3.1. Physiological and biochemical statuses in each group were nearly identical before each round of the experiment
As described in the experimental protocol, the dogs were subjected to a severe hypoxic attack—nearly lethal—which resulted in damage to important organs, such as the heart, liver, and kidneys. Therefore, we included a 1-week interval between each round of the experiment to allow for physical recovery from the hypoxic attacks and to eliminate any lingering effects of the previous experiment. According to the Latin Square ANOVA results, all dogs were in a good state of health; no significant differences were observed among treatment groups with regard to HR, MAP, SpO2, CO, PaO2, PaCO2, pH, other indexes of ABG, or hepatic and renal functions before each round of experiments (Table 2). These findings suggested that the physiological and biochemical statuses of the dogs returned to baseline during the1-week intervals; additionally, there were no significant differences between experimental times or between individual dogs in terms of these indexes(Table 2).
Table 2. Physiological and Biochemical Statuses in Each Treatment Group Before Each Round ofthe Experiment
Indexes
|
Con
|
Pneu
|
P+1
|
P+5
|
Ptreatment
|
Ptime
|
Pdog
|
HR(bpm)
|
94±10.4
|
96±16.4
|
95±15.6
|
94±14.2
|
0.969
|
0.645
|
0.439
|
MAP(mmHg)
|
86.6±5.24
|
81.0±10.50
|
83.0±10.42
|
83.4±9.05
|
0.225
|
0.228
|
0.062
|
CO(l/min)
|
8.10±0.76
|
7.85±1.27
|
8.55±1.11
|
7.95±0.94
|
0.509
|
0.164
|
0.383
|
SpO2(%)
|
100±0
|
100±0
|
100±0
|
100±0
|
----
|
----
|
----
|
PaO2(mmHg)
|
587.00±43.55
|
598.25±40.44
|
578.38±48.77
|
600.00±56.81
|
0.780
|
0.379
|
0.463
|
PaCO2(mmHg)
|
33.13±2.850
|
31.13±2.696
|
32.00±2.390
|
32.88±3.563
|
0.439
|
0.075
|
0.405
|
pH
|
7.411±0.038
|
7.411±0.035
|
7.410±0.030
|
7.405±0.040
|
0.964
|
0.660
|
0.114
|
THbc(g/dl)
|
11.06±0.64
|
11.45±1.17
|
11.49±0.68
|
11.73±0.93
|
0.487
|
0.118
|
0.571
|
Na+(mmol/l)
|
145.75±1.39
|
144.75±0.71
|
145.38±1.60
|
144.38±1.69
|
0.209
|
0.101
|
0.697
|
K+(mmol/l)
|
2.76±0.220
|
2.71±0.327
|
2.80±0.177
|
2.75±0.239
|
0.902
|
0.059
|
0.751
|
Ca++(mmol/l)
|
1.18±0.023
|
1.19±0.037
|
1.22±0.034
|
1.19±0.049
|
0.251
|
0.219
|
0.699
|
ALT(IU/l)
|
31.39±7.795
|
28.84±11.839
|
27.04±4.892
|
29.60±7.419
|
0.728
|
0.388
|
0.172
|
AST(IU/l)
|
39.33±11.981
|
34.35±7.197
|
35.00±9.249
|
40.10±6.983
|
0.438
|
0.819
|
0.143
|
Crea(μmol/l)
|
49.29±8.115
|
50.74±5.711
|
52.11±5.499
|
49.08±9.137
|
0.736
|
0.098
|
0.103
|
BUN(mmol/l)
|
3.37±0.454
|
3.44±0.535
|
3.69±0.654
|
3.36±0.465
|
0.423
|
0.627
|
0.073
|
Glu(mmol/l)
|
4.54±0.555
|
4.11±0.714
|
4.60±0.576
|
5.29±1.917
|
0.305
|
0.427
|
0.932
|
LDH(IU/l)
|
141.4±47.08
|
129.6±35.44
|
145.8±55.13
|
128.1±31.21
|
0.225
|
0.591
|
0.063
|
GGT(IU/l)
|
2.975±0.492
|
2.862±0.447
|
3.188±0.710
|
3.350±0.678
|
0.727
|
0.427
|
0.560
|
CK-MB(IU/l)
|
322.33±38.32
|
294.70±27.79
|
308.93±26.48
|
318.20±26.66
|
0.317
|
0.612
|
0.460
|
SOD(IU/ml)
|
130.28±33.34
|
145.91±25.61
|
150.33±33.05
|
138.65±38.51
|
0.679
|
0.420
|
0.736
|
MAO(IU/ml)
|
3.925±1.025
