90006-6.
5. Rowe CR. An atlas of anatomy and treatment of midclavicular fractures.
Clin Orthop Relate Res. 1968;(58):29-42.
6. Andersen K, Jensen PO, Lauritzen J. Treatment of clavicular fractures.
Figure-of eight bandage versus a simple sling. Acta Orthop Scand. 1987;
58(1):71-4, http://dx.doi.org/10.3109/17453678709146346.
7. Eskola A, Vainionp S, Myllynen P, Ptil H, Rokkanen P. Outcome of
clavicular fracture in 89 patients. Arch Orthop Trauma Surg. 1986;
105(6):337-8, http://dx.doi.org/10.1007/BF00449938.
8. Nordqvist A, Petersson CJ, Redlund-Johnell I. Mid-clavicle fractures in
adults: end result study after conservative treatment. J Orthop Trauma.
1998;2(8):572-6, http://dx.doi.org/10.1097/00005131-199811000-00008.
9. Hill JM, McGuire MH, Crosby LA. Closed treatment of displaced middlethird fractures of the clavicle gives poor results. J Bone Joint Surg Br.
1997;79(4):537-9, http://dx.doi.org/10.1302/0301-620X.79B4.7529.
10. McKee MD, Pedersen EM, Jones C, Stephen DJ, Kreder HJ, Schemitsch
EH, et al. Deficits following nonoperative treatment of displaced midshaft
591
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
CLINICS 2015;70(8):584-592
30. Wijdicks FJG, Van der Meijden OAJ, Millett PJ, Verleisdonk E, Houwert
RM. Systematic review of the complications of plate fixation of clavicle
fractures. Arch Orthop Trauma Surg. 2012;132(5):617-25, http://dx.doi.
org/10.1007/s00402-011-1456-5.
31. Eden L, Doht S, Frey SP, Ziegler D, Stoyhe J, Fehske K, et al. Meffert RH:
Biomechanical comparison of the locking compression superior anterior
clavicle plate with seven and ten hole reconstruction plates in midshaft
clavicle fracture stabilisation Int Orthop. 2012;36(12):2537-43.
32. Drosdowech DS, Manwell SEE, Ferreira LM, Goel DP, Faber KJ, Johnson
JA. Biomechanical analysis of fixation of middle third fractures of the
clavicle. J Orthop Trauma. 2011;25(1):39-43, http://dx.doi.org/10.1097/
BOT.0b013e3181d8893a.
33. Favre P, Kloen P, Helfet DL, Werner CML: Superior versus anteroinferior
plating of the clavicle: a finite element study. J Orthop Trauma. 2011;
25(11):661-5.
34. Will R, Englund R, Lubahn J, Cooney TE. Locking plates have increased
torsional stiffness compared to standard plates in a segmental defect
model of clavicle fracture. Arch Orthop Trauma Surg. 2011;131(6):841-7,
http://dx.doi.org/10.1007/s00402-010-1240-y.
35. Iannotti MR, Crosby LA, Stafford P, Grayson G, Goulet R. Effects of plate
location and selection on the stability of midshaft clavicle osteotomies: A
biomechanical study. J Shoulder Elbow Surg. 2002;11(5):457-62, http://dx.
doi.org/10.1067/mse.2002.125805.
36. Collinge C, Devinney S, Herscovici D, DiPasquale T, Sanders R. Anteriorinferior plate fixation of middle-third fractures and nonunions of the
clavicle. J OrthopTrauma 2006;20(10):680-6.
37. Kloen P, Werner CML, Stufkens SAS, Helfet DL. Anteroinferior plating of
midshaft clavicle nonunions and fractures. Oper Orthop Traumatol.
2009;21(2):170-9, http://dx.doi.org/10.1007/s00064-009-1705-8.
38. Galdi B, Yoon RS, Choung EW, Reilly MC, Sirkin M, Smith WR, et al.
Anteroinferior 2.7-mm versus 3.5-mm plating for AO/OTA type B clavicle
fractures: a comparative cohort clinical outcomes study. J Orthop Trauma.
