Sepsis from Gram-negative bacterial lipopolysaccharide (LPS) or endotoxin is a major cause of morbidity and mortality in critically ill patients (1). When LPS is released into the circulation, it activates a variety of host defense mechanisms, including proinflammatory cytokines, arachidonic acid metabolites, and polymorphonuclear neutrophils (leukocytes), to eliminate the pathogen (2,3). During LPS-induced inflammation, leukocytes roll along the lumen of inflamed venules before they adhere and emigrate into the site of inflammation (4). These leukocyte-endothelial interactions result in the release of reactive oxygen species and other mediators that destroy the bacteria, but they also inflict damage to the endothelium and vascular smooth muscle and affect blood vessel composition, function, and integrity (5).
Pretreatment with volatile anesthetics may protect the endothelium by decreasing leukocyte-endothelial interactions. Previous in vitro studies have shown that prolonged exposure to volatile anesthetics decreases the expression of mannose-specific receptors on leukocytes (6), slows the movement of human leukocytes across an endothelial monolayer toward a chemoattractant (7), and decreases the expression of adhesion molecules on leukocytes in response to oxidative stress, thus decreasing leukocyte adhesion to endothelial cells (8). In addition, prolonged exposure to volatile anesthetics in vivo has been reported to decrease the number of trapped leukocytes in the heart and/or coronary arteries during reperfusion (9) and to decrease cytokine-induced leukocyte accumulation in rat mesenteric venules (10).
Our laboratory recently showed that pretreatment with 30 min of 1.4% isoflurane (ISO) protected cultured endothelial and vascular smooth muscle cells from cytokine-induced and hydrogen peroxide-induced cell death (11). In addition, we showed that ISO pretreatment in vivo with 30 min of 1.4% ISO protected the endothelial structure, increased endothelial-dependent vasodilation, and attenuated the acidosis and hemodynamic deterioration associated with LPS-induced inflammation (12). However, this later study did not investigate whether the improvement in endothelial function could be the result of decreased leukocyte-endothelial interactions (12). To test the hypothesis that the protective effects of ISO pretreatment on the vasculature may, in part, involve decreasing leukocyte-endothelial interactions, rats were randomized to receive or not receive ISO pretreatment before LPS. Specifically, rats were evaluated to determine whether ISO pretreatment would decrease leukocyte-endothelial interactions and support the hemodynamics of the mesentery microcirculation during LPS-induced inflammation.
This study was approved by the institutional animal care and use committee at the University of Virginia. Male Sprague-Dawley rats (270 ± 21 g) were anesthetized with pentobarbital (50 mg/kg IM), and a tracheostomy tube was inserted for maintaining a patent airway. The lungs were ventilated with 95% oxygen/5% CO2 at a respiratory rate of 70–90 breaths/min and tidal volume of 0.8–0.9 mL/100 g body weight. Catheters were placed into the carotid artery for measuring mean arterial blood pressure (MAP), into the jugular vein for continuous infusion of pentobarbital (rate ∼5.6 mg/h), and into the femoral vein for administering LPS.
A midline laparotomy was made for exteriorizing the bowel and for cannulating the bladder, thus permitting its drainage throughout the experimental procedure. The rat was placed right side down on a heated water blanket and then transferred to the intravital microscope, which had a platform with an elevated pedestal to hold the mesentery preparation. A segment of ileum (with good vascular projections into the mesentery) was removed from the abdomen, spread onto the optical pedestal, and covered with sterile gauze presoaked in physiological salt solution (millimolar composition: NaCl 132, KCl 4.7, CaCL2 2.0, MgCL2 1.2, and NaHCO3 18.0). The mesentery was covered with an oxygen-impermeable plastic membrane (Saran Wrap) and kept warm and hydrated by continuous superfusion with physiological salt solution gassed with 95% nitrogen/5% CO2 (pH 7.4). To minimize caliber changes of postcapillary venules and arterioles, body temperature and mesenteric temperatures were controlled at 37.5°C ± 0.3°C.