|
3.500±1.232
|
3.688±0.958
|
3.600±0.853
|
0.833
|
0.676
|
0.144
|
Hct(%)
|
31.25±2.435
|
32.50±6.164
|
31.75±3.576
|
33.86±4.190
|
0.688
|
0.242
|
0.935
|
HCO3-std(mmol/l)
|
21.94±1.570
|
22.09±1.229
|
22.76±0.656
|
22.18±1.091
|
0.958
|
0.694
|
0.689
|
Lac(mmol/l)
|
0.313±0.083
|
0.375±0.128
|
0.413±0.146
|
0.400±0.131
|
0.325
|
0.631
|
0.110
|
BE(mmol/l)
|
-3.96±1.859
|
-3.85±2.088
|
-2.68±1.787
|
-2.94±2.001
|
0.416
|
0.677
|
0.170
|
Data are presented as mean ± SD. Ptreatment indicates P values of inter-groups comparison; Ptime indicates P values of inter experimental time comparison; Pdog indicates P values of individuals of beagles comparison.
Con: control group, Pneu: pneumoperitoneum group, P+1:early pneumoperitoneum plus abdominal lifting and compression group, P+5: late pneumoperitoneum plus abdominal lifting and compression group
ALT, alanine aminotransferase; AST, aspartate aminotransferase; Crea, serum creatinine; BUN, blood urea nitrogen; Glu, blood glucose; LDH, lactate dehydrogenase; GGT, γ-glutamyl transpeptidase; CK-MB, creatine kinase isoenzymes-B; SOD, superoxide dismutase; MAO, monoamine oxidase; Hct, red blood cell specific volume; Lac, lactate; BE, buffer exces
3.2. PaO2in each group was approximately identical at the time of asphyxia termination
It was critical to determine when to restore mechanical ventilation during the asphyxia model. We aimed to achieve the maximum possible extent at which the dogs exhibited the greatest tolerance to hypoxia. If restoration of mechanical ventilation was performed too early, the effect of artificial oxygen pneumoperitoneum on hypoxia tolerance could not be fully explored. If it was performed too late, hypoxia could cause irreversible damage or lead to failed resuscitation. PaO2 measurement is undoubtedly the gold standard for assessing internal oxygen content. However, ABG analysis could only be performed intermittently; thus, we used continuous indexes. Based on pre-experimental observations, we restored mechanical ventilation when one of the following conditions was met: 1)SpO2 was <50%, continually; 2)MAP was twice the baseline value; or 3) malignant arrhythmia occurred. We simultaneously performed ABG testing to measure PaO2 at the termination time. The above practices were implemented because: 1) occasionally,SpO2could not be read due to poor peripheral circulation caused by severe hypoxia; 2) CO was reliable when hemodynamic status was stable, but could not be measured during severe fluctuations in hemodynamic status due to hypoxia; and 3) dogs exhibited different levels of intolerance to hypoxia in each experiment, indicating that HR or MAP alone could not accurately reflect pathological progress. Therefore, we combined these indexes to determine an accurate and reversible pathological point. Consistent with our pre-experiments, PaO2 measurements were not significantly different among groups when asphyxia was terminated (Ptreatment=0.762, Ptime=0.700, Pdog=0.960, Fig. 1A),indicating that hypoxic states were similar among dogs at this time.