2013;27(3):121-5, http://dx.doi.org/10.1097/BOT.0b013e3182693f32.
39. Gilde AK, Jones CB, Sietsema DL, Hoffmann MF. Does plate type influence
the clinical outcomes and implant removal in midclavicular fractures fixed
with 2.7-mm anteroinferior plates? A retrospective cohort study. J Orthop
Surg Res. 2014;9:55, http://dx.doi.org/10.1186/s13018-014-0055-x.
40. Golish SR, Oliviero JA, Francke EI, Miller MD. A biomechanical study of
plate versus intramedullary devices for midshaft clavicle fixation. J Orthop
Surg Res. 2008;16(3):28, http://dx.doi.org/10.1186/1749-799X-3-28.
41. Ferran NA, Hodgson P, Vannet N, Williams R, Evans RO. Locked intramedullary fixation vs. plating for displaced and shortened mid-shaft
clavicle fractures: a randomized clinical trial. J Shoulder Elbow Surg.
2010;19(6):783-9, http://dx.doi.org/10.1016/j.jse.2010.05.002.
42. Narsaria N, Singh AK, Arun GR, Seth RRS. Surgical fixation of displaced
midshaft clavicle fractures: elastic intramedullary nailing versus precontoured plating. J Orthopaed Traumatol. 2014;15(3):165-71, http://dx.
doi.org/10.1007/s10195-014-0298-7.
43. Assobhi JEH. Reconstruction plate versus minimal invasive retrograde
titanium elastic nail fixation for displaced midclavicular fractures.
J Orthopaed Traumatol. 2011;12(4):185-92, http://dx.doi.org/10.1007/
s10195-011-0158-7.
44. Alves K, Jupiter J. Clavicle Fractures: Plate Versus Intramedullary Fixation. Tech Should Surg. 2014;15:55-9, http://dx.doi.org/10.1097/
BTE.0000000000000007.
45. Duan X, Zhong G, Cen SQ, Huang FG, Xiang Z. Plating versus intramedullary pin or conservative treatment for midshaft fracture of clavicle:
a meta-analysis of randomized controlled trials. J Shoulder Elbow Surg.
2011;20(6):1008-15, http://dx.doi.org/10.1016/j.jse.2011.01.018.
46. Virtanen K, Malmivaara AOV, Remes VM, Paavola MP. Operative and
nonoperative treatment of clavicle fractures in adults: A systematic review
of 1,190 patients from the literature. Acta Orthopaedica. 2012;83(1):65-73,
http://dx.doi.org/10.3109/17453674.2011.652884.
47. Xu J, Xu L, Xu WD, Gu YD, Xu JG. Operative versus nonoperative
treatment in the management of midshaft clavicular fractures: a metaanalysis of randomized controlled trials. J Shoulder Elbow Surg. 2014;
23(2):173-81, http://dx.doi.org/10.1016/j.jse.2013.06.025.
48. Liu GD, Tong SL, Ou S, Zhou LS, Fei J, Nan GX, et al. Operative versus
non-operative treatment for clavicle fracture: a meta-analysis. Int Orthop.
2013;37(8):1495-500, http://dx.doi.org/10.1007/s00264-013-1871-z.
49. Shin SJ, Do NH, Jang KY. Risk factors for postoperative complications of
displaced clavicular midshaft fractures. J Trauma Acute Care Surg.
2012;72(4):1046-50.
592
REVIEW
The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes in
the Chinese Han population: a meta-analysis
Enting Ma,I Huili Wang,II Jing Guo,I Ruirui Tian,III Li WeiIV,*
I
General Hospital of Tianjin Medical University, Department of Pediatric Ward, Tianjin, China. II Xian International University, Department of Nursing,
Xian, China. III Tianjin Medical University, Department of Nursing, Tianjin, China. IV General Hospital of Tianjin Medical University, Department of Surgery,
Tianjin, China.
Transcription factor 7-like 2 has been shown to be associated with type 2 diabetes mellitus in multiple ethnic groups
in recent years. In the Chinese Han population in particular, numerous studies have evaluated the association
between the rs11196218A/G polymorphism of the transcription factor 7-like 2 gene and type 2 diabetes mellitus.
