1 Department of Anaesthesia, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. 2 University Department of Anaesthesia, University of Liverpool, L69 3GA, UK
*Corresponding author. E-mail: schuerholz.tobias{at}mh-hannover.de
Accepted for publication: December 5, 2003
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Abstract |
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Methods. Anaesthetized, mechanically ventilated and multi-catheterized pigs received 1 g kg1 body weight faeces into the abdominal cavity to induce sepsis and were observed over 8 h. Five animals in each group received volume replacement therapy with modified fluid gelatin 4% or 8% (MFG4%, MFG8%), 6% hydroxyethylstarch (HES) 200/0.5 or Ringers solution (RS) to maintain a central venous pressure of 12 mm Hg. Flow cytometry was used for determination of microvesicles before induction of sepsis (baseline) and after 8 h. Platelets and microvesicles were identified with an anti-platelet monoclonal Ab and a secondary antibody. Microvesicles were determined as the smallest 13% positive cells in forward scatter. Intergroup comparisons were performed using WilksLambda and RyanEinotGabrielWelsh F-test. Differences within groups were compared using a two-tailed Students t-test.
Results. Baseline values were considered as 100%. While microvesicle formation was reduced in HES (73 (SD 19)%), MFG4% (63 (41)%) and MFG8% groups (53 (17)%), an increase in the RS-group (210 (121)%) was observed. Eight hours after induction of sepsis, formation of microvesicles was significantly higher in the RS group compared to all colloid-treated groups.
Conclusion. In this porcine septic shock model the formation of platelet-derived microvesicles was significantly increased by volume replacement with Ringers solution in comparison to colloid solutions.
Br J Anaesth 2004; 92: 71621
Keywords: blood, plasma substitutes; blood, platelets; complications, sepsis; complications, septic shock; microvesicles
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Introduction |
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Larson and colleagues7 have described the induction of platelet microvesicle formation in gram-negative induced human septic shock and experimental endotoxaemia. Increased microvesicle formation has been associated with a higher mortality in a porcine model of endotoxaemia,8 and has been related to fatal outcome in patients with activated coagulation.9
Our study is based on the assumption that the choice of volume replacement therapy may also have an important impact on the coagulation process, as it has been recently demonstrated that the use of crystalloids resulted in an enhancement of coagulation.10 This might be particularly important in patients with severe sepsis/septic shock who generally present with large fluid deficits and therefore need volume replacement therapy.11 Although it is widely accepted that volume replacement therapy is important for restoration and maintenance of the intravascular volume in order to improve organ perfusion and nutritive microcirculatory flow, the type of solution which should be used in the treatment of sepsis remains controversial.12
Given the lack of data in respect to the effect of different volume replacement solutions on microvesicle formation during sepsis the aim of our study was to investigate the effects of 4 and 8% modified fluid gelatin, hydroxyethyl starch with a mean molecular weight (MW) of 200 kD and a substitution ratio of 0.5 and Ringers solution on platelet-derived microvesicle formation in a porcine septic shock model.
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Methods |
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Anaesthesia
As previously described,13 animals received a premedication of of Azaperon 5 mg kg1 (Stressnyl®, Jansen, Neuss, Germany) intramuscularly. Thereafter a peripheral venous catheter was placed in an ear vein and anaesthesia was induced by intravenous injection of propofol (Disoprivan®, Zeneca, Plankstadt, Germany) until intubating conditions were achieved. Pigs were orally intubated and placed in the supine position. Anaesthesia was maintained with infusion of thiopentone 5 mg kg1 h1 (Trapanal®, Byk Gulden, Konstanz, Germany), and fentanyl 0.01 mg kg1 h1 (Fentanyl Curamed®, Curamed, Karlsruhe, Germany). Controlled mode ventilation was chosen to ventilate the animals (Servo 900, Siemens-Elema, Solna, Sweden) with an inspiratory oxygen fraction of 0.4, an inspiratory/expiratory ratio of 1:2, and a respiratory rate of 16 bpm. The tidal volume was adjusted to maintain a PCO2 of 35 to 45 torr (4.6 to 5.9 kPa). The core body temperature was maintained at 3739°C using an infrared lamp, a circulating water-mattress and warmed solutions.
Surgical procedures
Right jugular vein and right carotid artery, as well as the left femoral artery were surgically exposed. For drug and fluid administration a central venous catheter was inserted into the superior vena cava, and a balloon-tipped thermodilution pulmonary artery catheter (131F7, Baxter Healthcare, Irvine, CA, USA) was inserted via the right jugular vein. Catheters and infusion systems were not lined with heparin.
