Esophageal capnometry during hemorrhagic shock and after resuscitation in rats

Background Splanchnic perfusion following hypovolemic shock is an important marker of adequate resuscitation. We tested whether the gap between esophageal partial carbon dioxide tension (PeCO2) and arterial partial carbon dioxide tension (PaCO2) is increased during graded hemorrhagic hypotension and reversed after blood reinfusion, using a fiberoptic carbon dioxide sensor. Materials and method Ten Sprague–Dawley rats were anesthetized, tracheotomized, and cannulated in one femoral artery and vein. A calibrated fiberoptic PCO2 probe was inserted into the distal third of the esophagus for determination of luminal PeCO2 during maintained anesthesia (pentobarbital 15 mg/kg per hour), normothermia (38 ± 0.5°C), and fluid balance (saline 5 ml/kg per hour). Three out of 10 rats were used to determine the limits of hemodynamic stability during gradual hemorrhage. Seven of the 10 rats were then subjected to mild and severe hemorrhage (15 and 20–25 ml/kg, respectively). Thirty minutes after severe hemorrhage, these rats were resuscitated by reinfusion of the shed blood. Arterial gas exchange, hemodynamic variables, and PeCO2 were recorded at each steady-state level of hemorrhage (at 30 and 60 min) and after resuscitation. Results The PeCO2–PaCO2 gap was significantly increased after mild and severe hemorrhage and returned to baseline (prehemorrhagic) values following blood reinfusion. Base deficit increased significantly following severe hemorrhage and remained significantly elevated after blood reinfusion. Significant correlations were found between base deficit and PeCO2–PaCO2 (P < 0.002) and PeCO2 (P < 0.022). Blood bicarbonate concentration decreased significantly following mild and severe hemorrhage, but its recovery was not complete at 60 min after blood reinfusion. Conclusion Esophageal–arterial PCO2 gap increases during graded hemorrhagic hypotension and returns to baseline value after resuscitation without complete reversal of the base deficit. These data suggest that esophageal capnometry could be used as an alternative for gastric tonometry during management of hypovolemic shock.

Studies have demonstrated that an increase in veno-arterial PCO 2 gradient could be a reliable marker of tissue hypoperfusion [28][29][30][31][32]. Knichwitz and coworkers [25] demonstrated that continuous intramucosal PCO 2 measurement allows early detection of regional intestinal ischemia before the onset of changes in global hemodynamic or metabolic variables. Furthermore, measurement of tissue PCO 2 in several organs has been shown to correlate with gastrointestinal perfusion [8,26,27,33]. Sato and coworkers [8] studied the relationship between gastric wall PCO 2 and esophageal PCO 2 (PeCO 2 ) before, during, and after reversal of hemorrhagic shock in five spontaneously breathing rats, using an ion-sensitive fieldeffect transistor. They found a high correlation (r = 0.9) between the gastric wall PCO 2 and PeCO 2 during hemorrhagic hypotension induced reduction in splanchnic blood flow. The use of tissue PCO 2 and arterial PCO 2 (PaCO 2 ) difference is a better marker of ischemia than is either gastric intramucosal pH or intramucosal PCO 2 [34] because the gap is not influenced by alveolar ventilation [35]. Therefore, in the present study we measured intraluminal PeCO 2 using a rapidly responsive fiberoptic sensor [25,35,36]. The arterial blood gases were periodically measured for determination of the PeCO 2 -PaCO 2 gap. Our hypothesis is that the PeCO 2 -PaCO 2 gap could be significantly increased during graded hemorrhagic hypotension and will return to baseline shortly after resuscitation.

Surgical procedures
The experimental protocol for the present study was approved by the Institutional Animal Care and Use Committee of Miami Children's Hospital. Ten young, albino Sprague-Dawley rats (250-350 g) were initially anesthetized with 60 mg/kg pentobarbital intraperitoneally. In a supine position, a tracheostomy was performed and an endotracheal tube (3.5 cm of a polyethylene tube, 2.4 mm diameter) was advanced to a position approximately 1 cm above the carina. Subsequently, a femoral vein and a femoral artery were exposed and cannulated. Each rat then was placed over an electric heating blanket. Rectal temperature (TH-5; Physitemp Thermalert, Clifton, NJ, USA; with a rat size thermal probe), mean arterial blood pressure (MABP), and heart rate (HR; 2001A, Datascope Corp, Paramus, NJ, USA) were continuously monitored. Normothermia (38 ± 0.5°C) was established while anesthesia (pentobarbital 15 mg/kg per hour) and fluid balance (saline 5 ml/kg per hour) were strictly maintained (Medfusion pump 2010; Medex, Duluth, CA, USA). Rats breathed room air, spontaneously, during the experiments.

