The current study was undertaken to investigate the effects of pretreatment with isoflurane and sevoflurane on the development of neurogenic pulmonary edema in an animal model.
Rats were exposed to room air (control), 1.5% isoflurane, or 2.5% sevoflurane for 4 h. They were then anesthetized with intraperitoneal injections of pentobarbital sodium, and fibrinogen and thrombin were injected into the cisterna magna to induce neurogenic pulmonary edema.
Consecutive injections of fibrinogen and thrombin caused increases in blood pressure, with the peak values obtained in the isoflurane and sevoflurane groups being lower than the control values. The incidence of significant neurogenic pulmonary edema was 58%, 100%, and 8% in the control, isoflurane, and sevoflurane groups, respectively. The lung water ratio, an index of severity of edema, was 4.86 +/- 0.78, 6.15 +/- 0.64, and 4.40 +/- 0.32 in the control, isoflurane, and sevoflurane groups, respectively. Furthermore, immunohistochemical staining for vascular endothelial growth factor demonstrated an increase of expression in the rat lungs exposed to isoflurane. Treatment with an anti-vascular endothelial growth factor antibody during exposure to isoflurane completely inhibited the effect of isoflurane to promote neurogenic pulmonary edema in this model.
Exposure to 1.5% isoflurane enhances the development of neurogenic pulmonary edema development in this animal model, most likely via release of vascular endothelial growth factor from bronchial epithelial cells, an effect not observed with sevoflurane.
THE pathogenesis of neurogenic pulmonary edema (NPE) is not completely understood, but seems to be associated with enhanced sympathetic nerve activity.1Consistent with this concept, NPE is associated with an increased intravascular pressure and enhanced vascular permeability in the pulmonary circulation, possibly mediated by sympathetic nerve neurotransmitters released from nerve terminals, e.g. , neuropeptide Y.2–4Synergistic interaction between neurotransmitters in vascular beds has also been shown to play a role in enhancing increases in vascular pressure and permeability2,5when sympathetic nerve activity is increased. Because anesthetics have been thought to inhibit the activities of autonomic nerves, they have not been considered to affect NPE. However, it has been reported that some inhaled anesthetic agents affect the vascular permeability of biologic membranes to water and electrolytes6; isoflurane increases the permeability of alveolar epithelial cells7and the alveolar–capillary membrane,8and halothane increases fluid conductance across the pulmonary capillary bed,9perhaps by interacting with oxidants. In addition to direct actions on endothelial cells, anesthetic effects on interstitial lung fluid clearance in the lungs could contribute to edema.10Although autonomic nerve activity may be enhanced by surgical maneuvers11and contribute to the development of NPE, it is difficult to explain the development of NPE during anesthesia solely in relation to the actions of neurotransmitters.
Vasoactive substances released from vascular endothelial cells, such as nitric oxide, bradykinin, and vascular endothelial growth factor (VEGF), and agents released by mast cells, such as histamine, can affect vascular smooth muscle and endothelial cells. In particular, VEGF promotes endothelial cell viability, mitogenesis, chemotaxis, and vascular permeability. Recent in vitro vascular permeability studies have shown that it has the ability to increase the microvascular permeability to a level 50,000 times higher than with histamine.4,12,13The current study was undertaken to evaluate the effects of inhaled anesthetics on VEGF expression and NPE development and to determine whether VEGF may mediate NPE development in rats anesthetized with volatile anesthetics. Because sevoflurane has not been reported to increase vascular permeability, the effects of isoflurane on NPE development were compared to those of sevoflurane.
Materials and Methods
All procedures were performed in accordance with the “Guiding Principles in the Care and Use of Animals in the Field of Physiologic Sciences” published by the Physiologic Society of Japan14and with the previous approval of the Animal Care Committee of Aichi Medical University (Aichi-gun, Aichi Prefecture, Japan). Wistar male rats weighing 180–200 g (8–10 weeks old) were used.
