Stress failure plays an major role in development of High Altitude
Pulmonary Edema in rats
Jun She1，2#, Jun Shen3, Arnaud Goolaerts4, Yuanlin Song5, Ce Shen1,and Chunxue
Bai2*
1
Department of Pulmonary Medicine, Shanghai Sixth Hospital of Shanghai Jiaotong
University; 2Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University,
Shanghai, China; 3Navy Medical Institute, Shanghai, China; 4Deaprtment of Anesthesia,
San Francisco General Hospital, 5Department of Anesthesia and Perioperative Care,
University of California San Francisco, San Francisco, California, USA
# The institutions are not one after another.
*
Corresponding Author:
Chunxue Bai MD, PhD
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 180 Feng
Lin Road, Shanghai 200032, China
Tel: +86-21-64041990-3077,
E-mail: bai.chunxue@zs-hospital.sh.cn
1
SUPPLEMENTARY DATA
1. Experimental groups and their abbreviations
Of the thirty-nine Sprague-Dawley rats, seven were excluded for not much of a hand at
walking on treadmill. All animals did not develop an increase in body temperature
indicative of any potential infection. We proposed to establish an animal model of
HAPE by exposing exercising animals to hypobaric hypoxia. The experimental groups
were randomly assigned to as follows:
① NR: rats exposed to normobaric normoxia at rest (control);
② NE: rats exposed to normobaric normaxia with exercise;
③ HR: rats exposed to hypobaric hypoxia at rest;
④ HE: rats exposed to hypobaric hypoxia with exercise.
There were 8 rats in each group.
2. Arterial blood gases data
The PaO2, PaCO2, pH and PA-aO2 in arterial blood gases data were obtained from the
survival animals (table S1). The period of exposure was 48 hours. Five of the eight
exercising rats exposed to hypobaric hypoxia chamber survived. Three rats died, one
after 27 hours, and two after 35.5hours. Without exercising rats exposed to hypobaric
hypoxia chamber, exercising rats exposed to normobaric normoxia and the control
tolerated the procedures well (NR, N = 8; NE, N = 8; HR, N = 8; HE, N = 5).
3. Lung wet-to-dry weight ratio data
The lung wet weight, dry weight and wet-to-dry weight ratio were shown in table S2. N
= 8 in each group.
2
4. Bronchoalveolar lavage data
The data of thirty-two rats were given here in the present study. The total protein,
albumin, red and white blood cell counts, IgM and LTB4 were shown in table S3. N = 8
in each group.
5. Data Summary
The arterial blood gases, bronchoalveolar lavage, lung wet-to-dry weight ratio, lung
injury score and electron breaks in the experiment groups and the control after 48 hours
were summarized, and showed in table S4.
6. Hypobaric hypoxia chamber and exercising rats
In the present study, we designed the experimental protocol by using the hypobaric
hypoxia chamber to simulate the altitude of 4700 m above sea level. There were some
pictures about hypobaric hypoxia chamber and exercising rats walked on treadmill,
(figure S1 A, B, C).
7. Gross appearance
Bloody fluid was noticed in the nose and mouth from the died animal of HE group,
(figure S2).
8. Data correlation analysis
Correlation matrix among the main variables of the study. Variables are including: red
blood cell, total protein, albumin, lung wet to dry weight ratio, alveolar edema score,
leukotriene, neutrophil and macrophage infiltration score, electron microscopy breaks,
alveolar-arterial difference of oxygen partial pressure (figure S3).
3
TABLES
Table S1.
PaO2, PaCO2, PH and PA-aO2in arterial blood gases.
Number
1
2
NR
95.2
94.1
NE
94.1
HR
HE
3
4
5
87.6
91.6
90.5
88.6
91.2
89.6
61.2
56.8
51.6
51.2
53.5
6
7
8
91.3
93.4
89.4
87.4
97.1
93.7
91.5
60.4
58.9
55.7
63.7
57.4
54.4
55.8
60.1
*
*
*
PaO21, (mmHg)
PaCO22, (mmHg)
NR
40.1
32.7
39.5
34.9
36.2
30.1
34.2
39.8
NE
32.9
34.6
32.1
25.4
33.2
31
34.4
30.2
HR
26.4
30.1
20.3
28.8
32.7
19.5
33.8
22.7
HE
28.1
18.3
24.6
26.8
25.4
*
*
*
NR
7.35
7.37
7.42
7.43
7.41
7.54
7.36
7.36
NE
7.35
7.39
7.39
7.4
7.38
7.36
7.41
7.39
HR
7.48
7.43
7.52
7.45
7.37
7.51
7.42
7.51
HE
7.38
7.48
7.32
7.33
7.41
*
*
*
PH
PA-aO23
NR
14.43 22.93 22.63 23.23 23.03 28.33 22.13 20.53
NE
22.73 26.53 26.43 34.73 29.13 21.63 21.63 28.03
HR
24.96 25.66 40.66 23.36 20.96 37.36 15.06 32.46
4
HE
33.26 40.76 33.56 29.96 27.06
*
*
*
1. PaO2 = arterial partial pressure of oxygen
2. PaCO2 = arterial partial pressure of carbon dioxide
3. PA-aO2 = alveolar-arterial difference in oxygen
* Three of the eight exercising rats exposed to hypobaric hypoxia chamber (HE) died,
one after 27 hours, and two after 35.5hours.
