Effect of Endotoxemia on Intestinal Villus Microcirculation in Rats

JOURNAL OF SURGICAL RESEARCH ARTICLE NO.

61, 521–526 (1996)

0157

Effect of Endotoxemia on Intestinal Villus Microcirculation in Rats HEINFRIED SCHMIDT,* M.D., DEAA, ANDREAS SECCHI,* M.D., RUTH WELLMANN,* M.D., ALFONS BACH,* M.D., HUBERT BO¨HRER,* M.D., DEAA, MARTHA MARIA GEBHARD,† M.D., AND EIKE MARTIN,* M.D., FANZCA *Department of Anesthesiology and †Department of Experimental Surgery, University of Heidelberg, D-69120 Heidelberg, Germany Submitted for publication May 31, 1995

Intestinal mucosal hypoperfusion with subsequent ischemia during endotoxemia might cause a breakdown of the gut barrier with translocation of bacteria and their toxins into the systemic circulation, thus maintaining a ‘‘gut-derived’’ septic state. The aim of this study was to investigate the influence of endotoxin on the microcirculation of intestinal villi, which represent the most vulnerable part of the mucosa. The changes in blood flow and in the diameters of the central villus arterioles located in the distal ileum were monitored in control rats without lipopolysaccharide (LPS) exposure (n Å 7), and in rats receiving 1.5 mg/ kg b.w. LPS (n Å 7) or 15 mg/kg b.w. LPS (n Å 7) over 60 min. The blood flow and the arteriolar diameters were determined using in vivo videomicroscopy at baseline, and 60 min and 120 min later. In control animals, no changes in blood flow and arteriolar diameters were observed during the entire experiment. Administration of 1.5 mg/kg b.w. LPS reduced the blood flow to 69.5 { 9.0% of the baseline value at the end of the study period. This decrease in blood flow was associated with a decrease in the villus arteriolar diameters by 17.4 { 2.5% from the baseline values. In animals exposed to 15 mg/kg b.w. LPS, the decrease in villus blood flow at 60 min was 64.8 { 10.9% of baseline, and at 120 min 66.9 { 12.6% of baseline. The diameters of the villus arterioles were reduced by 11.5 { 2.4% and 15.1 { 1.7%, respectively. In the control group and in the 1.5-mg/kg LPS group, the mean arterial blood pressure did not change during the entire study period. In the 15-mg/kg LPS group, the mean arterial pressure tended to decrease after 60 min. These data suggest a reduction of villus blood flow due to vasoconstriction in the central villus arterioles during normotensive endotoxmia, which might represent the mechanism for the mucosal ischemia observed in critically ill patients. q 1996 Academic Press, Inc.

INTRODUCTION

The gut has been viewed as the ‘‘motor of multiple organ failure’’ [1]. In general, the intact gut mucosa serves as a barrier that prevents the translocation of bacteria and bacterial toxins from the gut into the systemic circulation [2]. Trauma [3], sepsis [4], and endo-