The rats were randomly divided into three groups: group 1, exposed to room air as the control (28 animals); group 2, exposed to 1.5% isoflurane (1.09 minimum alveolar concentration [MAC]) plus room air for 4 h (26 animals); and group 3, exposed to 2.5% sevoflurane (1.05 MAC) plus room air for 4 h (26 animals). Rats were placed in transparent plastic boxes (depth 50 cm × height 50 cm × width 80 cm) into which anesthetic gases were pumped at the desired partial pressure through inlet tubes and an outdoor outlet tube. The concentrations of the anesthetic agents in the boxes were monitored along with the gases in the air using an isoflurane, sevoflurane, and respiratory oxygen/carbon dioxide analyzer (Capnomac; Datex Instrumentarium Co. Ltd., Helsinki, Finland). In 12 animals in groups 2 and 3, 0.1 ml anti-VEGF antibody (rabbit polyclonal anti-mouse VEGF immunoglobulin G; VEGF Ab-1, 1 mg/ml; NeoMarkers, Fremont, CA) was injected into the external cervical vein before the exposure to 1.5% isoflurane or 2.5% sevoflurane.
After this 4-h period, animals were moved outside of the plastic boxes used for exposure to inhaled anesthetics and then anesthetized with intraperitoneal injections of pentobarbital sodium (35 mg/kg body weight) for the induction of NPE (see next two paragraphs). Tracheal tubes were inserted after a tracheotomy was performed in the midcervical region. Catheters were introduced into the right femoral vein and artery, the former for blood sampling, and the latter for measurement of arterial blood pressure and heart rates (Multipurpose Polygram, W-1100; Nihon Kohden, Tokyo, Japan). To measure arterial oxygen tension immediately before the induction of NPE, 0.1 ml arterial blood was collected from rat femoral arteries. Arterial oxygen tension was measured with a blood gas analyzer (i -STAT and cartridge EG6+; Abbott Laboratories Ltd., South Pasadena, CA).
In an additional 18 rats, after 4 h of exposure to 1.5% isoflurane, NPE was induced during continued isoflurane administration, rather than during pentobarbital anesthesia. Nine of these animals were pretreated with intravenous injections of anti-VEGF antibody. In these animals, an endotracheal tube was inserted during the anesthesia through a mask (plastic cylinder; 5 cm diameter and 15 cm length), which was connected to the outlet tube from the plastic gas-boxes mentioned above. Thereafter, isoflurane was continuously introduced through the endotracheal tube connected to the mask while NPE was induced.
Neurogenic pulmonary edema was induced as previously described.15Briefly, the animals were fixed in a prone position with a stereotaxic instrument, and the cisterna magna was accessed at the base of the dorsal side of the cranium using a needle (26 gauge, 10 mm long). The vagus nerves were left intact. Rats were consecutively treated with intracisternal injections of fibrinogen and thrombin, 0.075 ml each, at concentrations of 100 mg/ml and 200 U/ml, respectively. The severity of pulmonary edema was graded from 0 to 3, with 0 for none and 3 for severe.15When edema fluid appeared in the tracheal tubes within 10 min, it was collected in plastic tubes for later analysis, and the grade of edema formation was classified as grade 3. When edema fluid did not appear in the tracheal tubes within 10 min, the chest walls were opened. Sometimes edema fluid then spontaneously appeared in the tracheal tubes, and this was classified as grade 2. However, in a few cases, edema fluid only appeared in the tracheal tubes when the lungs were gently compressed, and this was classified as grade 1. When edema fluid did not appear even with such compression, the grade was 0. Finally, in all rats, lungs were dissected out, weighed, and dried at 80°C overnight. The difference between wet and dry weights, relative to the dried lung weight, was used as the lung water ratio, an index of the severity of pulmonary edema.
Immunohistochemistry VEGF mRNA Analysis
Immunohistochemical and VEGF messenger RNA (mRNA) expression studies were performed in two rats from groups 2 and 3 and four rats from group 1 (controls). After opening the chest by a central incision with the aid of mechanical ventilation, a plastic catheter for infusion was inserted into the pulmonary artery through the right ventricle. Blood was washed out with saline, and then paraform aldehyde, dissolved with phosphate buffer (pH 7.4) at a concentration of 4%, was infused at a constant pressure of 80 cm H2O. Thereafter, one lung from each animal was excised, immersed in the same fixative for 12 h, and embedded in paraffin with an auto–paraffin-embedding apparatus (Thermolyne Histo-Center; Shiraimatsu Co. Ltd., Osaka, Japan).