In all animals a midline laparotomy and cystotomy were performed using standardized sterile surgical techniques. A urinary catheter was placed to drain and determine urine output. An opening of 2 cm was made in the caecum and 1 g kg1 body weight of its content was aspirated. The caecotomy was then closed and a sterile catheter positioned intra-abdominally before the abdomen was closed with a suture. The catheter was tunnelled out through the subcutaneous tissue of the lateral flank. Following surgical preparation animals were allowed to recover for 120 min before baseline measurements (baseline) were performed. A continuous infusion of Ringers solution (RS; 10 ml kg1 h1 i.v.) was given during surgery and the postsurgical period. To induce sepsis, autologous faeces (1 g kg1) suspended in 150 ml of warm isotonic NaCl (37°C) was injected through the abdominal catheter.
Monitoring and laboratory measurements
Mean arterial pressure (MAP), central venous pressure (CVP) and pulmonary arterial occlusion pressure (PAOP) were recorded from calibrated pressure transducers (Peter v. Berg, Medizintechnik, Engelharting, Germany). Cardiac output (CO) was determined by thermodilution with a CO computer (Vigilance®; Baxter Healthcare, Irvine, CA, USA). Data reported being the mean of three injections of 5 ml ice-cold 5% glucose randomly spread over the respiratory cycle. Arterial and mixed venous blood was sampled in heparinized syringes and immediately analyzed (System 860 including co-oximetry system 835, Bayer Diagnostics, Fernwald, Germany) to determine blood gases, pH, PCO2, PO2, and the calculated variables base excess, standard bicarbonate, and oxygen saturation. This also allowed calculation of systemic oxygen delivery (DO2) and oxygen consumption (VO2) from haemoglobin, oxygen saturation, PO2 and CO according to standard formulae.
Haematocrit (Hct) and platelet count were determined using an automatic cell counter. Cumulative fluid balance was calculated as fluid input minus urinary output over the observation period (8 h).
Experimental procedure
According to a predefined random list, five animals in each group were allocated for fluid therapy with 4% modified fluid gelatin (MFG4%) (Gelafundin, B Braun, Melsungen, Germany) or 8% modified fluid gelatin (MFG8%) (Braun Medical AG, St Gallen, Switzerland), 6% hydroxyethyl starch (HES) (MW 200 kD/0.5 degree of substitution, Hemohes, B Braun, Melsungen, Germany), and Ringers solution (DAB 7, B Braun, Melsungen, Germany). The pH of the Ringers solution used varies between 5.0 and 7.0 (manufacturers information). In our study we did not check individual bag pH values. Haemodynamic treatment scheme was aimed at maintenance of CVP of 12 mm Hg. After induction of sepsis, animals were observed for 8 h. All variables and blood samples were measured simultaneously before induction of sepsis (baseline) and 8 h after induction. Blood samples for determination of microvesicles were collected in 1.4 ml sodium citrate tubes (No. 06.1668.001, Sarstedt, Nuembrecht, Germany).
Measurement of plasma volume and albumin escape rate
For measurement of the red blood cell volume (RBV) 10 ml of blood was withdrawn and erythrocytes in the plasma-free suspension were labelled with chromium-51-tagged erythrocytes (51Cr) (Berthold MAG 312, Germany). After central venous reinjection of the labelled erythrocytes, arterial blood samples (2 ml) were taken after 30, 45, 240 and 480 min. RBV was calculated from the radioactivity of the isotope before injection and after distribution in the blood: RBV=Total 51Cr activity injectedxHaematocrit (Hct)xf/Blood 51Cr activity of the samplex100. Concentration with f=correction factor (0.91) for whole body Hct. Plasma volume (PV) was determined using a standard formula: PV=(100Hct)xfxRBV/Hctxf.
Preparation of samples for flow cytometry
Platelet-rich plasma (5 µl) was added to polystyrene tubes containing 30 µl HEPES-diluted anti-platelet antibody (mouse-anti-pig, DH Sachs, Charlestown, PA, USA). The samples were incubated for 20 min at room temperature. After washing with HEPES-buffer the second fluorescent antibody (goat-anti-mouse-Phycoerythrin, DAKO, Denmark) was added and incubated for 15 min at room temperature. Again the samples were washed with HEPES and then diluted and fixed with 500 µl ice-cold PBS, containing 1% p-formaldehyde. Flow cytometry-analysis was performed within 1 h of sampling.
Flow cytometry
Porcine platelets were analyzed using an Epics XL® cytometer (Beckman Coulter, Krefeld, Germany). Data processing from 20 000 platelets was carried out using WinMDI software.14 The cytometer gives information related to mean cell size (forward scatter), complexity (side scatter) and percentage of positive antibody-stained cells based on their light-scattering properties. Each single cell is represented by a point in a coordinate system. Analytical markers were set in the fluorescence channel to divide a negative control sample into fractions containing 9799% of platelets and 13% of the smallest platelets. Platelets with forward scatter lower than the marker were identified as microvesicles. All measurements were run in duplicate. Microvesicles were counted as a percentage of the platelets smaller than a predetermined size in forward scatter in flow cytometry.7
Statistical analysis
Data were analysed using SPSS for Windows (release 10.07) and all results are presented as mean (SD). After verifying normal distribution (skewness <1.5),15 first a multivariate analysis of variance for repeated measurements using WilksLambda16 was performed, followed by a multiple test strategy of RyanEinotGabrielWelsh F-test17 for pairwise intergroup comparison. Differences within groups were compared by two-tailed Students t-test for paired samples. In order to test the formation of microvesicles and platelets, count baseline values were set as 100% and differences to baseline were calculated.