toxemia [5, 6] causes the translocation of gut-derived endotoxin, which might be the result of intestinal mucosal injury secondary to intestinal hypoperfusion and hypoxia. Endotoxin has been demonstrated to increase the intestinal permeability even in healthy volunteers [7], indicating that endotoxemia itself might induce further translocation of bacteria and endotoxin and may thus maintain sepsis and septic shock in a self-perpetuating process. Increased mucosal permeability, however, represents an early sign of intestinal mucosal injury [8]. The severity of the tissue injury is dependent on the extent and duration of ischemia. Intestinal ischemia as seen, for example, in the septic critically ill patient probably does not cause damage to the gut beyond the superficial part of the mucosa [8]. As a consequence of its peculiar vascular anatomy and the countercurrent oxygen exchange mechanism, the tip of the intestinal villus represents the most vulnerable structure to regional hypoxia in the intestinal wall. Furthermore, the first histological sign of intestinal mucosal injury due to ischemia is the lifting of the epithelial cells at the tip of the intestinal villi [9]. Because no in vivo data about the microcirculatory alterations in the intestinal villus during sepsis and endotoxemia are available, we investigated the effects of endotoxin on the blood flow and the arteriolar diameters in intestinal villi using in vivo videomicroscopy. MATERIALS AND METHODS Animal preparation. All experimental procedures and protocols used in this investigation were reviewed and approved by the Governmental Animal Protection Committee. Male Wistar rats weighing 250–300 g were used. All animals were kept on a diet of standard rat chow and water ad libitum until the day before the experiment. The rats were fasted 12 hr before the experiment; free access to water was maintained. Animals were initially anesthetized with intraperitoneal injection of 60 mg/kg b.w. pentobarbital (Nembutal; Sanofi, Germany) combined with an intramuscular injection of 30 mg/kg b.w. ketamine (Ketanest; Parke Davies, Germany) and supplemented with an intramuscular injection of 30 mg/kg b.w. pentobarbital 30 min prior to the start of the microscopy when necessary. Polyethylene catheters (OD 0.8 mm, ID 0.5 mm) were inserted into the right jugular vein for venous access and into the left carotid artery for blood sampling and monitoring of cardiovascular variables. The arterial line was connected to a pressure transducer switched to a monitor (Servomed; Hellige, Germany). A tracheostomy was performed for airway control, and the animals were breathing room 0022-4804/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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air. Rectal temperature was measured with a thermistor probe and maintained at 377C using a heating lamp. The preparation of the intestinal mucosa for intravital microscopy was performed using a modified procedure according to Bohlen [10] and Gore [11]. A loop of the small intestine was cautiously exteriorized through a 2-cm abdominal midline incision. A small segment (2 cm) of the terminal ileum supplied by a single vascular arcade was opened along its antimesenteric border using electrocautery. Stool on the surface of the gut was removed by cautiously flushing the mucosa with thermostated (37.07C) saline solution. With the mucosal surface facing upward, the bowel was placed on a transparent viewing pedestal. Thereafter, the edges of the opened intestinal segment were fixed to a frame by four sutures (Ethilon II, 0.7 metric; Ethicon, Germany) on each side. During this procedure great care was taken to avoid trauma to the exposed bowel. The animal was then transferred to the stage of the microscope. The mucosal surface was superfused with a thermostated (37.0 { 1.07C) bicarbonate buffered salt solution (132 mM NaCl, 4.7 mM Kcl, 2 mM CaCl2 , 1.2 mM MgCl2 , 18 mM NaHCO3) equilibrated with 5% CO2 in N2 to adjust the pH to 7.35, the PO2 at 25–30 mm Hg, and the PCO2 at 35–45 mm Hg. Intravital microscopy. Intravital microscopy was performed using a fluorescence microscope (Leica, Germany) equipped with a 25-fold water immersion objective (PL Fluotar 25/0.75 W; Leitz, Germany), a 10-fold eyepiece, and a transfer lense. The individual villi were visualized by transillumination using a 100-W cold light fountain (KL 1500 electronic; Schott, Germany) in order to measure the diameters of the central arteriolar or by epi-illumination using a epifluorescence illuminator (Type 307-148.002 514687; Leitz, Germany) in order to count the numbers of fluorescein isothiocyanate (FITC)-labeled erythrocytes. This illuminator consisted of an XBO 100 W/2 short arc mercury lamp and a bypass filter (450–490 nm) for the excitation fluorescence wavelength. A dichroic mirror with a 510-nm cutoff wavelength was located in the body of the microscope. Further rejection of FITC emission was achieved using a barrier filter at 520 nm located in front of the eyepiece. Images were transferred to a monitor (PVM/444QM; Sony, Japan) by a lowlight camera (CF 8/1; Kappa, Germany) and simultaneously recorded on videotape using a videorecorder (AG 7350; Panasonic, Japan) for subsequent off-line analysis. FITC-labeling of erythrocytes. Erythrocytes from separate donor animals were labeled with fluorescein isothiocyanate (FITC, Isomer I, No. F-7250; Sigma Chemicals, Germany) using a modified procedure according to Butcher [12] and Sarelius [13]. Normal rheologic behavior of FITC-labeled erythrocytes in vivo in the rat has been confirmed by Sarelius et al. [13]. The hematocrit of the stained erythrocytes was about 50%. Prior to the microscopic studies, all animals received an intravenous injection of 0.5 ml/kg b.w. FITC-labeled erythrocytes. Villus microcirculatory alterations. In each animal five villi per observation period were microscopied and videotaped. Each villus receives its arterial supply from a single main arteriole that runs through the center of the villus. Main arterioles were identified according to a descriptive classification [11] and the assessment of the flow direction in the vessels. Videotapes were analyzed off-line by a blinded observer, who counted the number of labeled erythrocytes circulating through the main arteriole near the tip of the villus during a 30-sec period. The flux of labeled erythrocytes (FFITC) in five villi per observation period was averaged. Simultaneously, arterial blood samples were drawn and the number of fluorescent erythrocytes per unit volume of arterial blood (NFITC) was measured by counting the number of fluorescent erythrocytes in 800 different fields of a Neubauer chamber (0.1-mm thickness; 0.0025-mm2 area per field). The systemic hematocrit (HctSYS) exceeds the hematocrit in microcirculatory vessels (HctMICRO) due to the Fahraeus effect [14]. Thus, volumetric blood flow has to be corrected by a correction factor. It has been shown that the discharge hematocrit (HctDISCH), which is defined as the portion of erythrocytes flowing out of a vessel, equals HctSYS [15]. Because previous studies on the Fahraeus effect in narrow capillaries described a ratio of HctMICRO/HctDISCH of 0.76 for vessel sizes in the range of those we measured in villi main arterioles [16]. Volumetric blood flow (V) was then calculated by the following formula: V Å (FFITC/NFITC)/0.76 (nl/min)