Paraffin blocks were made and sections were cut and subjected to deparaffinization, hydration, and incubation with blocking serum (3% normal goat serum; Vector Laboratories, Burlingame, CA) before exposure to the primary antibody or preimmune serum. For negative controls, preimmune rabbit immunoglobulin G was used at the same concentration. After 2 h of incubation at room temperature, the slides were washed in phosphate-buffered saline and incubated for 30 min with a biotinylated goat anti-rabbit antibody diluted 1:300 in phosphate-buffered saline. We then used the avidin-biotin-complex detection method (Vectastain Elite ABC kit; Vector Laboratories), with DAB as the substrate (DAKO Liquid DAB substrate-chromogen System; DAKO Corp., Carpinteria, CA), to detect VEGF localization. Sections were subsequently dehydrated, mounted, and analyzed by observers blinded to treatment assignment.
Lung homogenates and cultured rat aortic endothelial cells (see next section) exposed to isoflurane or sevoflurane were used for mRNA analyses to estimate VEGF expression. Reverse transcription polymerase chain reaction was performed with commercially provided reagents on Ready-To-Go reverse transcription polymerase chain reaction beads (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The program for the thermal cycler (TaKaRa PCR Thermal Cycler MP; Takara Co. Ltd., Tokyo, Japan) was as follows: 95°C for 1 min, 50°C for 1 min, 72°C for 2 min, for 30 cycles, with 0.1 μg poly(A )+mRNA as the template and pd(T )12–18as the first-strand primer. The polymerase chain reaction primers for VEGF were as follows: sense, 5′-TTTCGTAATTGAGGACATGG -3′; antisense, 5′-AGAAAAGCCTATGATCTGAG -3′. The polymerase chain reaction products, analyzed by electrophoresis on agarose gels, were normalized for expression of the β-actin housekeeping gene as an internal control: The polymerase chain reaction products obtained for VEGF and β-actin were 448 and 764 base pairs, respectively.
Isolation and Culture of Rat Aortic Endothelial Cells
Rat aortic endothelial cells were isolated from Wistar rats (150–200 g, 7–9 weeks old, male) and cultured according to the methods of Suh et al. 16Briefly, rats were anesthetized with intraperitoneal injections of pentobarbital sodium, 35 mg/kg body weight, and their aortas were removed and placed in phosphate-buffered saline without Ca2+or Mg2+. The vessels were cleaned, opened longitudinally, cut into two or three small pieces, and placed with their intimal side down on Matrigel-coated plates in the growth medium. The growth medium contained 10% FCS, 75 μg/ml ECGS, 10 U/ml heparin, 100 U/ml penicillin–streptomycin, 1% l-glutamine, and 100 μm MEM nonessential amino acids, in Dulbecco’s modified eagle medium. After 4–7 days, the pieces were removed and cells harvested (more than 90% viability of primary cultured cells was obtained when assessed with trypan blue). The cells were identified as endothelial cells when they exhibited positive binding of anti–von Willebrand factor antibody (DakoCytomation, Carpinteria, CA), after fixation with 7:3 methanol and acetone.
Culture plates containing rat aortic endothelial cells were moved to another carbon dioxide incubator to allow incubation under a desired partial pressure (1.5% for isoflurane and 2.5% for sevoflurane) of inhaled anesthetics at 37°C. The incubator was connected to a vinyl bag in which 5% CO2, 20% O2, and desired amounts of nitrogen gas and inhaled anesthetics had been mixed and pumped at an appropriate velocity. At the outlet, partial pressures of carbon dioxide and oxygen were monitored along with inhaled anesthetics (isoflurane and sevoflurane) using a respiratory oxygen/carbon dioxide analyzer (Capnomac). Exposure was for 4 h before sampling of cells for reverse transcription polymerase chain reaction.
