1

2

3Q1

45

6

789

1011121314151617

18

19

3536

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

Microvascular Research xxx (2014) xxx–xxx

YMVRE-03459; No. of pages: 6; 4C:

Contents lists available at ScienceDirect

Microvascular Research

j ourna l homepage: www.e lsev ie r .com/ locate /ymvre

Short time impact of different hydroxyethyl starch solutions on themesenteric microcirculation in experimental sepsis in rats

OFK. Wafa a, A. Herrmann b, T. Kuhnert b, A. Wegner b, M. Gründling b, D. Pavlovic a,b, C. Lehmann a,⁎

a Department of Anesthesia, Pain Management and Perioperative Medicine, Dalhousie University, Halifax, NS, Canadab Department of Anesthesiology and Intensive Care Medicine, Ernst Moritz Arndt University Greifswald, Germany

⁎ Corresponding author at: Department of AnesthPerioperative Medicine, QE II Health Sciences Centre, 10St., Halifax, NS, B3H 2Y9, Canada. Fax: +1 902 423 9454.

E-mail address: chlehmann@dal.ca (C. Lehmann).

http://dx.doi.org/10.1016/j.mvr.2014.07.0120026-2862/© 2014 Published by Elsevier Inc.

Please cite this article as: Wafa, K., et al., Shexperimental sepsis in rats, Microvasc. Res. (

Oa b s t r a c t

a r t i c l e i n f o

Article history:

20

21

22

23

24

25

26

27

28

ED P

RAccepted 28 July 2014Available online xxxx

Keywords:SepsisCrystalloidsColloidsHydroxyethyl starchIntravital microscopyFluid extravasationLeukocyte adherence

Background: Fluid resuscitation plays a crucial role in the therapy of severe sepsis and septic shock. The useof colloids in sepsis is controversial at present. The aim of our studywas to evaluate the effects of second andthird generation colloids on the mesenteric microcirculation in early experimental sepsis.Methods: Male Lewis rats (n = 64) were used. Animals underwent sham surgery or colon ascendens stentinsertion for sepsis induction by peritonitis. Sixteen hours after the surgery animals were randomlyassigned to receive one of the following fluid regimens intravenously: 16 ml/kg Ringer's lactate, 64 ml/kgRinger's lactate, 16 ml/kg 130/0.4 hydroxyethyl starch, and 16 ml/kg 200/0.5 hydroxyethyl starch. Intravi-tal microscopy of the mesenteric microcirculation (plasma extravasation; leukocyte–endothelial interac-tions) and arterial blood gas analysis were performed before and after fluid resuscitation.Results: In animals with experimental sepsis plasma extravasation was significantly increased compared tocontrol animals (p b 0.05). There were no significant differences in plasma extravasation between septic

29

30

31

32

33

CTanimals receiving crystalloids and or colloid. Furthermore, the type of administered fluid did not influence

the number of adhering leucocytes during the observation period.Conclusion: The short time impact of different hydroxyethyl starch solutions on the microcirculation of themesentery is not different from crystalloids in colon ascendens stent peritonitis-induced experimentalsepsis in rats.

34

© 2014 Published by Elsevier Inc. E

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

UNCO

RRIntroduction

Fluid resuscitation is essential for the management of critically illpatients with sepsis. The choice of fluids has been widely debatedregarding the use of crystalloids versus colloids. The selection of anappropriate fluid to manage fluid resuscitation is influenced by sev-eral factors such as the extent of capillary leakage and adverse sideeffects of specific fluids, e.g. impact on kidney function (Myburghand Mythen, 2013; Vercueil et al., 2005). In sepsis and septic shockthe release of inflammatory mediators causes vasodilation and anincrease of vascular permeability (Lee and Slutsky, 2010). As a result,relative hypovolemia develops and in turn increasesfluid requirements.Insufficientfluid resuscitation could lead to inadequate organ perfusion,whereas fluid overload may increase tissue edema in sepsis. Thus, theselection of the appropriate intravenous fluid to administer in sepsis

68

69

70

71

72

73

74

esia, Pain Management andWest Victoria, 1276 South Park

ort time impact of different2014), http://dx.doi.org/10.1

and septic shock is critical (Myburgh and Mythen, 2013; Vercueilet al., 2005).

