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Anesth Analg 2000;91:517-521
© 2000 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

Thoracic Aorta Occlusion-Reperfusion Decreases Hemostasis as Assessed by Thromboelastography in Rabbits

Vance G. Nielsen, MD, and Brian T. Geary, BS

Department of Anesthesiology, Division of Cardiothoracic Anesthesia, The University of Alabama at Birmingham, Birmingham, Alabama

Address correspondence and reprint requests to Vance G. Nielsen, MD, Department of Anesthesiology, The University of Alabama at Birmingham, 619 South 19th St., Birmingham, AL 35249. Address e-mail to vance.nielsen{at}ccc.uab.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Perioperative hemorrhage and thrombosis are serious complications associated with major vascular surgery. We hypothesized that thoracic aortic occlusion-reperfusion in rabbits would adversely affect hemostasis as assessed by thromboelastographic variables (reaction time, {alpha} angle and G [a measure of clot strength]). Isoflurane-anesthetized rabbits underwent either sham operation (n = 10) or 30 min of aortic occlusion followed by 90 min of reperfusion (n = 10). Blood samples (350 µL) were exposed to 10 µL of either 0.9% NaCl or cytochalasin D (a platelet inhibitor, 10 µM final concentration) and analyzed for 1 h by using thromboelastography after 30 min of postpreparation equilibration and at 30 and 90 min of reperfusion. Aortic occlusion-reperfusion resulted in a significant (P < 0.05) increase in reaction time, decrease in {alpha} angle, and decrease in G at 30 and 90 min of reperfusion compared with the sham-operated group. The decrease in hemostatic function after aortic occlusion-reperfusion was observed to the same degree in samples with or without platelet inhibition. There were no significant differences in platelet concentration between the sham-operated and aortic occlusion-reperfusion groups. Aortic occlusion-reperfusion decreased hemostatic function in rabbits primarily by decreasing the coagulation factor-dependent, platelet-independent contribution to clotting.

Implications: Thoracic aortic occlusion-reperfusion decreased hemostatic function in rabbits primarily by decreasing the coagulation factor-dependent, platelet-independent contribution to clotting. This decrease in hemostatic function may contribute to hemorrhagic complications associated with major vascular surgery.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
In the setting of aortic cross-clamping, blood is the first tissue to initiate and endure the effects of reperfusion injury. Among the many functions of blood, hemostasis is of great clinical interest after major vascular and trauma surgery, as hemorrhage and thrombosis are major sources of morbidity and mortality (1–6). Further, the site of the aorta cross-clamp can profoundly affect hemostasis, with infrarenal ischemia associated with enhanced clot strength in humans (3), whereas supraceliac cross-clamping has been associated with fibrinolysis (7). Multiple factors may have an impact on hemostasis in the setting of major vascular surgery (e.g., duration of ischemia, temperature change, blood loss, hemodilution, hepatic ischemia, and tissue factor release), making the investigation of coagulopathy problematic. Consequently, it would be of benefit to develop an animal model of aortic cross-clamping to better characterize the role played by ischemia-reperfusion injury in the evolution of coagulopathy.

We previously described a rabbit model of thoracic aorta occlusion-reperfusion used to investigate multiple organ injury (8–11). Further, we recently demonstrated that hemostatic function in the rabbit, as assessed by thromboelastography, is similar to that noted in humans (12,13). Thus, the purpose of the present study was to test the hypothesis that thoracic aorta occlusion-reperfusion would adversely affect hemostatic function in rabbits as assessed by thromboelastography.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The study was approved by our animal review committee. New Zealand White rabbits (2–3 kg) were anesthetized with 10 mg/kg IV ketamine via a marginal ear vein and subsequently administered inhaled 1% isoflurane carried in 99% oxygen. Isoflurane administration (inspired concentration) was monitored with an anesthetic specific monitor (model 8100TM; BCI International, Waukesha, WI). After tracheotomy and placement of a 3.5-mm outside diameter endotracheal tube, mechanical ventilation with a Harvard Apparatus ventilator was performed with PaCO2 maintained at 32–45 mm Hg. Pancuronium was administered 0.3 mg · kg-1 · h-1 IV to facilitate mechanical ventilation. Arterial pressure was monitored by placement of a 22-gauge central ear artery catheter and a right femoral arterial catheter for blood sampling. Central venous access was obtained via the right internal jugular vein for pressure monitoring and fluid administration. All pressures were recorded on a Grass Model 7D polygraphTM (Grass Instruments, Quincy, MA). All rabbits received a maintenance infusion of lactated Ringer’s solution at 4 mL · kg-1 · h-1, and esophageal temperatures were maintained at 38°–39°C with a heating pad. A 30-min equilibration period followed completion of the surgical preparation.