Intravital measurements of postcapillary venules and arterioles (25–50 μm in diameter; length, 100 μm) were obtained by using an upright epifluorescence microscope (Model BX51; Olympus) with a water-immersion objective lens (20× numerical aperture = 0.5; 40× numerical aperture = 0.8) and a 10× eyepiece. Images were obtained with a charge-coupled device camera (DXC 390; Sony) and viewed on a video monitor (Model PVM 1943MD; Sony). Video scans of the mesenteric microvasculature were time-stamped (Model VTG-33S; FOR.A Corp.) and recorded on videotape (Model HR-S9800U; JVC) for later playback and analysis.
Heart rate and MAP were determined from strip-chart recordings. For microcirculatory measurements, erythrocyte centerline blood flow velocity in arterioles and postcapillary venules was measured during the experimental procedure by using a dual photodiode with a digital cross-correlation program (CircuSoft Instrumentation, Hockessin, DE). Centerline blood flow velocity was converted to mean blood flow velocity by multiplying by 0.625 (13). Leukocyte dynamics in postcapillary venules (the number of rolling and adherent leukocytes and leukocyte rolling velocities) were determined off-line during playback of videotaped images by using a digital image-processing system that consisted of a personal computer (Macintosh G4), a downloadable digital image software program (NIH Image), and a frame-grabber board (RTMac; Matrox Electronic System Ltd., Quebec, Canada). Rolling leukocytes were defined as cells that moved with a velocity less than that of erythrocytes in a given venule per 60-s recording period. Adherent leukocytes were expressed per unit surface area (μm2) of vessel, assuming cylindrical geometry over a 100-μm length of vessel. Leukocyte rolling velocities were determined by tracking the distance individual leukocytes traveled in 2 s. Leukocyte rolling velocities are reported singularly or as the average of at least five leukocytes per venule at each recording period.
One hour after the rats had stabilized from surgery, baseline temperatures (esophageal and mesentery) and hemodynamic and microcirculatory measurements were recorded (see above). After baseline measurements, the pentobarbital-anesthetized rats were randomized into one of four groups (Fig. 1): control (CON; n = 7), ISO-CON (n = 7), LPS alone (n = 10), and ISO-LPS (n = 10).
The CON and LPS groups received a continuous infusion of pentobarbital for maintenance of anesthesia for 5 h after baseline measurements. The ISO groups received 1.4% ISO for 30 min delivered through the breathing circuit by using an agent-specific vaporizer; it was monitored with a gas-specific analyzer (Capnomac Ultima; Datex, Helsinki, Finland). Saline or LPS (10 mg/kg; Salmonella typhimurium trichloroacetic acid extract; Sigma, St. Louis, MO) dissolved in 0.5 mL of saline was filtered through a 0.45-μm pore filter (Cameo 25NS; MSI, Westboro, MA) and then injected over 1 min through the femoral venous catheter. The experiments were conducted such that the conclusion of LPS administration (or no LPS) was Time 0 (Fig. 1).
Statistical analysis was performed by using SigmaStat 2.03 (SPSS Inc., Chicago, IL). Data are reported as mean ± se. MAP, heart rate, temperature, microcirculatory variables (including the number of rolling and adherent leukocytes and leukocyte rolling velocities between baseline measurements), groups, and treatments were compared by using a one-way and two-way analysis of variance. If statistical significance was indicated by analysis of variance, then multigroup comparisons were made by using the Student-Newman-Keuls test. Statistical significance was assumed at P < 0.05.
There was no significant difference in temperature (esophageal and mesenteric) or heart rate between any groups of rats at any time period or in MAP during baseline measurements. ISO pretreatment did not alter MAP in CON rats. However, rats given LPS showed a significant decrease in MAP when compared with CON rats 1–4 h after LPS. ISO pretreatment significantly attenuated the decrease in MAP from baseline measurements when compared with rats receiving only LPS by 9.4% ± 2.5% (ISO-LPS) versus 16.8% ± 2.7% (LPS) at 2 h and by 19% ± 5% versus 35% ± 5%, respectively, 4 h after LPS (Fig. 2).
Group baseline measurements for mean arteriolar and postcapillary venular blood flow velocities were not significantly different (Fig. 3). ISO pretreatment did not alter arteriolar blood flow velocities in CON rats. Arteriolar and postcapillary venular blood flow velocities decreased significantly from CON after LPS was given at 2–4 h. There was no significant difference between LPS and ISO-LPS at any time. After 4 h of LPS, arteriolar blood flow velocity in the ISO-LPS and LPS groups decreased by 65.9% ± 9.4% and 63.8% ± 7.5%, whereas postcapillary venular blood flow velocity decreased by 74.7% ± 4.4% and 79.2% ± 5.3%, respectively, of their baseline values.