The VISEP study was one of the first studies to investigate theuse of crystalloids versus colloids in septic patients (Brunkhorstet al., 2008). The study raised concerns about first generationhydroxyethyl starch (HES) solutions, since the use of HES solutionscompared to crystalloids was associated with increased acute renalfailure. The development of novel starch solutions prompted newstudies to test the usefulness of second and third generation starchsolutions. Investigations of HES use in the intensive care unit (ICU)determined that there was no difference in 90-day mortality be-tween patients resuscitated with 6% HES or saline (Myburgh et al.,2012). However, patients with severe sepsis resuscitated withHES were found to have an increased risk of death at day 90 andwere more likely to require renal-replacement therapy comparedto patients that received Ringer's acetate (Perner et al., 2011).

To date, all clinical trials investigating the use of colloids versuscrystalloids in sepsis have focused on later stages of sepsis. In thisstudy, we investigated the effects of crystalloids versus colloids onthe mesenteric microcirculation in early experimental sepsis usinga colon ascendens stent insertion for sepsis induction by peritonitis(CASP) model in rats.

hydroxyethyl starch solutions on the mesenteric microcirculation in016/j.mvr.2014.07.012

F

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

A

2 K. Wafa et al. / Microvascular Research xxx (2014) xxx–xxx

Materials and methods

Animals

Sixty-four male Lewis rats (body weight 280 ± 70 g) were pur-chased fromCharles River Laboratories (Sulzfeld, Germany) and housedin chip-bedded cages at the Animal Care Facility of Greifswald Universi-ty (Greifswald, Germany). Animals were kept on a 12 hour light/darkcycle with ad lib food (rat chow, Altromin, Lage, Germany) and water.Housing conditions were maintained at 22 °C with a 55–60% humidityenvironment. All animal procedures were performed according to theguidelines set by the German animal safety legislation and wereapproved by the institutional Animal Care Committee (Landesamt fürLandwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern (LALLF). Protocol number: LALLF M-V/TSD/7221.3-1.1-043/08). Upon completion of the experiments, rats were sacrificed byreceiving an overdose of pentobarbital by intravenous administration.

ED P

RO

O

Surgery

Rats underwent either sham surgery or CASP surgery as describedpreviously (Lustig et al., 2007). Briefly, surgery was performed underpentobarbital anesthesia (60 mg/kg body weight, i.p.; Sigma-AldrichChemie, Steinheim, Germany). Once the abdomen of animals wasshaved anddisinfected, a 2-cm-longmedian laparotomywasperformedabove the symphysis. The wall of the colon ascendens was perforatedusing an i.v. cannula (16G; Venflon, Ohmeda, Sweden) inserted 1.5 cmdistal to the ileocecal valve. The inner needle was removed and the can-nula was cut to form a stent. To ensure that the stent was positionedcorrectly the cecum was palpated until the stent was filled with feces.Sham control animals underwent the same surgery as CASP animalsbut the colon was not perforated or stented.

T

B

CO

RRECExperimental protocol

Fourteen hours (time= 0 min) after the surgery animals were ran-domly assigned to receive one of the following fluids intravenously:16 ml/kg Ringer's solution (Sham, n = 8), 16 ml/kg Ringer's solution(CASP16ring, n = 8), 64 ml/kg Ringer's solution (CASP 64ring, n = 8),16 ml/kg Volulyte® (6% HES 130/0.4 in an isotonic electrolyte solution)(V-CASP16, n=10), 16 ml/kg Tetraspan® (6% HES 130/0.42 in Ringer'sacetate) (T-CASP16, n= 10), 16 ml/kg Hemohes® (6% HES 200/0.5 so-lution in NaCl 0.9%) (H-CASP16, n = 10), and 16 ml/kg HAES-Steril®(6% HES 200/0.5 solution in NaCl 0.9%) (HS-CASP16, n= 10). Intravitalmicroscopy (IVM) was performed for 14 h (IVM1, time = 0 min) and16 h (IVM2, time = 120 min) post CASP surgery. Animals receivedthe assigned fluid at 15 h (time for fluid administration = 60 min)post CASP surgery.