After equilibration, rabbits were randomized to either the sham-operated group (n = 10) or the aortic occlusion-reperfusion group (n = 10). Sham-operated animals had the left femoral artery exposed, with sham aortic occlusion beginning with ligation of the femoral artery. The aortic occlusion groups also underwent a left femoral cutdown, with insertion of a 4F Fogarty embolectomy catheter into the thoracic aorta with the balloon placed 1–2 cm above the diaphragm as confirmed by postmortem examination. Aortic occlusion was achieved by inflation of the catheter balloon with saline. A femoral arterial pressure of 0–10 torr confirmed subdiaphragmatic ischemia. After 30 min of occlusion, the balloon was deflated and the catheter removed. Reperfusion was verified by return of pulsatile flow to the femoral arterial line and transient hypotension as measured by the ear arterial line. Postocclusion shock was treated as previously described (8–11), according to the following algorithm. At the beginning of reperfusion, an IV bolus of lactated Ringer’s solution (20 mL/kg) was administered for 2 min and the infusion rate adjusted to maintain central venous pressure (CVP) at the 30-min equilibration value ±2 mm Hg. Next, phenylephrine administration began at reperfusion and was adjusted as follows: if the CVP = 30-min equilibration value ±2 mm Hg, and the mean arterial blood pressure (MAP) was <80% of the 30-min equilibration value, phenylephrine was administered. Lastly, sodium bicarbonate 8.4% was infused IV to maintain the arterial base deficit near zero.

Arterial blood samples were obtained after 30 min of equilibration and at 30 and 90 min of reperfusion for blood gas analysis with K+ and Ca+2 concentration determination. Platelet concentration and hematocrit were determined with a Sysmex K-800TM (TOA Medical Electronics Co., LTD, Kobe, Japan). Additional, nonheparinized 350-µL blood samples and 10 µL of either 0.9% NaCl or cytochalasin D (final concentration 10 µM) were placed into a disposable cup and inserted into a computer-controlled Thrombelastograph®TM (Model 5000; Haemoscope, Skokie, IL). Cytochalasin D inhibits microtubule formation (and glycoprotein IIb/IIIa receptor conformation changes required for activation) in platelets, resulting in a thromboelastographic signature caused only by coagulation proteins in whole blood (13,14). The final concentration of dimethylsulfoxide in each sample exposed to 0.9% NaCl or cytochalasin D was 0.28% (vol/vol). The proper functioning of the Thrombelastograph® was confirmed daily with level I and level II quality control standards purchased from Haemoscope per the instruction manual. The following thromoboelastographic variables were measured for each sample for 1 h at 39°C (the normal temperature of the rabbit): reaction time (R, min), angle ({alpha}, degrees), maximum amplitude (MA, mm), and G (dyne/cm2). A detailed description of the methodology of thromboelastography has been presented in great detail elsewhere (14,15). In brief, R is defined as the time from when the blood sample is placed into the Thrombelastograph® cuvette until initial fibrin formation occurs as noted by a signal of 2-mm amplitude. {alpha} is the angle formed from R to the inflection point of the thromboelastographic signal as clot strength stabilizes; it is a measure of the kinetics of clot formation. MA is the largest amplitude of the thromboelastographic signal and is a measure of clot strength. Finally, G (dyne/cm2) is a measure of clot strength (15) calculated from MA as follows: G = (5000 x MA)/(100 - MA). The relationship between MA and G is curvilinear. As MA varies from 0 to 100, G concordantly varies from 0 to infinity. Given this relationship, it is conceptually and statistically important to express clot strength as G (14). Consequently, although MA was determined, we reported G. The contribution of platelets to G (GP) was defined by the total G of whole blood not exposed to cytochalasin D (GT) minus the G of blood exposed to cytochalasin D, which is attributable to the soluble components of the coagulation pathway (GSC).

