Dobutamine maintains intestinal villus blood flow during normotensive endotoxemia: An intravital microscopic study in the rat

Dobutamine Maintains Intestinal Villus Blood Flow During Normotensive Endotoxemia: An Intravital Microscopic Study in the Rat Andreas

Secchi,

Ruth Wellmann,

Eike Martin,

and Heinfried

Schmidt

Purpose:The gut plays a pivotal role in sepsis. Intestinal hypoperfusion with subsequent ischemia leads to translocation of endotoxin. Dobutamine has been demonstrated to increase mesenteric blood flow during endotoxic shock; however, its effects on mucosal blood flow especially in intestinal villi is not known. Therefore, we investigated its influence on the blood flow and the arteriolar diameters in intestinal villi in a model of normotensive endotoxemia. Materials and Methods: Twenty-one male Wistar rats were divided into three groups: (I) control, saline; (2) endotoxin, endotoxin 1.5 mg/kg during 60 minutes; and (3) dobutamine, endotoxin 1.5 mg/kg (60 minutes) and dobutamine 2.5 pglkglmin during 120 minutes. Villus blood flow and arteriolar diameters were deter-

mined at 0 minutes, 60 minutes, and 120 minutes in each group using intravital microscopy. Results: Villus blood flow was constant in the control group, significantly reduced at 120 minutes in the endotoxin group (120 minutes, 55.1 f 7.4%). and remained at baseline values in the dobutamine group. The arteriolar diameters remained constant in the control and the dobutamine groups, but they were significantly reduced in the endotoxin group at 120 minutes (7.8 + 0.2 to 6.5 f 0.7 pm). Conclusion: Our results indicate that in rats with normotensive endotoxemia, arteriolar diameters and blood flow in intestinal villi were reduced. Dobutamine prevented arteriolar constriction and maintained villus blood flow at preendotoxemic values. Copyright o 1997 by W.B. Saunders Company

I

because of their vascular anatomy and their countercurrent oxygen-exchange mechanism.9 Thus, we measured blood flow and arteriolar diameters in arterioles at the tip of intestinal villi using intravital microscopy.

N THE PATHOPHYSIOLOGY of the systemic inflammatory response syndrome, which often leads to multiple organ failure, the gut is considered “the motor of multiple organ failure.” 1 Normally, the gut mucosa prevents the translocation of intraluminal bacteria and toxins to extraintestinal sites and into the portal circulation. However, under septic conditions, disruption of the mucosal barrier might occur resulting in a translocation of bacteria and toxins to the portal circulation and lymphatic system.2 Hypoperfusion of the gut mucosa is considered an important factor leading to alterations in gut epithelial permeability in endotoxemia and sepsis.3x4 Dobutamine is a synthetic catecholamine, which acts mainly on Pi-adrenergic receptors and poorly on p2- and a-adrenergic receptors.5 Dobutamine improves cardiac output during heart failure6 and appears to be beneficial in septic shock. Further, septic patients showed increases in gastric mucosal pH during the infusion of dobutamine, whereas in patients who have undergone cardiac surgery, dobutamine induces a dissociation between splanchnic oxygen delivery and gastric mucosal tissue oxygenation, suggesting inappropriate distribution of blood flow within the splanchnic region.7J The aim of this study was to investigate the effects of dobutamine on endotoxin-induced microcirculatory alterations in the gut mucosa. The tips of the intestinal villi are the most susceptible to regional hypoxemia and hypoperfusion in the gut Journal

of Critical

Care, Vol 12, No 3 (September),

1997: pp 137-141

MATERIALS

AND

METHODS

Animal Preparation The study protocol and all experimental procedures used in this investigation were approved by the Governmental Animal Protection Committee. Male Wistar rats (250-300 g body weight [b.w.]) were used for the experiment. All animals were kept on a diet of standard rat chow until the day before the experiment. Twelve hours before the experiment the rats had only free excess to water. Anaesthesia was performed by intraperitoneal injection of pentobarbital(20 mgikg b.w.; Nembutal; Sanofi, Germany) and intramuscular injection of 30 mg/kg b.w. ketamine (Ketanest; Park Davies, Germany). After cannulation of the left carotid arteria for monitoring of the mean arterial pressure and the right jugular vein for drug administration with polyethylene catheters (outer diameter, 0.8; inner diameter, 0.5 mm) a tracheotomy was performed for airway control. The rectal temperature was measured with a thermistor probe and maintained at 37°C using a heating lamp. For intravital microscopy of the intestinal mucosa we used a modified procedure according to Bohlen and Gore.‘O,” The