Fig. 1. Mean arterial pressure (MAP, A) and heart rate (HR, B) of rats which underwentsham surgery (control) and received 16 ml/kg Ringer's solution (Sham) or CASP surgeryand received one of the following solutions: 16 ml/kg Ringer's solution (CASP 16ring),64 ml/kg Ringer's solution (CASP 64ring), 16 ml/kg Volulyte® (V-CASP16), 16 ml/kgTetraspan® (T-CASP16), 16 ml/kg Hemohes® (H-CASP16), and 16 ml/kg HAES-Steril®(HS-CASP16). Values are expressed as mean ± SEM. *p b 0.05 compared to sham control.

UNPreparation for intravital microscopy

Anesthesia was induced by pentobarbital (60 mg/kg body weight,i.p.; Sigma-Aldrich Chemie, Steinheim, Germany) andmaintained by in-travenous pentobarbital injections (5 mg/kg) when required. The ani-mals were positioned in a supine position and the neck was shavedand disinfected. To secure the airways, rats received a tracheostomyand were allowed to breath room air. Polyethylene catheters (vein:inner diameter 0.28 mm, outer diameter 0.61mm; artery: inner diame-ter 0.58 mm, outer diameter 0.96 mm; Smith Medical, Kent, UK) wereinserted in the left external jugular vein and common carotid arteryfor the administration of fluids and fluorescence dyes. The rats wereplaced on a heating table (LHG hotplate HAS 01/AL, Harry GestigkeitGmbH, Düsseldorf, Germany) to maintain a body temperature of 37± 0.5 °C. Temperature was monitored by using a rectal thermometer.

Please cite this article as: Wafa, K., et al., Short time impact of differentexperimental sepsis in rats, Microvasc. Res. (2014), http://dx.doi.org/10.1

Intravital microscopy

IVM was performed using an epifluorescent microscope (AxiotechVario, Carl Zeiss, Jena, Germany) with a light source (HBO 50, CarlZeiss, Jena, Germany), ocular (10×) and lens (20×/0.5 Achroplan, CarlZeiss, Jena, Germany). The microscope contained type #20 and #10filter sets (Carl Zeiss, Jena, Germany). Fluorescence dyes were usedto visualize leukocytes (Rhodamine 6G, 200 μl, 0.05%, Sigma) and

hydroxyethyl starch solutions on the mesenteric microcirculation in016/j.mvr.2014.07.012

UNCO

RRECTED P

RO

OF

138

139

140

141

142

143

144

145

146

147

148

149

A

B

C

Fig. 2. Plasma extravasation ratio (ratio of fluorescence intensity inside (Iv) related to outside (Ip) the vessel, A), number of temporary adherent (rolling) leukocytes (B) and number ofadhering leukocytes (C) in the mesenteric venules of rats which underwent sham surgery (control) and received 16 ml/kg Ringer's solution (Sham) or CASP surgery and received one ofthe following solutions: 16 ml/kg Ringer's solution (CASP 16 ring), 64 ml/kg Ringer's solution (CASP 64 ring), 16 ml/kg Volulyte® (V-CASP16), 16 ml/kg Tetraspan® (T-CASP16), 16 ml/kgHemohes® (H-CASP16), and 16 ml/kg HAES-Steril® (HS-CASP16). Parameters were analyzed at 14 h post CASP surgery (IVM1; A, B, C) and at 16 h post CASP surgery (IVM2; A, B, C).Fluidswere administered at 15 h post CASP surgery. Values are expressed as mean± SEM. *p b 0.05 compared to respective sham control. #p b 0.05 compared towithin group IVM1 con-trol. $ p b 0.05 compared to respective CASP 16 ring group (IVM1 or IVM2).

3K. Wafa et al. / Microvascular Research xxx (2014) xxx–xxx

microvessels (FITC, 200 μl, 5%, Sigma), on a segment of the mesen-tery of the terminal ileum 1 cm proximal from the ileocecal valve.Video images were captured by a black-and-white CCD video camera(BC-12, AVT-Horn, Aalen, Germany) connected to a black-and-whitemonitor (PM-159, Ikegami Electronics, Neuss, Germany) and record-ed by a Panasonic NV-SV120EG-S recorder (Matsushita Audio Video,

Please cite this article as: Wafa, K., et al., Short time impact of differentexperimental sepsis in rats, Microvasc. Res. (2014), http://dx.doi.org/10.1

Tokyo, Japan). Videos were reviewed at a later date for data analysis.All video analysis was blinded and took place offline on a videomonitor.