Table S2. Lung wet value, dry value and wet-to-dry weight ratio in the experiment
groups and the control after 48 hours.
Number
1
2
3
4
5
0.55
0.28
0.67
6
7
8
0.48
0.36
0.24
Lung wet weight, (g)
NR
0.1
0.3
NE
0.22
0.83
0.23
0.23
0.54
0.47
0.35
0.61
HR
0.55
0.34
0.75
0.47
0.65
0.38
0.46
0.43
HE
0.18
0.2
0.24
0.26
0.51
0.63
0.85
0.25
Lung dry weight, (g)
NR
0.04
0.08
0.16
0.07
0.28
0.11
0.1
0.1
NE
0.09
0.28
0.06
0.05
0.13
0.12
0.14
0.15
HR
0.11
0.08
0.13
0.1
0.17
0.07
0.14
0.09
HE
0.03
0.03
0.04
0.05
0.1
0.09
0.2
0.04
Lung wet-to-dry weight ratio, (g/g)
NR
2.5
3.75
3.44
4
2.39
4.36
3.6
2.4
5
NE
2.4
3
3.8
4.6
HR
5
4.25
5.77
4.7
HE
6
6.7
6
4.2
5.2
3.9
3.8
5.4
5.1
7
2.5
4.1
3.3
4.78
4.25
6.25
Table S3. The total protein, albumin, red and the white blood cell counts, IgM and
LTB4 in the Bronchoalveolar lavage.
Number
1
2
3
4
5
6
7
8
Total protein, (ug/ml)
NR
216
387
274
362
249
387
310
237
NE
258
253
323
407
405
310
249
341
HR
548
489
604
387
412
571
413
483
HE
528
732
614
516
453
746
532
706
Albumin, (ug/ml)
NR
98
276
169
233
154
281
192
103
NE
124 117
204
332
294
191
120
218
HR
349 328
441
307
207
384
243
316
HE
383 458
371
327
274
463
296
439
RBC1, (*105/ml)
NR
8
16
11
17
14
19
16
10
NE
12
9
19
33
24
21
10
28
HR
58
44
71
32
25
67
23
47
HE
62
73
62
61
57
94
56
68
6
WBC2, (*105/ml)
NR
4
5
6
5
2
3
2
6
NE
6
2
7
4
6
3
2
5
HR
9
8
6
7
5
8
7
6
HE
9
6
5
6
5
7
11
8
NR
0
0
0.4
0
0
0
0.2
0.1
NE
0.2
0
0
0
0
0.4
0
0
HR
0.1
0
0
0.5
0
0
0
0
HE
0
0.4
0
0.1
0
0
0
IgM, (mg/dl)
0
LTB43, (pg/ml)
NR
NE
0.024 0.046 0.021 0.159 0.027 0.045 0.199 0.022
0.027 0.054 0.014 0.129 0.175 0.045
0.05
0.071
HR
0.076 0.015 0.154 0.014 0.073 0.054 0.149 0.064
HE
0.056 0.088
0.07 0.059 0.091
0.05 0.095 0.071
1. RBC = red blood cell counts
2. WBC = white blood cell counts
3. LTB4 = leukotriene B4
Table S4. Data Summary
NR
NE
HR
HE
Arterial blood gases
PaO23, (mmHg)
91.6±0.9
91.7±1.1
58.2±1.3**
55±1.2**
7
PaCO24, (mmHg)
35.9±1.3
31.7±1.1
26.8±1.9*
24.6±1.3**
PH
7.39±0.01
7.39±0.01
7.46±0.02*
7.38±0.02
PA-aO2
22.16±1.36
26.36±1.57
27.56±3.05
3.6±0.29
4.6±0.29*
Lung wet-to-dry weight ratio 3.3±0.27
32.92±1.81*
5.8±0.32**
Bronchoalveolar lavage
Total protein, (ug/ml)
302.8±24.4
318.3±22.6
488.4±28.5**
603.4±39.8**
Albumin, (ug/ml)
188.3±25.1
200±28.6
321.9±26.2*
376.4±25.9**
RBC1, (×105/ml)
13.9±1.4
19.5±3.1
45.9±6.5*
66.6±4.4**#
WBC2, (×105/ml)
4.1±0.6
4.4±0.7
7±0.5*
7.1±0.7*
IgM, (mg/dl)
0.09±0.05
0.08±0.05
0.08±0.06
0.06±0.05
LTB4, (pg/ml)
0.07±0.02
0.07±0.02
0.07±0.02
0.07±0.006
Lung injury score
Alveolar edema
0.25±0.16
0.375±0.18
0.75±0.25
2.6±1.3**
intersititial edema
0.75±0.25
1±0.27
2.6±0.4**
3.4±0.3**
1±0.27
2±0.27
2.25±0.25
3.25±0.25**#
0.5±0.19
1.25±0.3
1.5±0.33
1.75±0.25*
0.25±1.63
0.63±0.26
1.88±0.4
4.25±0.56**
hemorrhage
Neutrophil/macrophage
infiltration
Electron breaks
Values are given as the mean ± SEM.
1. RBC = red blood cell counts