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This formula is based on the assumption that the ratio of labeled to native cells in capillary and arterial blood is identical, which was already demonstrated for the hamster cheek pouch and the cremaster muscle [13]. The diameters of the main arterioles were measured retrospectively by videotape replay on a monitor screen using a computerassisted microcirculation analysis system (Cap Image; Zeintl, Heidelberg). In each main arteriole, the diameters were measured at eight different places from the base to the tip of the villi. These values were averaged for each individual arteriole and the means were used for further calculations. In order to compare the vessel diameters at the different time points, the values of five villi were averaged in each case. Monitoring. The mean arterial blood pressure and the heart rate were recorded at baseline, and 15, 30, 45, 60, 75, 90, 105, and 120 min later. The systemic leukocyte count, platelet count, and hematocrit were determined at baseline, and after 60 and 120 min using an analyzing system that was calibrated for rat blood cells (Hematology Analyzer System CP 9000-3; Serono-Baker-Diagnostics Inc.). Furthermore, heparinized blood samples for blood gas analysis (ABL3; Radiometer, Denmark) were taken at baseline, and after 60 and 120 min. Experimental protocol. The rats were randomized into three groups of seven animals each. Following the administration of FITClabeled erythrocytes a stabilization period of 30 min was allowed. Animals of the test groups were then challenged with an i.v. infusion of endotoxin (1.5 mg/kg b.w. and 15 mg/kg b.w; lipopolysaccharide E. coli 026:B6; Sigma Chemicals, Germany) in 15 ml/kg b.w. saline 0.9% over a 60-min period. All animals were then treated with 15 ml/kg b.w. saline 0.9% for an additional 60 min. Animals in the control group received equivalent volumes of saline 0.9% (15 ml/kg/ hr) throughout the study. Videomicroscopy for the measurement of villus blood flow and the diameters of the villus central arterioles was performed at baseline, and 60 and 120 min later. Exclusion criteria. Animals with local trauma due to the preparation procedure as indicated by bleeding, edema, and primary nonperfusion of the villi were excluded from the study. According to these criteria, two animals were excluded from the study. Statistical analysis. For statistical analysis, group means and standard errors were calculated for baseline values and for percent changes. Significant differences within each group were determined by the sign rank test. Differences between pairs of groups were analyzed for statistical significance using the Wilcoxon’s matched pairs test. All data are presented as mean { SEM. Differences were considered to be significant at P õ 0.05.

RESULTS

None of the animals showed signs of infection or sepsis prior to the experiments. There were no differences between the groups concerning weight, heart rate, MAP, villus blood flow, and the diameters of the central villus arterioles at the start of the experiments (Table 1). In TABLE 1 Animal Weight, Heart Rate (HR), Mean Arterial Pressure (MAP), Villus Blood Flow, and Microvascular Diameters of the Central Villus Arterioles at Baseline NaCl 0.9%

Variable Weight (g) HR (bpm) MAP (mmHg) Villus blood flow (nl/min) Villus arteriolar diameter (mm)

277 368 125 7.0 7.0

Note. All data are mean { SEM.