All variables were expressed as mean ± SD. Parametric data were analyzed with one-way analysis of variance with or without repeated measures as appropriate. Nonparametric data were analyzed with Friedman repeated measures analysis of variance, or the Mann-Whitney U-test as appropriate. With regard to thromboelastography, it was decided a priori that blood samples that did not clot were to be assigned an R value of 60 min, an {alpha} value of 0°, an MA value of 0 mm, and a G value of 0 dyne/cm2. Post hoc analyses were conducted with the Student-Newman-Keuls test. An {alpha} error of <=0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Aortic occlusion-reperfusion significantly modified thromboelastographic variables in the rabbit as displayed in Table 1. Compared with sham-operated animals, whole blood without the addition of cytochalasin D obtained from rabbits exposed to aortic occlusion-reperfusion had a significant increase in R, decrease in {alpha}, and decrease in G. Blood samples exposed to cytochalasin D similarly demonstrated that aortic occlusion-reperfusion significantly increased R, decreased {alpha}, and decreased in G. The relative contributions of platelets and the soluble components of the coagulation pathway (GP and GSC, respectively) were significantly different between the two groups at 90 min of reperfusion.


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  		Table 1. Thromboelastographic Variables R (min), {alpha} (degrees), and G (dyne/cm2)
 
Hemodynamic, platelet concentration and hematocrit, and arterial blood gas analysis data are depicted in Tables 2 and 3. While there were no significant differences in MAP or CVP values between the groups, the aortic occlusion-reperfusion group demonstrated a significantly lower heart rate at 30 and 90 min of reperfusion compared with the sham-operated group. There was no significant difference in platelet concentration or hematocrit between the groups. Arterial pH and PaCO2 values of the aortic occlusion-reperfusion group at 30 min of reperfusion were significantly different from those of the sham group. The PaO2 values during reperfusion were significantly greater in the sham-operated group than in the aortic occlusion-reperfusion group. Although there was no significant differences in K+ between the groups, the aortic occlusion-reperfusion group had significantly lower Ca+2 values at 30 and 90 min of reperfusion compared with the sham-operated group. Lastly, the aortic occlusion-reperfusion group required 6.3 ± 3.0 mg · kg-1 · 1.5h-1 of phenylephrine and 7.8 ± 2.4 mEq · kg-1 · 1.5h-1 of sodium bicarbonate.


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  		Table 2. Hemodynamic, Platelet Concentration, and Hematocrit Data
 

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  		Table 3. Arterial Blood Gas and Electrolyte Data
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Thoracic aorta occlusion for 30 min followed by 90 min of reperfusion in rabbits resulted in a profound decrease in hemostatic function as assessed by changes in the thromboelastographic variables R, {alpha}, and G. These hemostatic changes in the aortic occlusion-reperfusion group occurred while circulating platelet concentrations did not differ from the sham-operated group. Of interest, the R, {alpha}, and G values observed after platelet inhibition with cytochalasin D were adversely affected by aortic occlusion-reperfusion. Further, the relative contribution of GSC to GT did significantly decrease after 90 min of reperfusion in the aortic occlusion group compared with the sham-operated group. These data support the concept that the primary etiology of the hemostatic defect observed after aortic occlusion-reperfusion in rabbits is a decrease in circulating coagulation protein function.

