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MUCOSAL BIOLOGY
Division of Physiology, Department of Neuroscience, Uppsala University, Uppsala, Sweden
Submitted 26 August 2006 ; accepted in final form 30 November 2006
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ABSTRACT |
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anesthesia; murine physiology; in vivo; duodenal mucosal protection
During recent years, the mouse has become the most commonly used species in molecular medicine. Increasing knowledge of the mouse genome and the exponentially increased availability of genetically modified mice make it an attractive species for use in physiological research. There is no doubt that the information gained from experiments in genetically modified mice has broadened our knowledge about physiological mechanisms. Unfortunately, however, most in vivo studies in the mouse have been performed without assessing arterial blood pressure or acid-base balance. Hence, the investigator of these studies has little knowledge about the condition of the animals except subjective estimates. This may lead to erroneous conclusions.
In mice, various types of anesthesia are available, such as ketamine, chloral hydrate, barbiturates, benzodiazepines, and urethane (5). Some of these drugs are short acting and have to be repetitively injected into the animal to maintain the depth of anesthesia. This, in turn, could influence the physiological parameters studied. Therefore, in experiments that last for several hours, it is important to keep the depth of the anesthesia as steady as possible. In small laboratory animals, including mice, isoflurane is the recommended inhalation anesthesia due to its long-term, safe, and easy-to-control properties (5). However, this anesthetic is not without side effects. A commonly reported side effect of isoflurane anesthesia in mice is disturbance of the acid-base balance, e.g., acidosis (8, 13, 24, 31).
Duodenal mucosal bicarbonate secretion (DMBS) is considered the main mechanism in duodenal protection against acid discharged from the stomach, and this secretion is impaired in patients with acute and chronic duodenal ulcer disease (14). Acid in the duodenal lumen is the main physiological stimulus of bicarbonate secretion, and the HCl-induced rise in secretion is in part mediated by endogenous prostaglandins and neural reflexes (25). In anesthetized animals, the in vivo induction of metabolic acidosis has been shown to reduce small intestinal bicarbonate secretion, demonstrating the importance of keeping acid-base variables as normal as possible (3, 28).
The aim of the present study was to investigate the impact of isoflurane-induced acidosis on DMBS in mice. The duodenum was perfused with isotonic saline, and DMBS was determined by back titration of the effluent. Basal DMBS and the effect of luminally applied PGE2 were examined in control animals and in animals treated with a constant infusion of Na2CO3. The acid-base status of the animal was determined by assessing gases, pH, bicarbonate concentration, and base excess in arterial blood.
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MATERIALS AND METHODS |
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All experiments were approved by the Uppsala Ethics Committee for Experiments with Animals. C57bl/6J mice (14 ± 2 wk of age) weighing 28–36 g were obtained from Scanbur B&K Universal (Sollentuna, Sweden). Animals were maintained under standardized temperature and light conditions (12:12-h light-dark cycle, temperature: 21–22°C). Mice were kept in cages and had access to tap water and pelleted food (Ewos, Södertälje, Sweden) ad libitum.
Surgical Procedures
Mice were anesthetized by a spontaneous inhalation of isoflurane (Forene, Abbott Scandinavia, Kista, Sweden). The inhalation gas contained a mixture of 
30–40% oxygen, 60–70% nitrogen, and 2.2 ± 0.2% isoflurane with the use of an isoflurane pump (Univentor 400 Anaesthesia Unit, AgnTho, Lidingö, Sweden) and was administered continuously through a breathing mask. Anesthesia was induced by a spontaneously inhalation of isoflurane, and the depth of the anesthesia was tested by probing the pedal withdrawal reflex, as recommended by Flecknell (5). The gas flow through the cylindrical mask (2 cm long with an inner diameter of 1.2 cm) was about 200 ml/min, which minimized animals' rebreathing of exhaled CO2. Body temperature was maintained at 37.5°C by means of a heating pad controlled by a rectal thermistor probe. A catheter was placed in the left carotid artery to monitor blood pressure and for the continuous infusion of either a standard Ringer solution [containing (in mM) 145 Na+, 124 Cl–, 2.5 K+, 0.75 Ca2+, and 25 HCO3–] or an isotonic Na2CO3 solution [containing (in mM) 200 Na+ and 100 CO
] at a rate of 0.35 ml/h.