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{ { { { {

1.5 mg/kg LPS 5 16 7 1.4 0.1

294 381 127 7.2 7.4

{ { { { {

9 9 3 0.7 0.2

15 mg/kg LPS 271 372 125 7.7 7.1

{ { { { {

13 24 3 0.7 0.1

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TABLE 2 Systemic White Blood Cell Count (WBC), Platelet Count (Plt), and Hematocrit (Hct) at Baseline, and 60 min and 120 min Later NaCl 0.9%

1.5 mg/kg LPS

15 mg/kg LPS

Variable

Baseline

60 min

120 min

Baseline

60 min

120 min

Baseline

60 min

120 min

WBC count (1109/liter) Plt count (1109/liter) Hct (%)

8.7 { 1.1 584 { 46 42.1 { 2.1

7.6 { 1.7 577 { 97 41.5 { 2.9

8.2 { 1.6 572 { 78 38.8 { 2.6

7.5 { 0.6 596 { 34 41.2 { 0.8

3.2 { 0.3*,** 486 { 40 43.0 { 0.7

2.3 { 0.2*,** 386 { 20*,** 41.2 { 1.3

4.9 { 0.5* 627 { 54 40.6 { 1.2

2.6 { 0.7*,** 397 { 46*,** 38.7 { 1.1

2.4 { 0.7*,** 379 { 68*,** 38.1 { 0.6

Note. All data are mean { SEM. * P õ 0.05 vs NaCl 0.9%. ** P õ 0.05 vs baseline.

control animals receiving NaCl 0.9%, no changes in the leukocyte and platelet counts were observed throughout the experiment. In contrast, administration of 1.5 and 15 mg/kg LPS resulted in a marked reduction of leukocyte and platelet counts, respectively (Table 2). The systemic hematocrit at the beginning of the study showed no differences between the groups (NaCl: 42.1 { 2.1%; 1.5 mg/ kg LPS: 41.2 { 0.8%; 15 mg/kg LPS: 40.6 { 1.2%) and remained unchanged in all groups. Arterial blood gases during and at the end of the experiment did not differ between groups and remained in the normal range. Macrohemodynamic Changes Changes in MAP are shown in Fig. 1. There were no changes in MAP throughout the study period in the animals receiving saline 0.9% and 1.5 mg/kg LPS. In animals receiving 15 mg/kg LPS, the mean arterial pressure tended to decrease after 60 min. However, this decrease did not reach statistical significance when compared to the baseline value (P Å 0.13) or to the saline group (P Å 0.4). Heart rate remained unaltered in animals receiving saline 0.9%. In animals exposed to 1.5 mg/kg LPS, the heart rate increased from 381 { 9 bpm at the start of the experiment to 433 { 9 bpm after 120 min (P õ 0.05). In animals treated with 15

FIG. 1. Mean arterial blood pressure (MAP) in control animals (NaCl) and in animals receiving different amounts of LPS (1.5 mg/ kg b.w. and 15 mg/kg b.w.) over a 60-min period. All data are mean { SEM.

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mg/kg LPS, the heart rate also increased (372 { 24 bpm to 418 { 15 bpm) after 120 min (P õ 0.05). Microhemodynamic Alterations in Intestinal Villi Changes in villus blood flow and villus arteriolar diameters are shown in Figs. 2 and 3. In control animals receiving saline 0.9%, the blood flow in villus central arterioles remained unchanged throughout the study (baseline: 7.0 { 1.4 nl/min; 60 min: 7.0 { 1.5 nl/min; 120 min: 6.9 { 1.3 nl/min). Similarly, the diameters of the villus central arterioles remained unaltered. Following the administration of 1.5 mg/kg LPS, the blood flow tended to decrease after 60 min (84.2 { 12.7% of baseline), and decreased to 4.7 { 0.4 nl/min (69.5 { 9.0% of baseline; P õ 0.05) after 120 min. The corresponding arteriolar diameters decreased by 10.9 { 2.1% and 17.4 { 2.5% (P õ 0.05) when compared to baseline values, respectively. The administration of 15 mg/kg LPS was associated with a significant decrease in villus blood flow to 64.8 { 10.9% of baseline (4.8 { 0.7 nl/min; P õ 0.05) at 60 min and to 66.9 { 12.6% of baseline (4.8 { 0.8 nl/min; P õ 0.05) at 120 min. These changes were accompanied by a nonsignificant reduction of the diameters of the arterioles of 11.5 { 2.4%