3.3. Asphyxia time was lengthened by artificial oxygen pneumoperitoneum combined with abdominal lifting and compression
As previously described, the dogs were subjected to multiple rounds of hypoxic states during the study period, which is harmful; however, this could be regarded as hypoxia tolerance training. Thus, we assessed whether the dogs showed enhanced tolerance to hypoxia in the later stages of the experiment, using Latin Square design. Notably, the effect of experimental time on hypoxia tolerance was not statistically significant (Ptime=0.247);individual dogs also did not affect hypoxia tolerance (Pdog=0.633). Asphyxia times of the Control, Pneu,P+1, and P+5 treatment groups were 9.65 ± 1.10min, 11.18 ± 0.50min, 11.45 ± 0.79min, and 11.70 ± 0.99min, respectively (Fig. 1C). Artificial oxygen pneumoperitoneum combined with abdominal lifting and compression lengthened asphyxia time (Pneu vs Control, mean difference 1.52min [95% confidence interval (95%CI)0.59, 2.45; P=0.003]; P+1 vs Control, mean difference 1.80min [95%CI 0.87, 2.72; P=0.001]; P+5 vs Control, mean difference 2.01min [95%CI 1.12, 2.97; P<0.001]; Fig. 1B and 1C).Finally, no significant differences were present in recovery time among groups(Fig. 1D).
3.4. Artificial oxygen pneumoperitoneum combined with abdominal lifting and compression increased PaO2
PaO2 is a reliable indicator of oxygen content. Based on our pre-experiments, PaO2 declined at a similar velocity among groups during the first 5 minutes of asphyxia, and the termination of asphyxia standard was met in some dogs after 10 minutes of asphyxia. Hence, PaO2 and PaCO2 were detected and compared at every minute beginning from the 6th to the 10th minute in this study (Fig. 2A and 2D). In Figure 2A and 2D, repeated measures two-way ANOVA revealed a difference in PaO2 levels among groups(P<0.05),and no difference in PaCO2levels among groups(P>0.05). PaO2reduction in the Control group was greater than that in other groups beginning from8 minutes after asphyxia; PaCO2 increased in a nearly linear fashion in all groups(Fig. 2A and 2D). Accordingly, we compared PaO2 values at the 9th and 10th minutes (Fig. 2B and 2C) and found that PaO2 levels in the Pneu, P+1, and P+5 groups were higher than that of the Control group(PaO2, 9th min: Pneu vs Control, 8.50mmHg [95%CI 3.50, 13.50]; P+1 vs Control, 11.5mmHg [95%CI 6.50, 16.50]; P+5 vs Control, 15.00mmHg [95% CI 10.00, 20.00]; 10th min: Pneu vs Control, 9.75mmHg [95%CI 6.34, 13.16]; P+1 vs Control, 10.50mmHg [95%CI 7.09, 13.91]; P+5 vs Control, 13.25mmHg [95%CI 9.84, 16.67]); additionally, PaO2 levels in the P+5 group were higher than that in the Pneu group(PaO2, 9th min: P+5 vs Pneu, 6.50mmHg [95%CI 1.50, 11.50]; 10th min: P+5 vs Pneu, 3.50mmHg [95%CI 0.09, 6.91];). These findings suggested that artificial oxygen pneumoperitoneum could increase PaO2 levels and that abdominal lifting and compression could further increase PaO2 levels during asphyxia.
3.5. Changes in HR and MAP during asphyxia
To investigate the safety and effect of this method on hemodynamics, we compared changes in HR and MAP in each group every 2 minutes, from baseline to the 10th min after asphyxia(Fig. 3). HR appeared to initially accelerate, then slowed(Fig.3A). No differences were observed among the groups (Fig. 3A and 3C),although HR in the Control group tended to be lower than that in other groups at 10min(Fig. 3A and 3C). MAP appeared to increase in the Pneu, P+1, and P+5 groups; in the Control group, it tended to initially increase, then decrease. Although no differences were observed among the groups(Fig. 3B and 3D), MAP at 10min in the Control group tended to be lower than that in other groups (Fig. 3B and 3D).
3.6. Artificial oxygen pneumoperitoneum combined with abdominal lifting and compression did not alleviate or aggravate the impact of hypoxia
To investigate the effects of hypoxia on hepatic and renal function, we assessed enzymes related to hepatic and renal function and oxidative stress at baseline, as well as at 2h and 24h after each experiment (Fig. 4). There were no differences in changes in ALT, AST, Crea, BUN, LDH, CK-MB, SOD, or MAO levels among groups. BUN, LDH, and SOD exhibited differences over time (Fig. 4D, 4E, 4G). After the experiment, BUN and LDH increased, while SOD decreased; however, all were within normal ranges. This indicates that the dogs had experienced hypoxic injuries, all of which were reversible. Moreover, the method did not appear to alleviate or aggravate this effect of hypoxia.