Duodenal Preparation
A laparotomy was performed, and the bile and pancreatic ducts were ligated very closely to its entrance into the duodenum to prevent pancreaticobiliary juice from entering the duodenum. Silicone tubing was introduced through a hole made in the forestomach, guided through the stomach and pylorus, and secured by a ligature 2–3 mm distal to the pylorus. A polyethylene-200 (Becton-Dickinson, Parsippany, NJ) cannula was inserted into the duodenum 1.5 cm distal to the pylorus and secured by ligatures. The duodenal proximal tubing was connected to a peristaltic pump (Gilson minipuls 3, Villiers, Le Bel, France), and the segment was perfused with isotonic saline (154 mM NaCl) at a rate of 0.25 ml/min.
To complete the duodenal preparation, the abdominal cavity was closed with sutures. After surgery, 30 min was allowed for cardiovascular, respiratory, and intestinal functions to stabilize before experiments were commenced.
Systemic blood acid-base parameters were determined (AVL Compact 3, Graz, Austria) in 40-µl arterial blood samples. The first blood sample [at time (t) = 0 min] was taken 30 min after the completion of surgery, which lasted about 20 min. The second blood sample was taken at the end of the experiment, i.e., 2 h after the first one (t = 120 min).
Measurement of Luminal Alkalinization
The rate of luminal alkalinization was determined by back titration of the perfusate to pH 4.90 with 10 mM HCl under continuous gassing (100% N2) using pH-stat equipment (Schott, TitroLine-easy, Mainz, Germany). The amount of titrated HCl was considered equivalent to DMBS. The pH electrode was routinely calibrated with standard buffers before the start of the titration. Rates of luminal alkalinization were expressed as micromoles of base secreted per centimeter of the intestine per hour.
Statistical Analysis
Descriptive statistics are expressed as means ± SE, with the numbers of experiments given in parentheses. The statistical significance of data was tested by repeated-measures ANOVA. To test differences within a group, one-factor repeated-measures ANOVA was used, followed by Fishers's protected least-significant difference (PLSD) post hoc test. Between groups, two-factor repeated-measures ANOVA followed by one-way ANOVA at each time point was used. If the ANOVA was significant at a given time point, a Fisher's PLSD post hoc analysis was used. All statistical analyses were performed on an IBM-compatible computer using StatView 5.0 software. P values of <0.05 were considered significant.
Experimental Protocols
Blood samples for acid-base analysis were taken at the start (t = 0) of the experiment and after 120 min. Mean arterial blood pressure (MAP; in mmHg) and body temperature (°C) were monitored continuously and recorded at 10-min intervals.
MAP and acid-base balance.
The carotid artery was catheterized but no laparotomy was performed. In group I (n = 6), mice were infused intra-arterially with Ringer solution [containing (in mM) 145 Na+, 124 Cl–, 2.5 K+, 0.75 Ca2+, and 25 HCO3–], and, in group II (n = 6), mice were given isotonic Na2CO3 solution [containing (in mM) 200 Na+ and 100 CO] at a rate of 0.35 ml/h.
Basal HCO3– secretion, MAP, and acid-base balance. The complete duodenal surgery was performed as described above. In group III (n = 6), mice were given Ringer solution, and animals in group IV (n = 6) were given Na2CO3 solution at a rate of 0.35 ml/h. Basal DMBS was measured in 10-min intervals.
PGE2-stimulated HCO3– secretion, MAP, and acid-base balance. The complete duodenal surgery was performed as described above. In group V (n = 6), mice were given Ringer solution, and animals in group VI (n = 6) were given Na2CO3 solution at a rate of 0.35 ml/h. Secretion was stimulated luminally with three concentrations of PGE2 (5.0 µM at t = 20–40 min, 10 µM at t = 60–80 min, and 50 µM at t = 100–120 min). DMBS was measured at 10-min intervals.