From the Department of Anaesthesia, University of Heidelberg, Heidelberg, Germany. Received February 3, 1997. Accepted May 23, 1997. Address reprint requests to Andreas See&, MD, Department of Anaesthesia, University of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany. Copyright 0 1997 by KB. Saunders Company O&W9441/97/1203-0005$5.00/O

137

138

SECCHI

ET AL

abdomen was opened by midline laparotomy, and a loop of the small intestine supplied by a single vascular arcade was exteriorized. It was opened along its antimesenteric border using electrocautery. Stool on the surface was cautiously flushed away with normal saline at 37°C. The intestinal loop was fixed on a Plexiglas stage, the mucosa being upside, with 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 mucosal surface was perfused during the entire experimental period with a buffered bicarbonate salt solution (132 mmol/L NaCl, 4.7 mmol/L KCl, 2 mmol/L CaC12, 1.2 mmol/L MgCIz, 18 mmol&. NaHCOs; equilibrated with 5% CO2 in Nz to adjust the pH to 7.35, the Pop at 25-30 mm Hg, and the Pcoz at 35-45 mm Hg) at 37°C.

counting the number of labeled erythrocytes (Fr& circulating through the central arteriole near the tip of the villus during a 30-second period. At each timepoint, arterial blood samples were also taken for counting the number of fluorescent erythrocytes per unit volume (NFITc) in a Neubauer Chamber (Neubauerbright line, Hecht, Germany). As it was shown in the hamster cheek pouch and the cremaster muscle that the ratio of labeled to native cells in capillary and arterial blood is identicaLi blood flow can be calculated from the ratio F~ro/N~~o. Because the systemic hematocrit exceeds the hematocrit in microcirculatory vessels due to the Fahraeus effect,i5 the calculated volumetric blood flow has to be corrected by a correction factor of 0.76i6 as follows:

Fluorescein Isothiocyanate Labeling of Erythrocytes

The diameters each recorded base to the tip lation analysis

Erythrocytes from separate donor rats were labeled with fluorescein isothiocyanate (FITC, Isomer I, No. F-7250; Sigma Chemicals, Germany) using a modified procedure according to Butcher and Weissmann’* and Sarelius and Duling.i3 Blood from donor rats was washed three times with Alsever’s-buffer solution and one time with bicine-saline-buffer solution to remove plasma. The washed erythocytes were diluted 1:2 with bicine-saline-buffer solution and incubated with FITC (9 mg/mL erythrocytes) for 180 minutes at 25°C. Labeled erythrocytes were washed five times in bicine-saline-buffer solution. Then, the erytbrocytes were diluted with saline until the hematocrit was 50%. Citrate-phosphate-dextrose solution (CPD; No. C-7165; Sigma Chemicals, Germany) was used for preservation. Thiiy minutes before the first microscopy, all animals received 0.5 mL/kg b.w. FITC-labeled erythrocytes.

Intravital Microscopy Intravital microscopy was performed using a specially designed intravital microscope (Leica, Germany) equipped with a 25.fold water immersion objective (PL Fluotar 25/0.75 W; Leitz, Germany), a lo-fold eyepiece, and a transfer lense. The FITC-labeled erythrocytes, which were used to calculate the blood flow in the villus central arterioles, were visualized using epifluorescence illumination. This was performed using an illuminator (Type 307-148.002 514687; Leitz, Germany) consisting of a XBO lOOW/2 short arc mercury lamp and a bypass filter (450490 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. The images from the microscope were transferred to a monitor (PVM444QM; Sony, Japan) by a low-light camera (CF 8/l; Kappa, Germany) and simultaneously recorded on videotape using a videorecorder (AG 7350; Panasonic, Japan) for off-line analysis.