Esophageal capnometry
The esophagus was intubated orally with a 22-gauge, 1.5-inch-long catheter. A fiberoptic carbon dioxide sensor (Paratrend 7; Diametrics Medical Inc, Roseville, MN, USA) was introduced through the oral catheter up to 8-10 cm from the incisor teeth into lower third of the esophagus (at 2-3 cm above the gastroesophageal junction). The fiberoptic sensor consisted of two optical fibers for the measurement of PCO 2 and pH, a miniature Clark electrode for determination of partial oxygen tension, and a thermocouple for measuring temperature. The sensor was automatically calibrated with precision gases under microprocessor control, as per the manufacturer's recommendations, before insertion into the esophagus.

Baseline measurements
Within 30-60 min after the insertion of the sensor, baseline values for PeCO 2 , core temperature, HR, and MABP were recorded. The rats then were heparinized with 200 U/kg per hour heparin and an arterial blood sample was taken for baseline (time 0) gas analysis (ABL-30 Blood Gas Analyzer; Radiometer, Copenhagen, Denmark), hemoglobin, and arterial oxygen saturation (OSM3 Hemoxymeter; Radiometer). Measurements of PaCO 2 and PeCO 2 , as well as partial arterial oxygen tension, were corrected for each animal's body temperature. Values for bicarbonate and base excess were automatically calculated by the blood gas analyzer's program.

Hemorrhagic hypotension
Three out of the 10 rats were used to test the limits of hemodynamic stability during hemorrhagic hypotension in this model. Gradual bleeding up to 15 ml/kg in these three rats led to a 30-40% reduction in MABP. Additional bleeding up to 25 ml/kg was tolerated as long as the MABP did not drop below 30 mmHg. Lower blood pressures, caused by removal of 25 ml/kg blood, created a deteriorating and irreversible systemic hypotension, accompanied by severe tachycardia. Therefore, in the actual experiments (n = 7) we considered 15 ml/kg bleeding over a 30-min period as mild hemorrhagic hypotension. Removal of 20-25 ml/kg blood, while maintaining a MABP equal to or higher than 35 mmHg, was considered severe hemorrhagic hypotension. The blood was collected in a heparinized (400 U) tube and incubated at 38°C. Thirty minutes after mild hemorrhagic hypotension, all the baseline variables were again measured. This procedure was repeated after removal of another 5-10 ml/kg blood (for generation of severe but reversible hemorrhage). All variables were recorded during severe hemorrhagic hypotension, and then the shed blood was reinfused over 20-30 min. All variables were measured again at 30 and 60 min following termination of blood reinfusion. At the end of the experiment, the animals were killed with intravenous pentobarbital and the exact position of the esophageal probe was verified.

Statistical analysis
Statistical evaluation was performed in the seven rats that completed mild and severe hemorrhage with resuscitation. All variables are presented as mean ± SD. The data were computed by repeated measures of analysis of variance followed by Dunnett multiple comparisons test, using the baseline values as controls. A linear regression analysis was also performed to evaluate association between PeCO 2 -PaCO 2 gap and the base deficit. P < 0.05 was considered statistically significant.

Hemodynamic and gas exchange variables
Mild and severe homorrhagic hypotension created average reductions of 33% and 53% in MABP, respectively. Reinfusion of the blood restored MABP to the normal range. Blood hemoglobin concentration followed a pattern similar to that of blood pressure ( Table 1). The HR was significantly increased following severe hemorrhage (29%). After blood reinfusion, the HR remained significantly higher than its prehemorrhagic baseline value ( Table 1). The partial arterial oxygen tension was increased significantly during both mild and severe hemorrhagic hypotension, apparently caused by hyperventilation. The latter also reduced the PaCO 2 significantly (Fig. 1). Arterial saturation following blood reinfusion was not significantly different from baseline. Blood bicarbonate concentrations decreased significantly following hemorrhage, but recovery was not complete at 60 min after blood reinfusion (Table 1).