All results are expressed as mean ± SD, with n denoting the number of preparations (the number of animals). Parametric data such as blood pressure, heart rate, and lung water ratio were analyzed using paired t tests or analyses of variance performed with Scheffé multiple comparison tests. When the SDs obtained in the parametric tests were significantly different among the groups, a nonparametric statistical method, the Kruskal-Wallis one-way analysis of variance with Dunnett tests, was performed. For nonparametric data such as incidence, the chi-square test was used. P < 0.05 was considered significant.
Both systemic blood pressure and heart rate in the isoflurane and sevoflurane groups were lower than in the rats maintained in room air (control group) (fig. 1and table 1). Consecutive injections of fibrinogen and thrombin into the fourth ventricle greatly increased systemic arterial pressure and heart rate (fig. 1). The peak values for average systemic arterial pressure and heart rate obtained in the isoflurane and sevoflurane groups were significantly lower than those obtained in the control group (both P < 0.001). Changes in systemic arterial pressure in the isoflurane group but not in the sevoflurane group were significantly lower than those observed in the control group (P < 0.001). Changes in heart rate in the isoflurane group, in contrast, were significantly greater than those obtained in the controls (P < 0.05). Changes in heart rate in the sevoflurane group were significantly smaller than with isoflurane (P < 0.05).
Arterial oxygen tension after 4 h of inhalation with anesthetic gases (n = 9, each group) were 87 ± 6 and 86 ± 6 mmHg for isoflurane and sevoflurane, respectively, these values being similar to those measured in rats anesthetized with pentobarbital alone (85 ± 3 mmHg).
Effects of Isoflurane and Sevoflurane on NPE Development
In the control group, 7 of 12 rats showed grade 2 or 3 fibrin-induced pulmonary edema, with 5 exhibiting grade 0 (table 2). Exposure to 1.5% isoflurane for 4 h resulted in a 100% incidence of grade 3 pulmonary edema, significantly higher than the incidence in the control group (P < 0.05). After exposure to 2.5% sevoflurane for 4 h, one showed grade 3, and 11 showed grade 1 or 0, the 8% incidence of grade 2–3 pulmonary edema being significantly lower than that in the isoflurane group (P < 0.001).
Effects of Anti-VEGF Antibody on NPE Development
Mean systemic arterial pressure obtained in the isoflurane group after exposure to the anti-VEGF antibody was significantly lower and the peak pressure after the injections of fibrinogen and thrombin was significantly higher than those obtained without the pretreatment (both P < 0.001; table 1). The heart rate in the isoflurane group, when compared with that without the antibody, was significantly greater (P < 0.001), as was that after fibrin treatment (P < 0.001). Changes in blood pressure and heart rate were significantly greater than without the antibody (P < 0.001 and P < 0.05, respectively). In the sevoflurane and room air groups, no significant differences in either systemic arterial pressure or heart rate were observed between the cases with and without anti-VEGF antibody pretreatment.
Pretreatment with antibody did not significantly affect arterial blood oxygen tension obtained after 4 h of exposure to isoflurane (89 ± 4 mmHg, n = 9).
All 12 rats exposed to 1.5% isoflurane for 4 h were successfully protected from fibrin-induced pulmonary edema development by pretreatment with the anti-VEGF antibody, the incidence being lower than that obtained without the antibody (P < 0.001; table 2). Furthermore, the lung water ratio in rats exposed to 1.5% isoflurane in the presence of the anti-VEGF antibody was significantly lower than that obtained without the antibody (P < 0.001). Rats maintained in room air or exposed to 2.5% sevoflurane were not significantly influenced by the pretreatment with the anti-VEGF antibody in terms of incidence.
VEGF mRNA Expression in Lungs and Cultured Aortic Endothelial Cells
As shown in the upper panel of figure 2, the lungs in isoflurane-exposed rats (lane 2) exhibited greater VEGF mRNA expression relative to β-actin mRNA compared with sevoflurane-exposed or control lungs (lanes 1 and 3, respectively). In contrast, aortic endothelial cells incubated in 2.5% sevoflurane plus 5% CO2or 1.5% isoflurane plus 5% CO2(lanes 4 and 5) demonstrated similar expression compared with cells exposed to room air plus 5% CO2(lane 6).