FIG. 2. Changes in intestinal villus blood flow in control animals (NaCl) and in animals receiving different amounts of LPS (1.5 mg/ kg b.w. and 15 mg/kg b.w.) over a 60-min period. Baseline values are given in Table 1. All data are mean { SEM. *P õ 0.05 vs NaCl 0.9%. #P õ 0.05 vs baseline.

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FIG. 3. Changes in villus arteriolar diameters in control animals (NaCl) and in animals receiving different amounts of LPS (1.5 mg/ kg b.w. and 15 mg/kg b.w.) over a 60-min period. Baseline values are given in Table 1. All data are mean { SEM. *P õ 0.05 vs NaCl 0.9%. #P õ 0.05 vs baseline.

at 60 min and a significant reduction of 15.1 { 1.7% (P õ 0.05) at 120 min. DISCUSSION

The mucosal layer of the small intestine plays a central role in the development of multiple organ failure [17]. In general, the gut mucosa prevents the translocation of intraluminal bacteria and endotoxin into the systemic circulation. However, this barrier function of the intestinal wall is deranged as a consequence of a number of pathologic events including hemorrhage [18], intestinal obstruction [19], trauma [3], sepsis [4], and endotoxicosis [5, 6]. During experimental endotoxemia, intestinal mucosal injury with tissue hypoxia and increased lactate production persists despite adequate volume resuscitation [20]. Similarly, Falk et al. [21] described the development of mucosal lesions during sepsis in animals with normal intestinal blood flow. These results suggest that regulatory responses exist in the intestinal microcirculation that reduce mucosal perfusion in spite of increased blood flow to the gut. Thus, the objective of our study was to evaluate the microcirculatory response in individual intestinal villi during endotoxemia in the rat. In order to investigate the effects of two different doses of endotoxin, we used a low-dose and a high-dose continuous infusion of endotoxin combined with a high volume load to maintain mean arterial blood pressure. The rats exposed to endotoxin in our study exhibited changes that were consistent with the development of sepsis. They showed an increase in heart rate and a marked reduction of the leukocyte and platelet count when compared to control rats. The endotoxin-treated rats in our study remained normotensive, probably because endotoxin was not given as a bolus, but was infused continuously over 60 min. This model therefore meets criteria of a chronic, resuscitated sepsis state [22, 23]. All animals used in the study were breathing

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spontaneously and maintained a normal arterial pH, PCO2 , and PCO2 , and PO2 throughout the study, which excludes the effects of these substances on the microcirculation. The anesthetics used in our study are not inert with regard to their actions on the microcirculation [24]. However, intramuscular ketamine in a dose we used in our study did not cause changes in secondorder arteriolar diameters in the wings of unanesthetized bats [25], and pentobarbital in a comparable dose maintains the diameters of small third-order arterioles in the rat cremaster muscle within the normal range of values observed in unanesthetized, decerebrated rats [26, 27]. Thus, the endotoxin-induced alterations of the villus microcirculation observed in our study should have only been minimally influenced by the anesthetics used. Because other methods used to determine tissue blood flow such as the H2 clearance method and laser Doppler velocitometry [28] are not able to distinguish between submucosal blood flow and blood flow in the villus arteriole and, furthermore, the microsphere method [29] cannot be used for on-line quantitation of villus blood flow; we used an in vivo fluorescence videomicroscopic method for the measurement of blood flow in the central arteriole near the tip of the intestinal villi. The absence of changes in villus blood flow and in the vessel diameters in our control animals during the entire observation period indicates microvascular stability of the preparation for at least 2 hr. The distribution of blood flow in the intestinal wall is not homogeneous. The mean mucosal blood flow is two to four times greater than muscularis blood flow [17] in order to provide a high oxygen delivery to the mucosa. The results of our study demonstrate a decrease in villus blood flow after administration of endotoxin during normotensive endotoxemia. This decrease was dependent on the dose of endotoxin and on the time after starting the endotoxin exposure. The tips of the villi represent the structures most vulnerable to hypoxia and decreased blood flow in the intestinal wall as a result of the countercurrent exchange mechanism for oxygen [30, 31]. The villus is supplied by a centrally located arteriole that does not arborise until the tip of the villus. The blood flow in the distal area is then drained through a subepithelial network of capillaries and venules. Because of the small distance between these two vessel systems an equilibration of oxygen tension might take place at the base of the villus, which is dependent on the time allowed for diffusion of oxygen. This time, however, is dependent on the blood flow in the central arteriole. Using a mathematical model of the countercurrent shunting of oxygen in the intestinal villus Shepherd et al. [31] showed a blood flow-dependent decrease of the PO2 in the villus tip. Because very low resting PO2 tensions have been recorded from the tip of the villus [30], an increase in the efficacy of the countercurrent exchanger during a reduction of blood flow in the central arteriole might well explain an almost anoxic environment at the tip of the villi. Thus, the reduction of villus blood flow observed in our study