However, the results have been inconsistent, so we performed a meta-analysis to assess the association. Odds ratio
and 95% confidence interval values were calculated using a random-effects model or a fixed-effects model based on
heterogeneity analysis. The quality of the included studies was evaluated using the Newcastle-Ottawa Scale.
Subgroup analyses were conducted based on conformity with Hardy-Weinberg equilibrium in the control group as
well as on other variables, such as age, sex and body mass index. Sensitivity analysis was also performed to detect
heterogeneity and to assess the stability of the results. In total, 10 case-control studies comprising 7,491 cases and
12,968 controls were included in this meta-analysis. The combined analysis indicated that the rs11196218A/G
polymorphism was not associated with type 2 diabetes mellitus (G vs. A, OR = 1.04, 95% CI = 0.97-1.13, p = 0.28). The
subgroup analyses also did not show any association between the rs11196218A/G polymorphism and the risk of type
2 diabetes mellitus. Furthermore, the results of the subgroup analyses indicated that the absence of an association
was not influenced by age, sex or body mass index. The results of the sensitivity analysis verified the reliability and
stability of this meta-analysis. In conclusion, this study indicated that there is no significant association between the
rs11196218A/G polymorphism and the risk of type 2 diabetes mellitus in the Chinese Han population.
KEYWORDS: Type 2 diabetes mellitus (T2DM); Transcription factor 7-like 2 (TCF7L2); rs11196218A/G polymorphism;
Meta-analysis.
Ma E, Wang H, Guo J, Tian R, Wei L. The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes in the
Chinese Han population: a meta-analysis. Clinics. 2015;70(8):593-599
Received for publication on January 29, 2015; First review completed on March 20, 2015; Accepted for publication on May 12, 2015
E-mail: wlykdxzyy@126.com
*Corresponding author
INTRODUCTION
Copyright & 2015 CLINICS This is an Open Access article distributed under the
terms of the Creative Commons License (http://creativecommons.org/licenses/by/
4.0/) which permits unrestricted use, distribution, and reproduction in any
medium or format, provided the original work is properly cited.
DOI: 10.6061/clinics/2015(08)10
593
A meta-analysis
Ma E et al.
CLINICS 2015;70(8):593-599
Ethics statement
Search strategy
In this meta-analysis, we searched articles in PubMed,
Elsevier, SpringerLink, Embase, the Cochrane Library, ISI
Web of Science, Google Scholar and the China National
Knowledge Infrastructure (CNKI). The search languages
included English and Chinese. The following key words and
subject terms were used: TCF7L2, transcription factor 7-like
2, rs11196218, diabetes mellitus, type 2, type 2 diabetes
mellitus, T2DM, and T2D. The reference lists of eligible
studies and relevant review papers were additionally
identified via a manual search on this topic. The last research
update was performed on July 15, 2014.
Statistical analysis
Quality assessment
The Newcastle-Ottawa Scale (NOS) (22) was used to assess
the quality of the studies included in our meta-analysis. The
NOS contains eight items and is categorized into three
dimensions: selection, comparability and exposure, for
case-control studies. In particular, the selection dimension
contains four items, the comparability dimension contains
one item, and the exposure dimension contains three items.
A star system is used to allow semi-quantitative assessment
of study quality, and a study can be awarded a maximum of
one star for each numbered item within the selection and
exposure categories. Meanwhile, a maximum of two stars
can be given for comparability. The NOS ranges from zero up
to nine stars, as follows: high-quality study: more than seven
stars; medium-quality study: between four and six stars;
poor-quality study: less than four stars.
Data extraction
For quality control, the data were extracted by two
reviewers using a standardized extraction form. If the
information on the genotype distribution was inadequate,
we tried to contact the authors by telephone or e-mail. The
following information was extracted from each article:
the last name of the first author, the year of publication,
the region, the numbers of cases and controls, the source of
the controls, the numbers of genotypes for cases and
controls, matching factors, and the Hardy-Weinberg equilibrium (HWE) in each control group. Disagreement was
resolved by consulting a third reviewer.