A P-value <0.05 was considered statistically significant. For this significance level of =0.05 an analysis for the prospective power of the experimental design was performed for a 2-sided test. A 50% difference in means of the microvesicle formation between two groups was regarded as clinically important. To achieve a power level of 90% five animals in each group were needed.
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Results |
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Discussion |
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Compared to baseline there was a reduction in the platelet count in RS, MFG8%, HES group, which was significant in the latter. The increase in the MFG4% group is due to one animal, which had a very low baseline platelet count, which increased to sub-normal values at the end of the study period. In our study the platelet count did not differ between the groups 8 h after induction of sepsis. Thus, our results confirm the findings of Larsson and colleagues who support the view that microvesicle formation is not a simple reflection of a decrease in platelet count.7
Similar to our results, increased formation of platelet-derived microvesicles has been reported in human gram-negative septic shock and experimental porcine sepsis. During clinical sepsis the activation of platelets and the coagulation system is well known.18 Recently, it has been suggested that activation of platelets and monocytes as well as adhesion of platelets to neutrophils may play a role in the development of organ dysfunction.19
In our model, we observed that the massive fluid input in the Ringers solution group comprising a total fluid volume of 11.5 (3.6) litre (576 (183) ml kg1), and a positive fluid balance after 8 h of 7.3 (1.0) litre (366 (49) ml kg1) was associated with a reduced plasma volume. In contrast, restoration of adequate intravascular volume was obtained in the colloid groups. This was combined with a metabolic acidosis and significant lower VO2 in the RS group compared to the colloid groups. This may indicate circulatory alterations and sequestration occurring in the RS group, thereby reducing functional capacity of vital organs and contributing further to an activation of the coagulation cascade. RS may increase tissue oedema compared to the colloids used. One effect of such an oedema would be to retard oxygen uptake by increasing the distance from blood vessels to the mitochondria; this in turn could reduce functional capacity and contribute to the development of coagulopathy and multiple organ failure.20 The composition of the different solutions may also be important. Recently, it has been shown that a crystalloid solution in which lactate was substituted by ethyl pyruvate ameliorated intestinal hyperpermeability in rats.21 Pyruvate probably serves as an endogenous scavenger of ROS, which have been implicated in the pathogenesis of coagulopathy and sepsis. In a murine model of lethal endotoxaemia, treatment with a Ringers ethyl pyruvate solution in place of a Ringers lactated solution prolonged survival and blunted the release of interleukin-6.22 According to our results, Ruttmann and colleagues23 could demonstrate enhanced coagulation measured by TEG with a volume load of about 1000 ml h1 for 3 h.
The underlying reasons for the enhanced activation of platelets using crystalloid Ringers solution remains speculative. It has been discussed that a direct endotoxin effect may cause the increased formation of platelet-derived microvesicles.7 Another explanation is that the release of platelet-derived microvesicles could be mediated by complement activation.24 Furthermore, platelet-derived microparticles formed during complement activation in vivo could provide a membrane surface that facilitates the assembly and dissemination of procoagulant enzyme complexes. In this respect, platelet-derived microvesicles might have a potential role in the pathogenesis of disseminated intravascular coagulation.24 Interestingly, we demonstrated previously, that the complement system is activated in our porcine septic shock model.25
Another interesting aspect may be the influence of crystalloids on coagulation. In vitro haemodilution with saline26 or other crystalloid solutions27 exerted a procoagulant effect, possibly by enhancement of thrombin formation. Recent clinical work demonstrated that haemodilution of up to 30% with saline can induce a hypercoagulable state.10 This increase in coagulation seems to be independent of the type of crystalloid used.28 The reduction in antithrombin caused by dilution has been postulated as a possible important mechanism for the enhancement of haemodilution-induced coagulation.29 In contrast, Innerhofer and colleagues, investigating primary platelet-mediated haemostasis and clot formation, demonstrated that perioperative administration of 6% HES 200/0.5 or MFG4% interfered more with fibrin polymerization and resulting total clot strength than did RS.30
There may be clinical implications in measuring the production of microvesicles: in order to elucidate the effects of different fluid types, appropriate study endpoints need to be developed, as mortality does not seem to be a useful endpoint for fluid resuscitation. Since activation of coagulation occurs in septic shock, measuring the formation of platelet-derived microvesicles might possibly be used as a guide towards therapy.
In conclusion, in a porcine model of septic shock we have shown that the formation of platelet-derived microvesicles was significantly increased in animals treated with Ringers solution, but not in animals treated with artificial colloids HES, MFG4% and MFG 8%. These results suggest that the intravascular activation of platelets in experimental sepsis may be enhanced by crystalloids.
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Acknowledgement |
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References |
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