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is in agreement with the well-known fact that the earliest histological changes of intestinal ischemic injury are characterized by patchy necrosis of the superficial epithelium located at the tip of the mucosal villus [32]. Furthermore, Gianotti et al. [33] demonstrated an inverse linear relationship between the blood flow in individual intestinal villi and the number of translocated Candida albicans per individual villus in a model of burned guinea pigs. A positive correlation between selective splanchnic vasoconstriction, mucosal hypoperfusion, intestinal mucosal acidosis, and an increased incidence of bacterial translocation and endotoxin absorption from the gut was described by Tokyay et al. [34] in minipigs with major burns. This data suggests that the mucosal blood flow and the blood flow in individual intestinal villi is an important determinant of the magnitude of microbial translocation. Furthermore, our results are consistent with those obtained by Vallet et al. [35] who described a marked redistribution of blood flow within the gut wall, away from the mucosa and toward the muscularis during shock. The reduction of villus perfusion in our study was associated with a decrease in the diameters of the main villus arterioles. These results are in agreement with those obtained by other groups [36, 37] who demonstrated a decrease of segmental intestinal blood flow measured in mesenteric arcade arteries in a normotensive model of live E. coli bacteremia in rats. In these studies, the reduction of segmental blood flow was accompanied by a decrease of diameters in first- through third-order arterioles of the previllus microcirculation in the submucosal layer of the bowel wall. Similar results indicating that intestinal vasoconstriction occurs during the compensatory stages of endotoxemia were obtained by Clark et al. [38] in horses challenged with a slow infusion of a low dosage of endotoxin. In their study, increased vascular resistance in the jejunum resulted from increased precapillary resistance. Thus, the reduction of villi blood flow observed in our study might be the result of a vasoconstriction of the previllus and the villus arteriolar microcirculation. In our view, this reaction in the microcirculatory bed of the gut might be interpreted as a redistribution of blood flow away from the splanchnic area in order to maintain cardiac output during the early state of sepsis. The regulatory mechanisms responsible for the vasoconstriction and hypoperfusion within the visceral microvasculature during sepsis are not yet fully understood. However, Spain et al. [39] demonstrated that the inhibition of nitric oxide synthase by L-NAME (Nv-nitroL-arginine methyl ester) aggravates the vasoconstriction in the renal microcirculation observed during bacteremia. Furthermore, the same investigators [40] described an attenuation of the pre- and postglomerular constriction during bacteremia by the topical application of L-arginine, the substrate of the nitric oxide synthase. These results suggest that there might be a substrate-dependent limitation of the nitric oxide synthase during sepsis. This might account for an imbalance between vasodilatory and vasoconstrictory mech-