594
A meta-analysis
Ma E et al.
CLINICS 2015;70(8):593-599
Publication
year
Region
Ng
2007
Hong Kong
Zhang
Luo
Ma
Lin
Wen
Zhu
Zheng
Qiao
Zhai
2008
2009
2010
2010
2010
2011
2012
2012
2014
Hunan
Beijing
Shanghai
Chengdu
Shanghai
Anhui
Chongqing
Harbin
Henan
Source of
controls
Community-based and
hospital staff
Hospital-based
Hospital-based
Hospital-based
Hospital-based
N/D
Hospital-based
Hospital-based
Hospital-based
Community-based
and Hospital-based
Sample sizes
Cases
Controls
HWE
Comparability
Case
Control
433
419
676
190
599
239
Yes
536
500
259
1529
1165
300
227
700
1842
475
500
200
1439
1136
300
152
560
7777
716
684
309
2171
1699
156
340
1025
2639
272
252
209
887
629
444
114
367
967
623
696
240
2015
1677
122
218
819
111287
221
240
168
863
595
478
86
295
4041
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Abbreviations: HWE, Hardy-Weinberg equilibrium; Yes, the genotype distribution conformed to HWE in the control group; No, the genotype distribution
not conform to HWE in the control group;
Hospital-based: subjects who were enrolled from health checks conducted at the hospital; community-based and hospital staff: subjects who were
enrolled from the community and hospital staff; N/D: no description; *: p40.05; **: po0.05.
RESULTS
Studies and data included in this meta-analysis
In total, 295 articles were relevant to our search terms, of
which 285 papers were excluded. Thus, 10 case-control
studies (15-19,30-34) comprising 7,491 cases of T2DM and
12,968 controls were ultimately included in this metaanalysis (Figure 1). All of these studies were published from
2007-2014.
The characteristics of these studies are summarized in
Table 1. The genotype frequency of the single nucleotide
polymorphism (SNP) was consistent with HWE in the
control group (p40.05) in all studies except for two (32,33).
The quality assessment of all included studies, evaluated
according to the NOS, is provided in Table 2. Most studies
were of high quality in terms of selection and exposure.
However, the quality of comparability was relativity low, as
only 3 studies (15,33,34) were comparable with the controls
regarding age, sex and BMI.
595
Publication year
Selection
Comparability
Exposure
2007
2008
2009
2010
2010
2010
2011
2012
2012
2014
$$$
$$
$$
$$
$$
$$
$$$$
$$$$
$$$
$$
$
$
$
$
$$
$
$$
$
$$
$
$
$$
$$
$$
$$$
$$
$$
$$
$
$$
A meta-analysis
Ma E et al.
CLINICS 2015;70(8):593-599
Figure 2 - Meta-analysis of the association between the rs11196218A/G polymorphism and T2DM risk (G vs. A). n indicates the total
number of G alleles, and N indicates the total number of G alleles plus A alleles.
Sensitivity analysis
A single study included in the meta-analysis was omitted
each time to reflect the influence of each dataset on the
pooled OR values. We found that no single study could
change the pooled results (Table 3), which indicated that the
results were relatively reliable.
Publication bias
The shape of the funnel plots was symmetrical, suggesting
that there was no evidence of publication bias for the
rs11196218A/G polymorphism (Figure 4).
DISCUSSION
Since the initial discovery that TCF7L2 is strongly associated
with an increased risk of T2DM in Icelandic populations in
2006 (4), many replication studies have confirmed the role of
TCF7L2 in conferring susceptibility to T2DM in different
populations and ethnic groups (5,7,11,14,38,39), especially for
the rs7903146C/T polymorphism. However, in the Chinese
Figure 3 - Meta-analysis of the association between the rs11196218A/G polymorphism and T2DM risk (subgroup analyses for HWE in the
control group: G vs. A). n indicates the total number of G alleles, and N indicates the total number of G alleles plus A alleles.