V = (F,,/N,c)/0.76[nL/min] of the villus arterioles were measured off line in central arteriole at eight different places from the of the villi using a computer-assisted microcircusystem (Cap Image, Zeintl, Heidelberg).

Monitoring Mean arterial pressure and heart rate were recorded every 15 minutes during the entire experimental period. Systemic leukocyte count, platelet count, and hematocrit were determined at baseline, and 60 and 120 minutes later using an analyzing system, which was calibrated for rat blood cells (Hematology Analyzer System CP 9000-3; Serono Baker Diagnostics Inc, Allentown, PA).

Experimental Protocol After a stabilization period of 30 minutes following administration of FlTC-labeled erythrocytes, 21 male Wistar rats (250-300 g) were randomized into three groups and baseline measurements were performed. Thereafter, the control group received saline 0.9% only; the endotoxin group received LPS (1.5 mg/kg b.w.; lipopolysaccharide Escherichia coli 026:B6; Sigma Chemicals, Germany) during the first hour, and the dobutamine group received LPS 1.5 mg/kg during the first hour and dobutamine 2.5 pg/kg/min during the entire experimental period. Total volume substitution in all groups was 15 mL/kg b.w./h. Further timepoints of microscopy and determination of blood cell count were at 60 minutes and 120 minutes.

Statistical Analysis Data between groups were compared with analysis of variance (ANOVA) followed by the Student’s t test with Bonferroni’s correction. Paired Student’s t test was used to assess differences within groups when comparing the variables at various timepoints. P s .05 was considered to be significant. All data are expressed as mean i SEM.

RESULTS

Measurement of Blood Flow in the Central Villus Arterioles A modified measurement animal and at recorded on a

method described by Chun et ali4 was used for the of the blood flow in the intestinal villi. In each each timepoint, 5 to 7 villi were microscoped and videotape. Blood flow was determined off line by

Macrohemodynamic Changes Baseline values of mean arterial blood pressure did not differ between groups. They also remained stable throughout the observation period in all groups (Fig 1). Whereas the heart rate was equal in

DOBUTAMINE

AND

INTESTINAL

I

I

I

VILLUS

I

I

BLOOD

I

FLOW

I

I

Table 1. Hematocrit,

II

Count

140

lime (minutes)

1 2 2 ;

Grout

0

I 120

60

110 130 I ,oo c I

120

-0-A

fB

+C

0

I

I

I

30

I

I

60

I

90

I

I

I

120

[min]

Fig I. Mean arterial blood pressure. All data are mean ir SEM. Group A: saline 0.9% from t = 0 minutes until t = 120 minutes; group B: endotoxin 1.5 mg/kg from t = 0 minutes until t = 120 minutes; group C: endotoxin 1.5 mg/kg from t = 0 minutes until t = 120 minutes and dobutamine 2.5 pglkglmin from t = 0 minutes until t = 120 minutes.

all groups at baseline measurement, in animals exposed to endotoxin the heart rate increased significantly from 377 + 13 bpm at baseline to 446 + 14 bpm at 120 minutes in the endotoxin group (P % .OS)and from 386 5 10 bpm to 441 2 14 bpm in the dobutamine group (P 5 .OS).At 120 minutes, heart rate was significantly higher in both groups then in the control group (P 5 .0.5), in which the heart rate was constant over the entire experimental period (baseline: 390 + 21 bpm; 120 minutes: 396 + 11 bpm).