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RESULTS |
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In animals where no laparotomy was performed, MAP was stable during the 120-min experimental period. Mean MAP was slightly higher (P < 0.05) in mice infused with Na2CO3 solution (88.0 ± 0.4 mmHg, n = 6) than in Ringer solution-infused mice (84.0 ± 0.3 mmHg, n = 6), as shown in Fig. 1. Blood gas analysis at 0 and 120 min in isoflurane-anesthetized mice infused with Ringer solution (group I) showed clear mixed respiratory and metabolic acidosis. The acidosis became more pronounced with time, i.e., blood pH and base excess were both lower (P < 0.05) at t = 120 min than at t = 0 min (Table 1). Blood gas values in Na2CO3 solution-infused animals (group II) were normal (Table 1).
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As shown in Fig. 2, basal bicarbonate secretion was significantly (P < 0.05) greater (60%) in group IV, which had a mean rate of 6.5 ± 0.53 µmol·cm–1·h–1 (n = 6), than in group III, which had a mean rate of 4.1 ± 0.55 µmol·cm–1·h–1 (n = 6).
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22 mM (t = 0 min) to 28 mM (t = 120 min), but all other values were within the normal range (Table 2).
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As in group IV, basal DMBS was significantly (P < 0.05) higher (75%) in group VI compared with that in groups III or V, as shown in Fig. 3. Basal DMBS in groups V and VI was 3.4 ± 0.40 (n = 6) and 6.0 ± 0.38 µmol·cm–1·h–1 (n = 6), respectively. As shown in Figs. 3 and 4, the rise in DMBS in response to the well-characterized physiological secretagogue PGE2 was significantly lower in animals infused with Ringer solution (group V) compared with animals infused with isotonic Na2CO3 solution (group VI). Mean MAP was stable in both group V (74.2 ± 1.3 mmHg, n = 6) and group VI (75.2 ± 1.4 mmHg, n = 6), as shown in Fig. 3. Mice in group V had respiratory and metabolic acidosis (Table 3), which became more pronounced with time. Blood gas values in group VI were within the normal range (Table 3).
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DISCUSSION |
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The present study demonstrates that arterial acid-base values in isoflurane-anesthetized mice clearly deviate from those in conscious humans and rats. A relevant question is whether the acid-base values in Ringer solution-treated mice are to be considered abnormal, i.e., acidemic, or not. Typical values for pH and PCO2 previously reported in isoflurane-anesthetized spontaneously breathing mice have ranged, respectively, from 7.35 and 39 mmHg (27) to 7.25 and 49 mmHg (8). These values are in fair agreement with those in the Ringer solution-infused mice found here. Since corresponding values in conscious mice have been reported to be 7.42 and 25 mmHg (22), one can conclude that isoflurane anesthesia induces acidosis. However, it should be kept in mind that the acid-base values reported from conscious mice might be confounded by acute respiratory alkalosis induced by the trauma of tail-nick blood sampling.
In the present study, we observed that isoflurane-anesthetized mice, infused with Ringer solution, develop acidosis that worsens with time in nonabdominal-operated mice and in those subjected to a duodenal operation. The Na2CO3 infusion effectively normalized acid-base parameters with the exception of arterial PCO2, which was elevated at t = 120 min, particularly in mice subjected to the abdominal operation. Thus, it appears that the abdominal operation worsens the ability of mice to maintain alveolar ventilation, thereby increasing arterial PCO2. Although the Na2CO3 infusion cannot prevent arterial PCO2 from rising, it is important to note that if the acidosis is not compensated for, there is a marked drop in plasma pH, particularly in mice subjected to the abdominal operation, which weakens the physical condition of the mouse even further.
Although there were very small differences in blood pressure between mice with acidosis and those with a normal acid-base balance, the general trend was lower MAP during acidosis. MAP in the present study (80 mmHg) was similar to values previously reported in isoflurane-anesthetized mice (8, 16, 24) but slightly lower than those in conscious mice (16, 29, 30). A mouse with a body weight of 20–30 g has a blood volume of 1–2 ml. It is thus very important to make volumes of blood samples as low as possible to avoid hemodynamic effects (31). In the present study, a total of two blood samples, 40 µl each, were taken, with the second one at the end of the experiment. The first blood sampling had no effect on mean MAP.
The results of the present investigation clearly demonstrate that isoflurane-induced acidosis compromises the capability of the duodenal mucosa to secrete bicarbonate. This view is substantiated by the finding that when the acidemia was compensated for by an intra-arterial infusion of 100 mM Na2CO3, basal DMBS increased by 60 to 100%.