596
A meta-analysis
Ma E et al.
CLINICS 2015;70(8):593-599
OR
95% CI
p-value
Lin et al.
Luo et al.
Ma et al.
Ng et al.
Qiao et al.
Wen et al.
Zhai et al.
Zhang, Y.
Zheng et al.
Zhu, H.
1.01
1.03
1.02
1.00
1.02
1.03
1.04
1.03
1.02
1.01
0.96-1.07
0.98-1.08
0.97-1.07
0.95-1.05
0.97-1.07
0.98-1.09
0.98-1.11
0.98-1.08
0.97-1.07
0.96-1.06
0.62
0.32
0.44
0.90
0.41
0.26
0.16
0.32
0.50
0.69
Figure 4 - Funnel plot analysis to detect publication bias (G vs. A of the rs11196218A/G polymorphism). Each point represents an
independent study on the indicated association. The dark point represents two overlapping articles.
597
A meta-analysis
Ma E et al.
CLINICS 2015;70(8):593-599
14.
15.
16.
17.
18.
19.
AUTHOR CONTRIBUTIONS
Ma ET and Wei L developed the idea for the study and drafted the
manuscript. Ma ET, Wei L, Wang HL and Tian RR were responsible for
conducting the search, the data collection and the study quality assessment.
Ma ET, Wei L Wang HL and Guo J analyzed and interpreted the data. All
of the authors read and approved the nal version of the manuscript.
20.
21.
22.
REFERENCES
1. Hansen L, Pedersen O. Genetics of type 2 diabetes mellitus: status and
perspective. Diabetes Obes Metab. 2005;7(1):122-35, http://dx.doi.org/
10.1111/dom.2005.7.issue-2.
2. Duval A, Busson-Leconiat M, Berger R, Hamelin R. Assignment of the
TCF-4 (TCF7L2) to human chromosome band 10q25.3. Cytogenet Cell
Genet. 2000;88(3-4):264-5, http://dx.doi.org/10.1159/000015534.
3. Yi FH, Brubaker PL, Jin TR. TCF-4 mediates cell type-specific regulation gene expression by b-catenin and glycogen synthase kinase-3b.
J Biol Chem. 2005;280(2):1457-64, http://dx.doi.org/10.1074/jbc.
M411487200.
4. Grant SF, Thorleifsson G, Reynisdottir I, Benediktsson R, Manolescu A,
Sainz J, et al. Variant of transcription factor 7-like 2 (TCF7L2) gene confers
risk of type 2 diabetes. Nat Genet. 2006;38(3):320-3, http://dx.doi.org/
10.1038/ng1732.
5. Cauchi S, Meyre D, Dina C, Choquet H, Samson C, Gallina S, et al. (2006)
Transcription factor TCF7L2 genetic study in the French population:
expression in human b-cells and adipose tissue and strong association
with type 2 diabetes. Diabetes.2006;55(10): 2903-8.
6. Groves CJ, Zeggini E, Minton J, Frayling TM, Weedon MN, Rayner NW,
et al. Association analysis of 6,736 U.K. subjects provides replication and
confirms TCF7L2 as a type 2 diabetes susceptibility gene with a substantial effect on individual risk. Diabetes. 2006;55(9):2640-4, http://dx.
doi.org/10.2337/db06-0355.
7. Lyssenko V, Lupi R, Marchetti P, Guerra SD, Orho-Melander M, Almgren
P, et al. Mechanisms by which common variants in the TCF7L2
gene increase risk of type 2 diabetes. J Clini Invest. 2007;117(8):2155-63,
http://dx.doi.org/10.1172/JCI30706.
8. Saxena R, Gianniny L, Burtt N, Lyssenko V, Giuducci C, Sjgrenet M, et al.
Common single nucleotide polymorphisms in TCF7L2 are reproducibly
associated with type 2 diabetes and reduce the insulin response to glucose
in nondiabetic individuals. Diabetes. 2006;55(10):2890-5, http://dx.doi.
org/10.2337/db06-0381.