Cell Count,

(%)

White Blood Cell Count (cells/nL)

Platelet Count (cells/nL)

A B

42.1 2 1.8 41.2 i 0.8

8.1 t 3.1 7.5 i 0.6

567 2 42 596 234

C A

38.3 i 1.3 41.4 i 2.4

6.0 5 0.9 7.2 2 1.5

537 2 48 572 582

B C

43.0 i 1.8 40.4 t 1.0

3.2 5 0.3t,* 2.3 1 0.4t,§

486 F 40" 445 i 35

A B

39.0 k 2.2 41.2 2 1.3

7.9 1 1.4 2.3 C 0.3t,§

517 282 386 -t- 20t

39.6 L 0.8

A: saline

0.9%

from

3.1 ? 0.4t,9 t = 0 minutes

until t = 120 minutes

.Ol vs group

430 + 44* until

t = 120

from t = 0 minutes until 1.5 mg/kg from t = 0

and dobutamine

from t = 0 minutes until t = 120 minutes. *P< .05 vs baseline; tP5 .Ol vs baseline; A; §Ps

and Platelet

and at 120 Minutes

minutes; group B: endotoxin 1.5 mg/kg t = 120 minutes; group C: endotoxin minutes

time

Blood

at 60 Minutes

Hematocrit

C Group

l

White

at Baseline,

*Ps

2.5 pgikglmin 0.05 vs group

A.

values and was greater than that of the endotoxin group at 60 minutes and at 120 minutes (Fig 2). No vasoconstriction in central villus arterioles was observed in the control group (Fig 3). In the endotoxin group a significant constriction was noted after 60 minutes and 120 minutes (8.0 + 0.3

1401,

Blood Cell Count All groups had comparable baseline values of white cell count and platelet count. The administration of endotoxin induced a marked depression in both counts. In contrast, the control group showed no changes in white cell or platelet count. Hematocrit was unaffected in all groups throughout the observation period (Table 1). Microhemodynamic Changes in Intestinal Villi No differences in the baseline values of the intestinal villus blood flow were observed. Whereas the villus blood flow remained at baseline values in the control group, it decreased significantly in the endotoxin-treated group. The villus blood flow of the dobutamine treated group remained at baseline

20'

' 0

I

60

I

120

t [min] Fig 2. Intestinal villus blood flow at 60 minutes and at 120 minutes, baseline It = 0 minutes) = 100%; baseline values: group A: 8.1 f 1.62 nL/min, group B: 8.35 r 0.81 nL/min, group C: 11.42 f 0.84 nL/min; the difference between baseline values is not significant. All data are mean f SEM. *P 5 .05 versus baseline; **P 5 .Ol; #P 5 .05 versus group C; “P 5 .Ol versus group C. Group A: saline 0.9% from t = 0 minutes until t = 120 minutes; group B: endotoxin 1.5 mglkg from t = 0 minutes until t = 120 minutes; group C: endotoxin 1.5 mglkg from t = 0 minutes until t = 120 minutes and dobutamine 2.5 pg/kg/min from t = 0 minutes until t = 120 minutes.

SECCHI

al rLn

E Lz

0

-15

-20 -25

* -I

t 60

0

120

t [min] Fig 3. Changes of arteriolar diameters in intestinal villi. All data are mean -+ SEM. *P 5 .05 versus baseline; ‘P 5 .05 versus group C. Group A: saline 0.9% from t = 0 minutes until t = 120 minutes; group B: endotoxin 1.5 mg/kg from t = 0 minutes until t = 120 minutes; group C: endotoxin 1.5 mg/kg from t = 0 minutes until t = 120 minutes and dobutamine 2.5 ug/kg/min from t = 0 minutes until t = 120 minutes.

pm; *P I .05 v baseline), whereas the dobutamine-treated group showed no changes of arteriolar diameter during the observation period and the arteriolar diameters were at 120 minutes significantly greater than in the endotoxin group.

to 6.9 + 0.3* to 6.6 +- 0.3”

DISCUSSION

The gastrointestinal tract has been considered the “undrained abscess” of multiple organ failure17 and the maintainance of sepsis by gut-derived endotoxin has been postulated.lg In our study, we assessed the effects of dobutamine on intestinal villus microcirculation in a model of normotensive endotoxemia in rats. The endotoxemic rats in our study exhibited changes that were comparable to clinical findings observed during compensated sepsis in humans,19 and they met the criteria for laboratory models of sepsis proposed by Fink and Heard.20 After endotoxin application we measured a significant reduction in intestinal villus blood flow, which was associated with a vasoconstriction of the villus arterioles. These results were in agreement with the findings of other authors. Thus, Whitworth et a121observed a decrease in the arteriolar diameters in first (Al), second (A2), and third (A3) order