There was no significant difference in the number of rolling leukocytes between any groups of rats during baseline measurements (Fig. 4A). ISO pretreatment resulted in a significant increase in the number of rolling leukocytes at 0, 2, 3, and 4 h when compared with the CON group. The number of rolling leukocytes increased from 31 ± 9/min at baseline to 161 ± 43/min at 4 h (Fig. 4A). In contrast, LPS resulted in a significant decrease in the number of rolling leukocytes 1–4 h after LPS when compared with CON rats. The number of rolling leukocytes was not significantly different in the ISO-LPS and LPS rats. The number of rolling leukocytes decreased from 36 ± 6/min and 43 ± 8/min, respectively, at baseline to 17 ± 5/min and 27 ± 5/min, respectively, after LPS (0 h). After 1 h, the number of rolling leukocytes decreased to 6 ± 2/min and 5 ± 2/min, respectively, and continued at this value throughout the study (Fig. 4A).
There was no significant difference in the number of adherent leukocytes per 100-μm length of postcapillary venule between any groups or at any time period. ISO pretreatment did not alter the number of adherent leukocytes. All groups showed a significant increase in the number of adherent leukocytes from baseline measurements at 4 h: CON, 1 ± 1 to 22 ± 6; ISO-CON, 2 ± 1 to 19 ± 5; LPS, 2 ± 1 to 13 ± 2; and ISO-LPS, 3 ± 1 to 11 ± 2 (Fig. 4B).
Leukocyte rolling velocity in CON and ISO-CON groups was not significantly different throughout the experimental procedure, averaging 83.9 ± 11.4 μm/s (range, 26–133 μm/s;Fig. 5B) and 87.0 ± 9.9 μm/s (range, 30–126 μm/s;Fig. 5B), respectively, at 4 h (Fig. 5A). ISO pretreatment in CON rats did not change the distribution of leukocyte rolling velocities. In contrast, LPS caused a significant decrease in leukocyte rolling velocities. ISO pretreatment increased leukocyte rolling velocities when compared with rats that received only LPS after 2–4 h. Leukocyte rolling velocities in the LPS and ISO-LPS groups averaged 19.8 ± 2.0 μm/s (∼77% decrease from baseline values; range, 12–31 μm/s) and 63.7 ± 8.7 μm/s (∼29% decrease from baseline values; range, 14–108 μm/s), respectively, 4 h after LPS (Fig. 5).
The major findings of this study were that ISO pretreatment attenuated the decrease in systemic MAP and leukocyte rolling velocities associated with LPS-induced inflammation and increased the number of rolling leukocytes in postcapillary venules of CON rats. This study demonstrated that pretreating rats for 30 min with 1.4% ISO significantly attenuated the decrease in MAP associated with LPS but had no effect on MAP in CON rats that received ISO. This indicates that ISO decreases the effects of LPS rather than acting through mechanisms that directly increase MAP. The concentration and duration of ISO pretreatment used in this study were based on the results from our earlier in vivo study, which showed that pretreating rats for 30 min with 1.4% ISO attenuated the decrease in MAP for six hours after LPS administration (12). Although the increased MAP we observed in ISO-pretreated rats suggests that ISO pretreatment may be protective of the vasculature during LPS-induced inflammation, it is also possible that the increase in MAP reflects an increase in cardiac output secondary to myocardial protection, an effect that is known to occur with anesthetic preconditioning (14,15).
ISO pretreatment at the concentration and duration used in this study did not alter arteriolar mesenteric blood flow velocity in CON rats. However, it was ineffective in preserving mesenteric microcirculatory blood flow velocity after LPS. This may be due to LPS causing an increase in mesenteric vascular resistance, resulting in decreased arteriolar and postcapillary venular blood flow velocity. This observation has been reported by other investigators after LPS. Navaratnam et al. (16), using a chronically instrumented ovine model, showed that IV administration of LPS (1.5 μg/kg) caused a >50% reduction in mesenteric blood flow within one hour by increasing mesenteric vascular resistance (16). An increase in mesenteric vascular resistance during LPS-induced inflammation (16,17) may cause a shunting of intestinal blood to nonintestinal systemic circulation. This may explain why ISO pretreatment attenuated the decrease in MAP associated with LPS but did not alter blood flow velocity in the mesentery.