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anisms in the intestinal microcirculation resulting in hypoperfusion of the gut mucosa during sepsis. In conclusion, the results of our study indicate that vasoconstriction and reduction of intestinal villus blood flow occur early during normotensive endotoxemia representing a mechanism for the redistribution of blood away from the gut. Because of the extreme sensitivity of the villus to reductions in blood flow, the observed microcirculatory response to endotoxin might be responsible for the intestinal mucosal ischemia observed in critically ill patients. Therefore, the prevention of villus vasoconstriction and the preservation of villus blood flow might be beneficial even in compensated, normotensive states of sepsis. REFERENCES 1. Meakins, J. L., and Marshall, J. C. The gastrointestinal tract: the ‘‘motor’’ of MOF. Arch. Surg. 121: 197, 1986. 2. Deitch, E. A. The role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure. Arch. Surg. 125: 403, 1990. 3. Deitch, E. A., and Bridges, R. M. Effect of stress and trauma on bacterial translocation from the gut. J. Surg. Res. 42: 536, 1987. 4. Baron, P., Traber, L. D., Nguyen, T., Hollyoak, M., Heggers, J. P., and Herndon, D. N. Gut failure and translocation following burn and sepsis. J. Surg. Res. 57: 197, 1994. 5. Fink, M. P., Antonsson, J. B., Wang, H., and Rothschild, H. R. Increased intestinal permeability in endotoxic pigs. Mesenteric hypoperfusion as an etiologic factor. Arch. Surg. 126: 211, 1991. 6. Deitch, E. A., Berg, R., and Specian, R. Endotoxin promotes the translocation of bacteria from the gut. Arch. Surg. 122: 185, 1987. 7. O’Dwyer, S. T., Mitchie, H. R., Ziegler, T. R., Revhaug, A., Smith, R. J., and Wilmore, D. W. A single dose of endotoxin increases intestinal permeability in healthy humans. Arch. Surg. 123: 1459, 1988. 8. Haglund, U. Gut ischemia. Gut (Suppl)1: S73, 1994. 9. Chiu, C. J., Mc Ardle, A. H., Brown, R., Scott, H. J., and Gurd, F. N. Intestinal mucosal lesion in low-flow states. Arch. Surg. 101: 478, 1970. 10. Bohlen, H. G., and Gore, R. W. Preparation of rat intestinal muscle and mucosa for quantitative microcirculatory studies. Microvasc. Res. 11: 103, 1976. 11. Gore, R. W., and Bohlen, H. G. Microvascular pressures in rat intestinal muscle and mucosal villi. Am. J. Physiol. 233: H685, 1977. 12. Butcher, E. C., and Weissmann, I. L. Direct fluorescent labelling of cells with fluorescein or rhodamin isocyanate. I. technical aspects. J. Immunol. Methods 37: 97, 1980. 13. Sarelius, I. H., and Duling, B. R. Direct measurement of microvessel hematocrit, red cell flux, velocity and transit time. Am. J. Physiol. 243: H1018, 1982. 14. Barbee, J. H., and Cokelet, G. R. The Fahraeus effect. Microvasc. Res. 3: 6, 1971. 15. Desjardins, C., and Duling, B. R. Microvessel hematocrit: measurement and implications for capillary oxygen transport. Am. J. Physiol. 252: H494, 1987. 16. Albrecht, K. H., Gaehtgens, P., Pries, A., and Heuser, M. The Fahraeus effect in narrow capillaries (i.d. 3.3 to 11.0 mm). Microvasc. Res. 18: 33, 1979. 17. Landow, L., and Andersen, L. W. Splanchnic ischaemia and its role in multiple organ failure. Acta Anaesthesiol. Scand. 38: 626, 1994.