ET AL

arterioles of the intestinal microcirculation together with a 56% decrease in microvascular blood flow during high cardiac output bacteraemia in rats2i Also, Theuer et alz2 reported a vasoconstriction of about 30% from baseline in Al and A3 arterioles associated with a decrease in mucosal perfusion by 40% of baseline during normotensive bacteremia.22 The regulatory mechanisms responsible for the vasoconstriction and hypoperfusion within the visceral microvasculature during sepsis are not yet fully understood. However, an imbalance between vasodilatory and vasoconstrictory mechanisms because of an increased release of vasoconstrictors such as endothelin or norepinephrine is discussed.23,24 Taken together, these data indicate a central role of the intestinal microcirculation in the development of mucosal hypoperfusion during endotoxemia. The reduction of villus blood flow to critical values is supposed to lead to hypoxic conditions at the tip of the villi. lo However, gut hypoxia might contribute to an increase in intestinal mucosal permeability as shown by Fink et al3 in endotoxemit pigs. In our study, dobutamine was able to prevent the endotoxin-induced decrease in mucosal blood flow. There are at least two reasons that might be responsible for this observation. First, the maintainance of villus blood flow might be caused by the effects of an increase in systemic flow. Even in a dose of 2.5 ug/kg/min dobutamine increases significantly cardiac output in patients with chronic congestive heart failure. 25 Second, a direct vasodilatory action on mucosal vessels might be responsible for the maintainance of mucosal perfusion. In our experiment, dobutamine was able to prevent villus arteriolar vasoconstriction. Silverman and Tuma26 postulated an o-antagonistic and thus a direct vasodilatory effect of dobutamine on the splanchnic circulation, which may be more pronounced when splanchnic vascular tone is increased under conditions of enhanced sympathetic stimulation. This would explain, that in septic states, where there is a release of vasoconstrictiveacting catecholamines, 21 dobutamine increases splanchnic blood how,7926whereas in patients with congestive heart failure, dobutamine does not significantly alter splanchnic perfusion.28 In our laboratory, we showed in the same model,

DOBUTAMINE

AND

INTESTINAL

VILLUS

BLOOD

FLOW

141

that low-dose dopamine prevented the arteriolar vasoconstriction, but did not completely prevent the decrease in villus blood flo~.~~ Therefore, we presume that a slight increase in cardiac output caused by P1-agonism of dobutamine might also be necessary to maintain villus blood flow. In summary, this study demonstrates the effectiveness of dobutamine in maintainance of the mucosal blood flow during endotoxemia in rats as directly measured by intravital microscopy. Thus, the re-

sults of our study confirm the results of other authors, who showed indirectly by gastric tonometry an increase in splanchnic perfusion in septic patients. 7,26The prophylactic use of dobutamine might probably have protective effects on intestinal microcirculation during compensated septic states. However, further studies are necessary to determine whether dobutamine can limit gut ischemia and is able to prevent gut-induced multiple organ failure in critically ill patients.

REFERENCES 1. Meakins JL, Marshall JC: The gastrointestinal tract: The “motor of MOE”Arch Surg 121:197-208, 1986 2. Baron P, Traber LD, Traber DL, et al: Gut failure and translocation following bum and sepsis. J Surg Res 57: 197-204, 1994 3. Fink MP, Antonsson JB, Wang H: Increased intestinal permeability in endotoxic pigs: Mesenteric hypoperfusion as an etiologic factor. Arch Surg 126:211-218, 1991 4. O’Dwyer ST, Michie HR, Ziegler TR, et al: A single dose of endotoxin increases intestinal permeability in healthy humans. Arch Surg 123:1459-1464,1988 5. Sonnenblick EH, Frishman WH, LeJemtel TH: Dobutamine: Anew synthetic cardioactive sympathetic amine. N Engl J Med 300:17-22, 1979 6. MacGregor DA, Butterworth JF, Zaloga ZP, et al: Hemodynamic and renal effects of dopexamine and dobutamine in patients with reduced cardiac output following coronary artery bypass grafting. Chest 106:835-841, 1994 7. Gutierrez G, Clark C, Brown SD, et al: Effect of dobutamine on oxygen consumption and gastric mucosal pH in septic patients. Am J Respir Crit Care Med 150:324-329, 1994 8. Parviainen L, Ruokonen E, Takala J: Dobutamine-induced dissociation between changes in splanchnic blood flow and gastric intramucosal pH after cardiac surgery. Br J Anaesth 74277-282, 1995 9. Sheperd AP, Kiel SW: A model