Esophageal-arterial partial carbon dioxide tension gap and base deficit
The PeCO 2 -Pa PCO 2 was significantly increased after mild and severe hemorrhage, and returned to baseline values following blood reinfusion (Fig. 1). The base deficit became slightly more negative after mild hemorrhage but was significantly reduced after severe hemorrhage (-5.5 mmol/l and -14.4 mmol/l, respectively). The base deficit remained significantly high after blood reinfusion (-7.2 mmol/l after 60 min). After blood reinfusion, unlike base deficit, the PaCO 2 rapidly normalized (Table 1). A significant correlation was found between base deficit and PeCO 2 -PaCO 2 gap during hemorrhagic hypotension ( Fig. 2; r 2 = 0.39, P < 0.002). At the same time, there was also a significant correlation between base deficit and PeCO 2 ( Fig. 3; r 2 = 0.24, P < 0.022).

Discussion
A correlation between PeCO 2 and gastric PCO 2 during hemorrhagic shock was previously demonstrated in spontaneously breathing rats [8]. Our results, using a fiberoptic carbon dioxide sensor, are generally in agreement with those of Sato and coworkers [8], who used an ion-sensitive field-effect transistor sensor. In the present study, unlike that of Sato and Available online http://ccforum.com/content/7/1/79 Table 1 Gas exchange variables, partial esophageal carbon dioxide tension, and hemodynamic variables during mild and severe hemorrhagic hypotension and following blood reinfusion in anesthetized, spontaneously breathing rats coworkers, PeCO 2 did not significantly increase during hemorrhage, whereas the PeCO 2 -PaCO 2 gap was significantly increased. The PeCO 2 -PaCO 2 gap returned to baseline immediately after resuscitation (Fig. 1). Our data also demonstrate a significant association between the PeCO 2 -PaCO 2 gap and the corresponding base deficit that occurred during hemorrhagic hypotension (Fig. 2). Whereas the PeCO 2 -PaCO 2 gap rapidly recovered after resuscitation (Fig. 1), the base deficit did not completely return to baseline after restoration of blood volume ( Table 1).
The animals in our study hyperventilated because of metabolic acidosis, presumably secondary to hypoperfusion. Arterial hypocapnia can impact on the expected rise in tissue PCO 2 that occurs as a result of decreased tissue perfusion. Therefore, intramucosal PCO 2 as an indicator of tissue hypoperfusion is not as accurate as PeCO 2 -PaCO 2 [34]. Moreover, the tissue PCO 2 and PaCO 2 gap is not influenced by alveolar ventilation [37]. However, when ventilation is controlled, the change in tissue PCO 2 by itself could become a reliable indicator of tissue perfusion. In our spontaneously breathing rats the PeCO 2 was lower after severe hemorrhage. We reason that the PeCO 2 would have been higher if the rats were mechanically ventilated to maintain a relative arterial normocapnia. In ventilated subjects, change in tissue PCO 2 is an indicator of changes in tissue perfusion before any other global parameters of perfusion are changed [25,38]. In spontaneously breathing subjects, continuous measurements of tissue PCO 2 and PaCO 2 gap can be used as an early indicator of tissue hypoperfusion.

Figure 1
Changes in partial arterial carbon dioxide tension (PaCO 2 ), partial esophageal carbon dioxide tension (PeCO 2 ) and esophageal-arterial PCO 2 gap in seven anesthetized, spontaneously breathing rats subjected to mild and severe hemorrhagic hypotension followed by blood reinfusion. *P < 0.05, by repeated measures of analysis of variance followed by Dunnett multiple comparison test, using baseline as controls.

Figure 2
Linear regression analysis of the association between partial esophageal carbon dioxide tension (PeCO 2 ) minus partial arterial carbon dioxide tension (PaCO 2 ; i.e. PeCO 2 -PaCO 2 gap) and base deficit in seven anesthetized, spontaneously breathing rats during mild and severe hemorrhagic hypotension. Broken lines represent the upper and lower limits of 95% confidence interval.