ISO pretreatment had no significant effect on leukocyte rolling velocities in CON rats, but rats that received LPS showed an immediate decrease in leukocyte rolling velocities. This is likely due both to a significant decrease in MAP, resulting in a concomitant reduction in mesenteric microcirculatory blood flow velocity, and to an increased expression of adhesion molecules along the surface of endothelial cells and leukocytes, enhancing the probability of leukocyte-endothelial interaction (18,19).
A decrease in MAP reduces the driving force that propels erythrocytes and leukocytes through the microcirculation. Erythrocytes, because of their smaller cross-sectional area and faster flow velocity, tend to push leukocytes to a position closer to the vessel wall, thereby increasing the likelihood that adhesion molecules on the microvilli of leukocytes will make contact with their counterligand receptors (selectins and integrins) on the surface of endothelial cells (20). Interaction of selectins with their counterligands initiates the leukocyte adhesion cascade. This cascade begins with leukocyte capture (first contact of a leukocyte with the activated endothelium) and proceeds with rolling, firm adhesion through activation of β2-integrins, and emigration of the leukocyte to the site of inflammation (4,18,21).
The faster rolling velocities observed in ISO-pretreated LPS rats (ISO-LPS versus LPS) may be due to a decrease in the number of microvilli, the number of activated receptors along the surface of the leukocyte, or both. Although no one has evaluated the effects of anesthetic pretreatment, prolonged exposure to inhaled anesthetics has been reported to decrease the expression of mannose-specific receptors on murine polymorphonuclear leukocytes (6,8). As a result, anesthetics may modify the physical characteristics of leukocytes by reducing the number of microvilli or the expression of adhesion molecule receptors on the surface of leukocytes. A decrease in the number of microvilli or activated receptors may shorten the transit time of leukocytes through the inflamed vessels and allow them less time to sample endothelial signaling molecules, such as chemokines or lipid autocoids, on the surface of endothelial cells (22). Therefore, the improvement in leukocyte rolling velocities after ISO pretreatment before LPS may be due to a decrease in the density or expression of adhesion molecule receptors on the surface of leukocytes or to the morphological changes in the microvilli of leukocytes from ISO exposure.
The number of rolling leukocytes increased in CON rats after ISO pretreatment. This is consistent with our previous study that showed in a rat cremasteric preparation that prolonged exposure to 1.5 minimum alveolar anesthetic concentration (MAC) ISO or sevoflurane anesthesia significantly (P < 0.01) increased the number of both rolling and adherent leukocytes in postcapillary venules, whereas decreasing the concentration of both inhaled anesthetics to 0.5 MAC significantly decreased the number of both rolling and adherent leukocytes toward baseline values. 1 These observations are consistent with those of Morisaki et al. (23), who showed that ISO and sevoflurane caused a significant dose-dependent increase in leukocyte rolling and adherence in rat mesenteric postcapillary venules through mechanisms involving P-selectin and the formation of superoxide anions. Several studies have reported that volatile anesthetics, including sevoflurane (23,24), halothane (23), and ISO (25), increase the production of superoxide anions. These anions interact with nitric oxide to form highly reactive species that may result in the activation of leukocytes to increase leukocyte rolling and adhesion (26).
The decrease in the number of rolling leukocytes after LPS is likely due to an increased expression of adhesion molecules on endothelial cells and leukocytes and to a significant decrease in microcirculatory blood flow velocity. Coughlan et al. (27) reported that LPS caused neutropenia through increased expression of P-selectin in kidney, liver, and lung endothelial cells. In our study, the significant decrease in the number of rolling leukocytes after LPS was consistent with the observations of Coughlan et al.; however, it was not prevented by ISO pretreatment. This is likely due to LPS being a more potent activator of adhesion molecules (22) when compared with ISO, promoting the upregulation of adhesion molecules on endothelial cells and leukocytes and resulting in an increased adherence of leukocytes on endothelial cells, and to LPS causing an increase in mesenteric vascular resistance, resulting in a decrease in postcapillary venular blood flow velocity.