of countercurrent shunting of oxygen in the intestinal villus. Am J Physiol 262:H1136H1142, 1992 10. Bohlen HG, Gore RW: Preparation of rat intestinal muscle and mucosa for quantitative microcirculatory sudies. Microvasc Res 11:103-110, 1976 11. Gore RW, Bohlen HG: Microvascular pressures in rat intestinal muscle and mucosal villi. Am J Physiol 233:H685-

693, 1977

12. Butcher EC, Weissmann IL: Direct fluorescent labelling of cells with fluorescein or rhodamin isocyanate. I. Technical aspects. J Immunol Methods 37:97-108, 1980 13. Sarelius IH, Duling BR: Direct measurement of microvesse1 hematocrit, red cell flux, velocity and transit time. Am J Physiol243:H1018-H1026,1982 14. Chun K, Dmgas D, Biewer J, et al: Intestinal villus microcirculatory response to hemorrhage in adult and immature rats. J Pediatr Surg 243:H1018-1026, 1992 15. Barbee JH, Cokelet GR: The Fahraeus effect. Microvasc Res 3:6-16, 1971

16. Albrecht KH, Gaehtgens P, Pries A, et al: The Fahraeus effect in narrow capillaries (i.d. 3.3 to 11.0 pm). Microvasc Res 18:33-47, 1979 17. Marshall JC, Christou NV, Meal&s JL: The gastrointestinal tract: The “undrained abscess” of multiple organ failure. AnnSurg218:111-119,1993 18. Deitch EA: The role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure. Arch Surg 125:403-404, 1990 19. Bone RC, Balk RA, Cerra FB, et al: Definition for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. accplsccm consensus conference: Chest 101:1644-1655, 1992 20. Fink MP, Heard SO: Laboratory models of sepsis and septic shock. J Surg Res 49:186-196,199O 21. Whitworth PW, Cryer HM, Garrison RN, et al: Hypoperfusion of the intestinal microcirculation without decreased cardiac output during live Escherichia coli sepsis in rats. Circ Shock27:111-122, 1989 22. Theuer CJ, Wilson MA, Steeb GD, et al: Microvascular vasoconstriction and mucosal hypoperfusion of the rat small intestine during bacteremia. Circ Shock 40:61-68, 1993 23. Fantinin GA, Shiono S: Bal BS, et al: Adrenergic mechanisms contribute to alterations in regional perfusion during normotensive E. coli bacteremia. J Trauma 29:12521257,1989 24. Wilson MA, Steeb GD, Garrison RN: Endothelins mediate intestinal hypoperfusion during bacteremia. J Surg Res 55:168-175, 1993 25. Baumann G, Felix SB, Filcek SAL: Usefulness of dopexamine hydrochloride versus dobutamine in chronic congestive heart failure and effects on hemodynamics and urine output. Am J Cardiol65:748-754, 1990 26. Silverman HJ, Tuma P: Gastric tonometry in patients with sepsis. Chest 102/1:184-188, 1992 27. Hahn PY, Wang P, Tait SM et al: Sustained elevation in circulating catecholamine levels during polymicrobial sepsis. Shock 4:269-273, 1995 28. Leier CV: Regional blood flow responses to vasodilators and inotropes in congestive heart failure. Am J Cardiol 62:86E93E, 1988 29. Schmidt H, Secchi A, Welhnann R, et al: Effect of low-dose dopamine on intestinal villus microcirculation during normotensive endotoxemia in rats. Br J Anaesth 76:707-712, 1996