9. Guinan KJ. Worldwide distribution of type II diabetes-associated TCF7L2
SNPs: Evidence for stratification in Europe. Biochem Genet. 2012;50(34):159-79, http://dx.doi.org/10.1007/s10528-011-9456-2.
10. Mayans S, Lackovic K, Lindgren P, Ruikka K, Agren A, Eliasson M, et al.
TCF7L2 poly-morphisms are associated with type 2 diabetes in northern
Sweden. Eur J Hum Genet. 2007;15(3):342-6, http://dx.doi.org/10.1038/
sj.ejhg.5201773.
11. Potapov VA, Shamkhalova MN, Smetanina SA, Belchikova LN,
Suplotova LA, Shestakova MV, et al. Polymorphic markers TCF7L2
rs12255372 and SLC30A8 rs13266634 confer susceptibility to type 2 diabetes in a Russian population. Genetika. 2010;46(8):1123-31.
12. Lehman DM, Hunt KJ, Leach RJ, Hamlington J, Arya R, Abboud HE, et al.
Haplotypes of transcription factor 7-like 2 (TCF7L2) gene and its
upstream region are associated with type 2 diabetes and age of onset in
Mexican Americans. Diabetes. 2007;56(2):389-93, http://dx.doi.org/10.2337/
db06-0860.
13. Chandak GR, Janipalli CS, Bhaskar S, Kulkarni SR, Mohankrishna P,
Hattersley AT, et al. Common variants in the TCF7L2 gene are strongly
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
598
associated with type 2 diabetes mellitus in the Indian population. Diabetologia. 2007;50(1):63-7, http://dx.doi.org/10.1007/s00125-006-0502-2.
Hayashi T, Iwamoto Y, Kaku K, Hirose H, Maeda S. Replication study for
the association of TCF7L2 with susceptibility to type 2 diabetes in a
Japanese population. Diabetologia. 2007;50(5):980-4, http://dx.doi.org/
10.1007/s00125-007-0618-z.
Zhu H. Common Variants of the TCF7L2 Gene with Type 2 Diabetes and
Impaired Glucose Regulation in a Chinese Han Population. Master thesis.
2011. [Article in Chinese].
Zhang Y. Study on polymorphisms of TCF7L2 gene association with type
2 diabetes in Chinese population. Master thesis. 2008. [Article in
Chinese].
Ng MC, Tam CH, Lam VK, So WY, Ma RC. Replication and identification
of novel variants at TCF7L2 associated with type 2 diabetes in Hong Kong
Chinese. J Clin Endocr Metab. 2007;92(9):3733-7, http://dx.doi.org/
10.1210/jc.2007-0849.
Lin Y, Li PQ, Cai L, Zhang B, Tang X, Zhang XJ, et al. Association study of
genetic variants in eight genes/loci with type 2 diabetes in a Han Chinese
population. BMC Med Genet. 2010;11:97, http://dx.doi.org/10.1186/
1471-2350-11-97.
Zhai YJ, Zhao JZ, You HF, Pang C, Yin L, Guo T, et al. Association of the
rs11196218 polymorphism in TCF7L2 with type 2 diabetes mellitus in
Asian population. Meta Gene.2014;2:332-41, http://dx.doi.org/10.1016/j.
mgene.2014.04.006.
Thakkinstian A, McElduff P, DEste C, Duffy D, Attia J. A method
for meta-analysis of molecular association studies. Stat Med. 2005;24(9):
1291-306, http://dx.doi.org/10.1002/(ISSN)1097-0258.
Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for
systematic reviews and meta-analyses: the PRISMA statement. Int J Surg.
2010;8(5):336-41, http://dx.doi.org/10.1016/j.ijsu.2010.02.007.
Wells GA, Shea B, OConnell D, Peterson J, Welch V, Losos M, et al. The
Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses.2011. http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp. Accessed 20 Aug 2012.
Lau J, Ioannidis JP, Schmid CH. Quantitative synthesis in systematic
reviews. Ann Intern Med. 1997;127(9):820-6, http://dx.doi.org/10.7326/
0003-4819-127-9-199711010-00008.