It might be argued that the increased DMBS is caused primarily by the rise in the plasma concentration of bicarbonate due to the constant infusion of Na2CO3 into the bloodstream. Theoretically, the higher plasma concentrations of bicarbonate should increase the concentration gradient between blood and lumen, increasing the diffusion of bicarbonate into the duodenal lumen via "leaky" paracellular pathways (1). However, this would seem most unlikely in the present in vivo experiments. First, the basal secretion in Na2CO3 solution-infused animals did not increase during the 120-min experimental period, although the plasma concentration of bicarbonate increased significantly from 22 to 28 mM during the same time. Second, previous data have strongly suggested that the proximal duodenal mucosa transports bicarbonate into the luminal solution by active, physiologically regulated transport, and evidence has been presented that this transport of bicarbonate involves apical Cl–/HCO3– exchangers and the CFTR (6, 9–11, 15, 26). In the rat duodenum, luminal perfusion with lidocaine, a local anesthetic, increases paracellular permeability to 51Cr-EDTA sevenfold and, at the same time, reduces DMBS by 80% (21). These results suggest that active transcellular bicarbonate transport is much greater than the paracellular transport of this ion and that an increase in the paracellular transport has little impact on total alkalinization. Finally, tissue acidosis has been shown to increase (23), whereas elevation of the serosal bicarbonate concentration decreases, paracellular permeability (19, 20), possibly suggesting that the Na2CO3 infusion would decrease rather than increase the paracellular permeability to bicarbonate.
Previous findings in the rat have shown that both ileal and colonic bicarbonate secretion increase in response to acute respiratory acidosis (4). This may in part relate to the increase in arterial PCO2. Supporting this is the finding, in the anesthetized rabbit, that artificial respiration reduces blood PCO2 and decreases DMBS (12). Furthermore, increasing PCO2 but keeping PO2 at normal levels in the respirator air increased DMBS by a mechanism that was abolished by the carbonic anhydrase inhibitor acetazolamide. These results suggest that the high arterial PCO2 is utilized by the duodenal epithelium to produce bicarbonate (1). However, this does not appear to be the case in isoflurane-anesthetized mice because DMBS remained stable despite the fact that arterial PCO2 increased with time.
The differences in secretory rates between mice with acidosis and those with normal acid-base balance were even greater when DMBS was stimulated with PGE2. Three different concentrations of PGE2 were used to stimulate the secretion (5, 10, and 50 µM) in both experimental groups. In mice with acidosis, the lowest concentrations of PGE2 tested did not increase DMBS. Although higher concentrations of PGE2 stimulated bicarbonate transport, the increments were much lower in mice with acidosis than in those with a normal acid-base balance.
We (18) have recently shown that the duodenal secretory response to PGE2 is completely dependent on cytosolic carbonic anhydrase II, i.e., an enzyme important for intracellular pH regulation (18). Mice that lack carbonic anhydrase II have a normal plasma concentration of bicarbonate, i.e., 25 mM, but a blood pH of 7.25 despite an intra-arterial infusion of Na2CO3. This hints that it is the decrease in tissue pH induced by isoflurane rather than the reduction of the plasma bicarbonate concentration that deteriorates the ability of the duodenal epithelium to secrete bicarbonate. The finding that mice with acidosis are less sensitive to PGE2-induced stimulation of bicarbonate secretion than those with a compensated acid-base balance supports the view of some sort of enterocyte malfunction in mice with acidosis.
It is concluded that isoflurane-induced acidosis markedly depresses basal and PGE2-stimulated DMBS as well as the responsiveness to PGE2, an effect that was prevented by a continuous infusion of Na2CO3. When performing in vivo experiments in mice kept anesthetized by a spontaneous inhalation of isoflurane, it is recommended to assess the acid-base balance and, if necessary, supplement with a Na2CO3 infusion to maintain a normal acid-base balance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Significant net increase in DMBS during PGE2 stimulation; *significant secretory difference between the Na2CO3 solution-infused group (n = 6) and the Ringer solution-infused group (n = 6). Bottom: no differences in MAP were observed.