Immunohistochemistry for VEGF Staining
Figure 3Ashows staining with unimmunized rabbit serum as the primary antibody. Immunohistochemical staining showed no apparent VEGF expression in the lungs of the sevoflurane group, as shown in figure 3B, whereas after isoflurane treatment (figs. 3C–F), bronchial epithelial cells (figs. 3C–E), and smooth muscles (fig. 3D) were strongly immunoreactive. Vascular endothelial cells were also positively stained, especially in the alveolar capillaries (fig. 3F).
Effects of Anti-VEGF Antibody on NPE Development in Rats Anesthetized with Isoflurane for NPE Induction
In the group with 4 h of isoflurane exposure with isoflurane anesthesia during experiments for NPE induction, injections of fibrinogen and thrombin into the fourth ventricle significantly increased the mean systemic arterial pressure from 97.8 ± 12.3 to 144.6 ± 15.3 mmHg and significantly increased the mean heart rate from 288.4 ± 29.6 to 358.4 ± 26.2 beats/min (both P < 0.001). The incidence of grade 2 and 3 fibrin-induced pulmonary edema and the lung water ratio were 100% (nine grade 3 in nine animals), and 4.46 ± 0.23, respectively. Intravenous injection of 0.1 ml anti-VEGF antibody before isoflurane exposure significantly decreased the incidence and lung water ratio to 0% (nine grade 0 in nine animals) and 3.40 ± 0.09 (fig. 4), respectively.
The results obtained in the current study showed the development of fibrin-induced pulmonary edema to be promoted by isoflurane and inhibited by sevoflurane. Because the systemic arterial blood pressure after 4 h of exposure to the two agents did not differ significantly, the difference in incidence cannot be explained simply by this parameter. The inhibitory effects of sevoflurane on NPE development may correspond well with the decrease in systemic arterial pressure,17in accord with the facts that inhaled anesthetics usually diminish nervous activity and that NPE development is associated with enhanced sympathetic nerve activity.3,11However, contrary to expectations, the incidence of fibrin-induced pulmonary edema was increased by exposure to isoflurane.
We examined the effects of isoflurane using two experimental designs. In the first, animals were pretreated with 4 h of isoflurane and then anesthetized with pentobarbital for the induction of NPE. It is possible that this experimental design, which involves two different anesthetics in a similar experimental animal, could complicate the interpretation of the results. For this reason, we performed an additional set of studies in which isoflurane was maintained during the induction of NPE to eliminate any possible confounding effects of pentobarbital. These results were qualitatively similar, with the exception that the absolute values of lung water ratio were less in the latter protocol.
Isoflurane is an inhaled anesthetic that primarily relaxes the vasculature in the systemic cardiovascular system to exert hypotensive effects, in contrast to sevoflurane, which mainly diminishes cardiac output.18Reports document increased alveolar epithelial and capillary permeability in humans exposed to 1.5% isoflurane for approximately 6 h.7,8,19In contrast, sevoflurane elicited an inhibitory action on the capillary filtration coefficient in the lower limbs when assessed with noninvasive computer-assisted venous congestion.20No significant difference between isoflurane and sevoflurane in terms of pulmonary vascular permeability was found with short-duration volatile anesthetic exposure; 30 min of exposure to either isoflurane or sevoflurane before ischemia in isolated rat lungs attenuated the increase of vascular permeability with ischemia–reperfusion injury.21Therefore, it is likely that long exposure to 1.5% isoflurane causes interstitial edema and thus enhances NPE development, unlike sevoflurane, but the exact mechanisms remain to be clarified in detail.