Mantel N, Haenszel W. Statistical aspects of the analysis of data from
retrospective studies of disease. J Natl Cancer Inst. 1959;22(4):719-48.
DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin
Trials. 1986;7(3):177-88, http://dx.doi.org/10.1016/0197-2456(86)90046-2.
Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency
in meta-analyses. BMJ. 2003;327(7414):557-60, http://dx.doi.org/10.1136/
bmj.327.7414.557.
Haupt A, Thamer C, Heni M, Ketterer C, Machann J, Schick F, et al. Gene
variants of TCF7L2 influence weight loss and body composition during
lifestyle intervention in a population at risk for type 2 diabetes. Diabetes.
2010;59(3):747-50, http://dx.doi.org/10.2337/db09-1050.
Gloyn AL, Braun M, Rorsman P. Type 2 diabetes susceptibility gene
TCF7L2 and its role in b-Cell function. Diabetes. 2009;58(4):800-2,
http://dx.doi.org/10.2337/db09-0099.
Xavier GS, Loder MK, McDonald A, Tarasov AI, Carzaniga R, et al. TCF7L2
regulates late events in insulin secretion from pancreatic islet b-Cells.
Diabetes. 2009;58(4):894-5, http://dx.doi.org/10.2337/db08-1187.
Ma C. Relationships of rs13266634 and rs11196218 polymorphisms in
SLC30A8 (solute carrier family 30, member 8) and TCF7L2 (transcription
factor 7-like 2 genes with type 2 diabetes in Southern Chinese Han
Population) Master thesis. 2010. [Article in Chinese].
Zheng XY, Ren W, Zhang SH, Liu JJ, Li SF, Li JC, et al. Association
of type 2 diabetes susceptibility genes (TCF7L2, SLC30A8, PCSK1 and
PCSK2) and proinsulin conversion in a Chinese population. Mol
Biol Rep. 2012; 39(1): 17-23, http://dx.doi.org/10.1007/s11033-011-0705-6.
Luo YY, Wang HY, Han XY, Ren Q, Wang F, Zhang XY, et al. Metaanalysis of the association between SNPs in TCF7L2 and type 2 diabetes
in East Asian population. Diabetes Research and Clinical Practice. 2009;
85: 139-46, http://dx.doi.org/10.1016/j.diabres.2009.04.024.
Wen J, Rnn T, Olsson A, Yang Z, Lu B, Du YP, et al. Investigation of type
2 diabetes risk alleles support CDKN2A/B, CDKAL1, and TCF7L2 as
susceptibility genes in a Han Chinese Cohort. PLoS ONE. 2010;5(2):e9153,
http://dx.doi.org/10.1371/journal.pone.0009153.
Qiao H, Zhang XL, Zhao XD, Zhao YL, Xu LD, Sun HM, et al. Genetic
variants of TCF7L2 are associated with type 2 diabetes in a northeastern
Chinese population. Gene. 2012;495(2):115-9, http://dx.doi.org/10.1016/
j.gene.2011.12.055.
Ma C, Sheng HG, Ma JJ. Relationship of rs13266634 and rs11196218
polymorphisms in solute carrier family 30, member 8 and transcription
factor 7-like 2 genes with type 2 diabetes in Han Population in Southern
China. Shanghai Med J. 2009;32(12):1086-91.
Zheng XY, Ren W, Zhang SH, Liu JJ, Li SF, Li JC, et al. Correlation
between single nucleotide polymorphisms of rs7903146 and rs11196218
at TCF7L2 gene and the early phase insulin secretion of newly
diagnosed patients with type 2 diabetes. Med J Chin PLA. 2011;
36(3):269-72.
A meta-analysis
Ma E et al.
CLINICS 2015;70(8):593-599
599
tornam-se grandes.
Av. Rebouas, 600 - 05402-000 - So Paulo - Brasil - Tel.: 55 11 3898-7850 / Fax: 55 11 3898-7878 - reboucas@hcnet.usp.br
www.convencoesreboucas.com.br