2. WBC = white blood cell counts
3. PaO2 = arterial partial pressure of oxygen
4. PaCO2 = arterial partial pressure of carbon dioxide
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5. PA-aO2 = alveolar-arterial difference in oxygen
Arterial blood gas. PaO2, ** p < 0.01 for the HE and HR groups vs. NE and NR
groups. PaCO2, ** p < 0.01 for the HE group vs. NR group; * p < 0.05 for the HR
group vs. NR group. pH, * p < 0.05 for the HR group vs. HE group, as well as NE vs.
NR group. PA-aO2, * p < 0.05 for the HE group vs. NR, NE and HR groups.
Lung wet-to-dry weight ratio. ** p < 0.01 for the HE vs. NE and NR groups; * p <
0.05 for the HR group vs. NR group..
Bronchoalveolar lavage. Total protein, ** p < 0.01 for the HE and HR group vs. NE
and NR group, respectively. Albumin, ** p < 0.01 for the HE group vs. NE and NR
group. * p < 0.05 for the HR group vs. NE and NR group. RBC, ** p < 0.01 for the HE
group vs. NE and NR group; * p < 0.05 for the HR group vs. NE and NR group. # p <
0.05 for the HE group vs. HR group. WBC, * p < 0.05 for the HE and HR groups vs.
NE and NR groups. BAL IgM concentrations, There was no significant differences
between groups. BAL LTB4 concentrations, There was no significant differences
between groups.
Lung injury scores from histology. Alveolar edema score, ** p < 0.01 for the HE
group vs. HR, NE and NR groups. Interstitial edema score, ** p < 0.01 for the HE and
HR groups vs. NE and NR groups. Hemorrhage score, ** p < 0.01 for the HE group vs.
NR group. # p < 0.05 for the HE vs. NE group. Neutrophil and macrophage infiltration
score, * p < 0.05 for the HE group vs. NR group.
Electron breaks. ** p < 0.01 for the HE vs. HR, NE and NR groups.
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FIGURES
Figure S1.
(A)
(B)
(C)
Figure S1 legend. There were some pictures about hypobaric hypoxia chamber and
exercising rats walked on treadmill. (A) Hypobaric hypoxia chamber. (B) The inner of
hypobaric hypoxia chamber. (C) Exercising rats walked on treadmill.
Figure S2.
10
Figure S2 legend. Bloodstained frothy fluid was seen in the nose and mouth (arrow)
from died animal (HE group).
Figure S3. Data correlation analysis
Figure S3 legend. Correlation matrix among the main variables of the study. R is
provided for each couple of variables. The p values have been calculated using the
Pearson test and are provided for each couple of variables. Variables: RBC, red blood
11
cell; TP, total protein; AL, albumin; WD, lung wet to dry weight ratio; ES, alveolar
edema score; LTB4, leukotriene B4; IS, neutrophil and macrophage infiltration score;
BREAKS, electron microscopy breaks; PA-aO2, alveolar-arterial difference of oxygen
partial pressure; R, correlation coefficient; p, p value of statistical analysis; NA, non
applicable.
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