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18. Deitch, E. A., Bridges, W., Baker, J., Ma, J. W., Ma, L., Grisham, M. B., Granger, D. N., Specian, R. D., and Berg, R. Hemorrhagic shock induced bacterial translocation is reduced by xanthine oxidase inhibition or inactivation. Surgery 104: 191, 1988. 19. Deitch, E. A., Bridges, W. M., Ma, J. W., Ma, L., Berg, R. D., and Specian, R. D. Obstructed intestine as a reservoir for systemic infection. Am. J. Surg. 159: 394, 1990. 20. Vallet, B., Curtis, S. E., Lund, N., and Cain, S. M. Intestinal mucosal injury during experimental endotoxic shock. Ann. Franc d’Anesthesie et de Reanimation 13: 675, 1994. 21. Falk, A., Redfors, S., Myrvold, H., and Haglund, U. Small intestinal mucosal lesions in feline septic shock: a study on the pathogenesis. Circ. Shock 17: 327, 1985. 22. Bone, R. C. Gram-negative sepsis. Background, clinical features, and intervention. Chest 100: 802, 1991. 23. Bone, R. C., Balk, R. A., Cerra, F. B., Dellinger, R. P., Fein, A. M., Knaus, W. A., Schein, R. M., and Sibbald, W. J. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee American College of Chest Physicians/ Society of Critical Care Medicine. Chest 101: 1644, 1992. 24. Longnecker, D. E., Harris, P. D. Microcirculatory actions of general anesthetics. Fed. Proc. 39: 1580, 1980. 25. Longnecker, D. E., Miller, F. N., Harris, P. D. Small artery and vein response to ketamine HCL in the bat wing. Anesth. Analg. 53: 64, 1974. 26. Asher, E. F., Alsip, N. L., Zhang, P. Y., and Harris, P. D. Prostaglandin-related microvascular dilation in pentobarbital- and etomidate-anesthetized rats. Anesthesiology 76: 271, 1992. 27. Asher, E. F., Alsip, N. L., Ping, Z. Y., and Harris, P. D. Halothane attenuates small arteriole vasodilation to serotonin (5HT) in skeletal muscle. Anesth. Analg. 73: 583, 1991. 28. Kvietys, P. R., Sheperd, A. P., and Granger, D. N. Laser-Doppler, H2-Clearance and microsphere estimates of mucosal blood flow. Am. J. Physiol. 249: G221, 1985. 29. Dregelid, E., Haukaas, S., Amundsen, S., Eide, G. E., Soreide, O., Lekven, J., and Svanes, K. Microsphere method in measure-

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30.

31.

32. 33.

34.

35.

36.

37.

38.

39.

40.

ment of blood flow to wall layers of small intestine. Am. J. Physiol. 250: G670, 1986. Bohlen, H. G. Intestinal tissue PO2 and microvascular responses during glucose exposure. Am. J. Physiol. 238: H164, 1980. Shepherd, A. P., and Kiel, J. W. A model of countercurrent shunting of oxygen in the intestinal villus. Am. J. Physiol. 262: H1136, 1992. Reilly, P. M., and Bulkley, G. B. Vasoactive mediators and splanchnic perfusion. Crit. Care Med. 21: 55, 1993. Gianotti, L., Alexander, J. W., Fukushima, R., and Childress, C. P. Translocation of Canida albicans is related to the blood flow of individual intestinal villi. Circ. Shock 40: 250, 1993. Tokyay, R., Zeigler, S. T., Traber, D. L., Stothert, J. C., Jr., Loick, H. M., Heggers, J. P., and Herndon, D. N. Postburn gastrointestinal vasoconstriction increases bacterial and endotoxin translocation. J. Appl. Physiol. 74: 1521, 1993. Vallet, B., Lund, N., Curtis, S. E., Kelly, D., and Cain, S. M. Gut and muscle tissue pO2 in endotoxemic dogs during shock and resuscitation. J. Appl. Physiol. 76: 793, 1994. Whitworth, P. W., Cryer, H. M., Garrison, R. N., Baumgarten, T. E., and Harris, P. D. Hypoperfusion of the intestinal microcirculation without decreased cardiac output during live escherichia coli sepsis in rats. Circ. Shock 27: 111, 1989. Theuer, C. J., Wilson, M. A., Steeb, G. D., and Garrison, R. N. Microvascular vasoconstriction and mucosal hypoperfusion of the rat small intestine during bacteremia. Circ. Shock 40: 61, 1993. Clark, E. S., and Collatos, C. Hypoperfusion of the small intestine during slow infusion of a low dosage of endotoxin in anesthetized horses. Cornell Vet. 80: 163, 1990. Spain, D. A., Wilson, M. A., and Garrison, R. N. Nitric oxide synthase inhibition exacerbates sepsis-induced renal hypoperfusion. Surgery 116: 322, 1994. Spain, D. A., Wilson, M. A., Bloom, I. T. M., and Garrison, R. N. Renal microvascular responses are dependent on nitric oxide. J. Surg. Res. 56: 524, 1994.

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