In our study, ISO pretreatment did not alter the number of adherent leukocytes when compared with CON rats. Both CON groups showed an increase in adherent leukocytes throughout the study, most likely due to surgical trauma. However, in rats given LPS (ISO-LPS and LPS), the number of adherent leukocytes was not significantly different from that in CON rats. This is in contrast to the study of Woodman et al. (28). They reported that in the presence of good postcapillary blood flow velocity, the number of adherent leukocytes in feline mesenteric postcapillary venules increased significantly over those recorded in CON animals after four hours of inflammation induced by topical application of LPS. In this study, the smaller number of adherent leukocytes after four hours of LPS (ISO-LPS and LPS groups), when compared with the same time period in the study of Woodman et al. (11 ± 2 and 13 ± 2 versus 33 ± 6 leukocytes per 100-μm length of vessel, respectively), is likely due to the systemic effects of LPS causing a significant decrease in postcapillary venule blood flow velocity. The decrease in postcapillary blood flow velocity combined with increased activation of adhesion molecules on endothelial cells (4,22) and leukocytes (2,18) from LPS exposure may result in a sequestering of leukocytes in key organs to effectively reduce the number of adherent leukocytes in the mesenteric microcirculation (27).
Our previous studies indicate that ISO pretreatment protects endothelial and vascular smooth muscle cells in vitro (11) and protects the vasculature in vivo from LPS-induced inflammation (12). This study demonstrated that ISO pretreatment attenuated the decrease in MAP and was the first study to show that ISO pretreatment increased leukocyte rolling velocities during LPS-induced inflammation. Even though the number of rolling leukocytes was similar in both LPS and ISO-LPS rats, the faster leukocyte rolling velocities in ISO-pretreated rats may confer protection to the mesenteric vasculature by decreasing the transit time of leukocytes through inflamed vessels. Faster leukocyte rolling velocities may reduce the incidence of inflammation by decreasing the probability of leukocyte-endothelial interactions and cellular injury as a consequence of leukocyte accumulation. The extent to which faster leukocyte rolling velocities contribute to the mechanism of protection secondary to ISO pretreatment requires further study.
In conclusion, we showed that ISO pretreatment supported hemodynamics and increased leukocyte rolling velocities during LPS-induced inflammation. However, at the concentration of LPS and the duration of ISO used in this study, ISO pretreatment before LPS was ineffective in maintaining mesenteric arteriolar and postcapillary blood flow velocities after LPS-induced inflammation.
1 Zhang Y, Hayes JK, Wong KC, et al. Leukocyte activation in the microcirculation of the rat cremaster muscle during isoflurane and sevoflurane anesthesia [abstract]. Anesthesiology 1999;91:A712.
1. Bone RC. The pathogenesis of sepsis. Ann Intern Med 1991; 115: 457–69.
2. Adams JM, Hauser CJ, Livingston DH, et al. Early trauma polymorphonuclear neutrophil responses to chemokines are associated with development of sepsis, pneumonia, and organ failure. J Trauma 2001; 51: 452–6.
3. Aldridge AJ. Role of the neutrophil in septic shock and the adult respiratory distress syndrome. Eur J Surg 2002; 168: 204–14.
4. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 1991; 67: 1033–6.
5. Darley-Usmar V, Halliwell B. Blood radicals: reactive nitrogen species, reactive oxygen species, transition metal ions, and the vascular system. Pharm Res 1996; 13: 649–62.
6. Bardosi L, Bardosi A, Hendrys M, et al. Reduced expression of mannose-specific receptors on murine peripheral blood PMNL following prolonged anesthesia with different inhalation agents. Acta Anaesthesiol Scand 1990; 34: 286–90.
7. Erskine R, James JF. Isoflurane but not halothane stimulates neutrophil chemotaxis. Br J Anaesth 1990; 64: 723–7.
8. Mobert J, Zahler S, Becker BF, et al. Inhibition of neutrophil activation by volatile anesthetics decreases adhesion to cultured human endothelial cells. Anesthesiology 1999; 90: 1372–81.