The current immunohistochemical study demonstrated expression of VEGF to be increased in the lungs obtained from rats anesthetized with isoflurane when compared with sevoflurane, suggesting that the increase in lung vascular permeability may be caused via up-regulation of VEGF, a potent inducer of increased permeability. As shown in figure 3, an increase in VEGF expression was found in bronchial epithelial and smooth muscle cells, as well as alveolar capillary endothelial cells. Nonetheless, no marked change was observed in primary cultured endothelial monolayers. This may suggest that bronchial epithelial cells or other cells found in the lung homogenate, rather than endothelial cells, may initially respond to isoflurane exposure. Alternatively, the cultured monolayers may not reflect the situation found in vivo . A wide variety of cell types may express VEGF and VEGF receptors, and therefore, activated macrophages, neutrophils, and alveolar type II epithelial cells cannot be ruled out as candidate targets.22It is possible that VEGF released from the lungs may affect both the alveolar epithelial and endothelial barriers, thereby resulting in gas exchange dysfunction.
Intravenous injection of an anti-VEGF antibody before exposure of rats to 1.5% isoflurane was found to inhibit NPE development completely, whereas in rats anesthetized with pentobarbital or sevoflurane, pretreatment with the antibody did not affect the incidence of NPE. These results suggested that VEGF plays a causative role in the enhancement of NPE development by isoflurane. It has been reported4that both VEGF and neuropeptide Y, a coneurotransmitter with norepinephrine in the sympathetic nerve terminals, enhance albumin permeability across rat aortic endothelial monolayers and that such an action is stronger under hypoxic (5% O2) than normoxic conditions. Neuropeptide Y has little effect on permeability under normoxic conditions but a potent action when the environment is hypoxic. It is possible that the release of VEGF during exposure to isoflurane may cause an interstitial edema in lungs that produces local hypoxia, which may synergistically enhance or cause a further increase in vascular permeability via release of neuropeptide Y, with its vasoconstrictive action and action on sympathetic nerves. Pretreatment with anti-VEGF antibodies could prevent local generation of hypoxic conditions. However, systemic hypoxia was not observed after isoflurane. Whatever the case, it is likely that the increase in VEGF expression observed with isoflurane exposure makes an essential contribution to enhanced NPE development.
Isoflurane is a commonly used volatile anesthetic agent because of its low toxicity.23It is known to increase the nitric oxide content in the rat brain cortex,24an effect abolished by nitric oxide synthase inhibition. Isoflurane increases intracellular calcium concentrations in cerebrocortical neurons.25Therefore, isoflurane may increase nitric oxide concentrations in the cortex, which may diminish autonomic nerve activity, especially sympathetic nerve activity.26It could also affect peripheral nitric oxide concentrations in the tissue, which are also important regulators of endothelial functions affecting vascular tone, endothelial cell permeability, and vascular cell proliferation.27Two studies3,26have shown that an increase in nitric oxide content in the nucleus tractus solitarii was associated with a decrease in the incidence of NPE. However, sevoflurane is also reported to activate nitric oxide synthase to increase central nitric oxide concentrations in the rat brain cortex,24making it less likely that this mechanism contributes to the observed effects of isoflurane.
Vascular endothelial growth factor exerts a direct influence on endothelial cells through VEGFR-2–coupled tyrosine kinase, activating protein kinase C28and increasing the activity of endothelial nitric oxide synthase.27It has been shown to increase the Ca2+influx across the plasma membrane in endothelial cells in vitro ,29,30and nitric oxide synthesized and released in response to VEGF may also contribute to increases of vascular permeability.31
In conclusion, exposure to isoflurane for more than 4 h may enhance vascular permeability across lung vascular endothelial cells via release of VEGF. This may cause interstitial edema, resulting in local hypoxia, which then enhances NPE development. In contrast, exposure to sevoflurane is not associated with such effects. Because NPE is linked to overexcitation of sympathetic nerves, neuropeptide Y, a coneurotransmitter with norepinephrine, may participate in its enhanced development, especially under conditions where VEGF expression is increased. It is still unclear whether anesthesia with isoflurane is closely related to pulmonary edema induced by surgical maneuvers, such as the acute respiratory distress syndrome,32where the involvement of neuropeptide Y and VEGF remains unclear.