Figure 3
Linear regression analysis of the association between partial esophageal carbon dioxide tension (PeCO 2 ) and base deficit in seven anesthetized, spontaneously breathing rats during mild and severe hemorrhagic hypotension. Dotted lines represent the upper and lower limits of 95% confidence interval.
tonometry, and therefore gastric acid suppression may be needed for reliable measurements [20]. Other limiting factors in gastric tonometry are related to feeding [22,23] and the large lumen of the stomach, requiring longer time for intraluminal contents to equilibrate with intramucosal PCO 2 . Moreover, in the presence of low gastric pH, secretion of bicarbonate leads to intraluminal production of carbon dioxide [39]. The above factors may prevent rapid detection of changes in intramucosal PCO 2 . Therefore, several other sites have been used for tonometry. In animals, ileum has been used to assess splanchnic perfusion [25,36] -a clinically impractical procedure. Studies have demonstrated that sublingual capnometry, a relatively noninvasive procedure, correlates with gastric tonometry [26,[40][41][42]. Practically, it may be difficult to lodge the sensor securely under the tongue in uncooperative patients, thereby preventing equilibration with tissue PCO 2 [27]. Esophageal intubation, which is commonly used in critically ill patients, can be utilized to secure placement of the esophageal sensor. Similar to the gastric environment, bicarbonate is secreted in the esophagus and may enter the esophagus from salivary secretions. However, a relative alkaline pH in the esophagus, in the absence of acid reflux, may not lead to generation of a significant amount of carbon dioxide. Currently available tonometers have equilibration periods ranging between 10 and 90 min [16][17][18][19] and are therefore not efficient for rapid detection of changes in tissue perfusion on a continuous basis. Fiberoptic sensors that are used in clinical medicine for automatic and continuous measurements of blood gases [43,44] have a rapid response time [45]. Experimental evaluation of a fiberoptic PCO 2 sensor, similar to that used in the present study, has shown a high degree of precision in detecting short-term changes in intramucosal PCO 2 [35].

Capnometry and end-points of resuscitation
An interesting observation in the present study was the delayed recovery of base deficit after resuscitation (Table 1), whereas PeCO 2 , PaCO 2 , and the gap between them were actually recovered (Fig. 1). Porter and Ivatury [46] demonstrated that the use of base deficit, lactate, and/or gastric intramucosal pH are appropriate end-points of resuscitation for trauma patients. They also recommended that one or all of the above markers of tissue perfusion be corrected to normal range within 24 hours after injury. Povoas and coworkers [42] reported persistently elevated blood lactate level after reinfusion of blood when all other parameters of tissue perfusion, such as sublingual PCO 2 , gastric PCO 2 , and veno-arterial PCO 2 gradient, were normalized. In the present study, the delay in normalization of the base deficit in the face of a rapid normalization of the PeCO 2 -PaCO 2 gap may suggest that the PeCO 2 -PaCO 2 gap can serve as an early indicator for resuscitation end-point rather than base deficit. Physiologically, it takes time for liver and kidneys to correct metabolic acidosis following tissue dysoxia. It is therefore anticipated that there will be a lag phase between restoration of blood volume and return of base deficit to normal.
Studies indicate that PeCO 2 -PaCO 2 gap can continue to increase or remain abnormally high after resuscitation [25,47,48]. In those experiments [47,48], severe hemorrhage (45-47 ml/kg versus 30 ml/kg) might have contributed to ischemia/reperfusion injury, leading to persistent mucosal hypoperfusion and elevated tissue PCO 2 -PaCO 2 gap. In the presence of ischemia/reperfusion mucosal injury, the PeCO 2 -PaCO 2 gap may not return to normal even after restoration of circulatory volume. In such instances, base deficit (or other global parameters of tissue perfusion) may be a better index for the end-point of resuscitation.

Conclusion
The data presented here demonstrate that PeCO 2 -PaCO 2 gap increases during hemorrhagic hypotension and reverses after resuscitation, without complete recovery of base deficit. We suggest that esophageal capnometry could be used as an alternative to gastric tonometry for assessing splanchnic hypoperfusion.

Key messages
• Esophageal capnometry could be used as an alternative for gastric tonometry during the management of hypovolemic shock • PeCO 2 -PaCO 2 gap increases during graded hemorrhagic hypotension and returns to baseline value after resuscitation, without complete reversal of the base deficit