There are likely several mechanisms by which aortic occlusion-reperfusion may decrease coagulation protein function. First, ischemia-reperfusion injury to the liver may decrease production of procoagulants, analogous to the early stages of reperfusion during hepatic transplantation (16). Indeed, supradiaphragmatic aortic cross-clamping in humans (7) and occlusion in rabbits (8,9,11) results in significant release of hepatocellular enzymes. Second, in addition to the effects of ischemia, per se, xanthine oxidase-derived radicals could further increase hepatic injury as previously described (8,9,11), contributing to coagulopathy. Third, reperfusing liver has been demonstrated to release a heparin-like substance into the circulation in the setting of hepatic transplantation (17–19). In addition to either decreasing procoagulant production or causing the release of endogenous anticoagulants, it is possible that aortic occlusion-reperfusion may result in an increase in removal of coagulation proteins from the circulation secondary to hemodilution during resuscitation or loss into the interstititial space secondary to increased capillary membrane permeability. Considered as a whole, there are several lines of investigation to be pursued in discerning the mechanisms responsible for the coagulopathy observed after aortic occlusion-reperfusion.

It is important to note the limitations of the present study. First, it is unable to provide mechanistic insight into the cause of the coagulopathy observed after aortic occlusion-reperfusion. Further, although it is possible to directly determine the thromoboelastographic profile of whole blood and coagulation protein function with our methodology, one cannot directly measure platelet function in isolation. That is to say, whole blood can clot in the absence of platelet function, but whole blood cannot clot in the absence of coagulation protein function. Nevertheless, one can infer changes in platelet function by noting changes in the relative contribution of GP and GSC to GT. Unlike platelet aggregometry, which usually involves separation of the platelets from whole blood with citrate-mediated anticoagulation, thromboelastography does not involve any significant delays from the time of blood sampling to the measurement of clotting function. Further, the effects of plasma-borne activators/inhibitors of hemostasis are not separated from the platelets during thromboelastography. Thus, whole-blood thromboelastography still may provide mechanistic insight into hemostatic disorders. Lastly, our study examines the effects of ischemia-reperfusion without all the concordant tissue injury associated with protracted thoracic and retroperitoneal dissection and occasional systemic heparinization associated with aortic surgery.

In conclusion, the present study demonstrated that thoracic aorta occlusion-reperfusion in rabbits results in a hypocoagulable state as assessed by using thromboelastography. The underlying mechanism responsible for this hemostatic abnormality likely involved a decrease in circulating coagulation protein function. The precise etiology of the loss of coagulation protein function (inhibition of activity or lack of protein) will be sought in future studies. Lastly, this decrease in coagulation protein function noted after aortic occlusion-reperfusion in rabbits may contribute to some of the hemorrhagic complications associated with major vascular surgery in the clinical arena.


   Acknowledgments
 
This study was supported, in part, by a grant from BioTime, Inc., Berkeley, CA, and the Department of Anesthesiology.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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  3. Gibbs NM, Crawford GPM, Michalopoulos N. Thrombelastographic patterns following abdominal aortic surgery. Anaesth Intensive Care 1994; 22: 534–8.[ISI][Medline]
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  8. Nielsen VG, Tan S, Baird MS, et al. Gastric intramucosal pH and multiple organ injury: impact of ischemia-reperfusion and xanthine oxidase. Crit Care Med 1996; 24: 1339–44.[ISI][Medline]
  9. Nielsen VG, Tan S, Weinbroum A, et al. Lung injury after hepatoenteric ischemia-reperfusion: role of xanthine oxidase. Am J Respir Crit Care Med 1996; 154: 1364–9.[Abstract]
  10. Nielsen VG, Tan S, Baird MS, et al. Xanthine oxidase mediates myocardial injury after hepatoenteric ischemia-reperfusion. Crit Care Med 1997; 25: 1044–50.[ISI][Medline]
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  12. Nielsen VG, Baird MS. Extreme hemodilution in rabbits: an in vitro and in vivo Thrombelastographic® analysis. Anesth Analg 2000; 90: 541–5.[Abstract/Free Full Text]
  13. Nielsen VG, Geary BT, Baird MS. Evaluation of the contribution of platelets to clot strength by thromboelastography in rabbits: role of tissue factor and cytochalasin D. Anesth Analg 2000; 91: 35–9.[Abstract/Free Full Text]
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Accepted for publication May 17, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press