9. Kowalski C, Zahler S, Becker BF, et al. Halothane, isoflurane, and sevoflurane reduce postischemic adhesion of neutrophils in the coronary system. Anesthesiology 1997; 86: 188–95.
10. Miller LS, Morita Y, Rangan U, et al. Suppression of cytokine-induced neutrophil accumulation in rat mesenteric venules in vivo
by general anesthesia. Int J Microcirc Clin Exp 1996; 16: 147–54.
11. de Klaver MJM, Palmer LA, Manning L, et al. Isoflurane pretreatment inhibits cytokine-induced cell death in rat smooth muscle cells and human endothelial cells. Anesthesiology 2002; 97: 24–32.
12. Plachinta RV, Hayes JK, Cerilli LA, et al. Isoflurane pretreatment inhibits lipopolysaccharide-induced inflammation in rats. Anesthesiology 2003; 98: 89–95.
13. Lipowsky HH, Zweifach BW. Application of the “two-slit” photometric technique to the measurement of microvascular volumetric flow rates. Microvasc Res 1978; 15: 93–101.
14. Kersten JR, Schemeling TJ, Pagel PS, et al. Isoflurane mimics ischemic preconditioning via KATP channels: reduction of myocardial infarct size with an acute memory phase. Anesthesiology 1997; 87: 361–70.
15. Li F, Hayes JK, Wong KC, et al. Administration of sevoflurane and isoflurane prior to prolonged global ischemia improves heart function in isolated rat heart. Acta Anaesthesiol Sin 2000; 38: 113–21.
16. Navaratnam RLN, Morris SE, Traber DL, et al. Endotoxin (LPS) increases mesenteric vascular resistance (MVR) and bacterial translocation (BT). J Trauma 1990; 30: 1104–15.
17. Baykal A, Kavuklu B, Iskit AP, et al. Experimental study of the effect of nitric oxide inhibition on mesenteric blood flow and interleukin-10 levels with a lipopolysaccharide challenge. World J Surg 2000; 24: 116–20.
18. Gotsch U, Jager U, Dominis M, et al. Expression of P-selectin on endothelial cells is up-regulated by LPS and TNF-alpha in vivo. Cell Adhes Commun 1994; 2: 7–14.
19. Weiss DJ, Evanson OA. Evaluation of lipopolysaccharide-induced activation of equine neutrophils. Am J Vet Res 2002; 63: 811–5.
20. Abbitt KB, Nash GB. Rheological properties of the blood influencing selectin-mediated adhesion of flowing leukocytes. Am J Physiol Heart Circ Physiol 2003; 285: H229–40.
21. Maroszynska I, Fiedor P. Leukocytes and endothelium interaction as rate limiting step in the inflammatory response and a key factor in the ischemia-reperfusion injury. Ann Transplant 2002; 5: 5–11.
22. Jung U, Norman KE, Scharffetter-Kochanek K, et al. Transit-time of leukocytes rolling through venules controls cytokine-induced inflammatory cell recruitment in vivo. J Clin Invest 1998; 102: 1526–33.
23. Morisaki H, Suematsu M, Wakabayashi Y, et al. Leukocyte-endothelium interaction in the rat mesenteric microcirculation during halothane or sevoflurane anesthesia. Anesthesiology 1997; 87: 591–8.
24. Arriero MM, Munoz Alameda L, Lopez-Farre A, et al. Sevoflurane reduces endothelium-dependent vaso-relaxation: role of superoxide anion and endothelin. Can J Anaesth 2002; 49: 471–6.
25. Park KW, Dai HB, Lowenstein E, et al. Oxygen-derived free radicals mediate isoflurane-induced vasoconstriction of rabbit coronary resistance arteries. Anesth Analg 1995; 80: 1163–7.
26. Carden DL, Granger DN. Pathophysiology of ischemia-reperfusion injury. J Pathol 2000; 190: 255–66.
27. Coughlan AF, Hau H, Dunlop LC, et al. P-selectin and platelet-activating factor mediate initial endotoxin-induced neutropenia. J Exp Med 1994; 179: 329–34.
28. Woodman RC, Teoh D, Payne D, et al. Thrombin and leukocyte recruitment in endotoxemia. Am J Physiol Heart Circ Physiol 2000; 279: H1338–45.