We analyzed the number of adherent leukocytes (number of leuko-cytes during an observation period which remained firmly attached tothe intestinal endothelial for at least 30 s), number of rolling leukocytes

hydroxyethyl starch solutions on the mesenteric microcirculation in016/j.mvr.2014.07.012

T

F

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

t1:1 Table 1t1:2 Laboratory parameters for rats which underwent sham surgery (control) and received 16 ml/kg Ringer's solution (Sham) or CASP surgery and received one of the following solutions:t1:3 16 ml/kg Ringer´s solution (CASP 16 ring), 64 ml/kg Ringer's solution (CASP 64 ring), 16 ml/kg Volulyte® (V-CASP16), 16 ml/kg Tetraspan® (T-CASP16), 16 ml/kg Hemohes® (H-t1:4 CASP16), and 16 ml/kg HAES-Steril® (HS-CASP16). Blood samples were collected prior to IVM1 (Pre) or post IVM2 (post).

t1:5 Sham CASP 16 ring CASP 64 ring

Pre Post Pre Post Pre Post

t1:6 pH 7.52 ± 0.06 7.48 ± 0.03 7.45 ± 0.04* 7.46 ± 0.02 7.45 ± 0.03* 7.49 ± 0.06t1:7 pO2 100.25 ± 20.64 113.07 ± 17.10 77.40 ± 10.92 94.87 ± 15.58 74.18 ± 11.40 96.08 ± 30.07t1:8 pCO2 30.56 ± 4.68 29.27 ± 2.25 35.58 ± 4.33 30.41 ± 3.09 35.82 ± 4.58 27.2 ± 6.77#

t1:9 HCO3 26.72 ± 1.34 23.82 ± 1.33# 25.11 ± 1.56 23.23 ± 0.89 24.42 ± 0.82 22.78 ± 1.32t1:10 SBE 2.36 ± 1.59 −1.07 ± 1.47# 0.857 ± 1.71 −1.66 ± 1.31 0.160 ± 0.66 −2.00 ± 1.34t1:11 sO2 93.52 ± 2.44 95.25 ± 1.42 86.42 ± 6.66* 92.77 ± 2.96# 85.74 ± 6.05* 92.68 ± 4.57#

t1:12 Lactate 2.35 ± 0.57 2.72 ± 0.68 2.08 ± 0.51 2.58 ± 0.54 3.26 ± 1.02 2.725 ± 0.47t1:13 Glucose 6.10 ± 1.35 5.59 ± 0.57 6.37 ± 0.47 5.59 ± 0.27 6.10 ± 0.62 5.58 ± 0.42t1:14 Hct 46.06 ± 1.89 43.91 ± 2.97 44.57 ± 3.83 43.23 ± 3.25 45.16 ± 1.51 43.08 ± 2.91

*p b 0.05 compared to control shamevalue; #p b 0.05 compared to time zero valuewithin each group, $p b 0.05 compared to respective CASP 16ring, %p b 0.05 compared to respective CASP64ring, n = 5–11 per group. Blood pH (pH); oxygen partial pressure (pO2); carbon dioxide partial pressure (pCO2); plasma bicarbonate (cHCO3); base excess (SBE); oxygen saturation(sO2); and hematocrit (Hct).

4 K. Wafa et al. / Microvascular Research xxx (2014) xxx–xxx

UNCO

RREC

(the number of leukocytes which during an observation period of 30 spass in a rolling motion through a vessel diameter) and plasma extrav-asation (ratio of fluorescence intensity inside related to outside thevessel).

Laboratory analysis

Prior to IVM1 (Pre) and post IVM2 (Post) 0.55 ml of arterial bloodwas collected for blood gas and hematocrit analysis (ABL 330, Radiom-eter, Hamburg, Germany). Values were obtained for blood pH, oxygenpartial pressure (pO2), carbon dioxide partial pressure (pCO2), standardbicarbonate (cHCO3

−), base excess (SBE), oxygen saturation (SO2),lactate concentration (cLactate), glucose concentration (cGlucose),and hematocrit (Hct).

Macrocirculation

Blood pressure and heart rate were continuously monitored (vitalmonitor: Hewlett Packard, Saronno, Italy).

Statistical analysis

Unless otherwise stated, all data comparisonswere completed usinga one-way or a two-way analysis of variance (ANOVA). Significance ob-tained by one-way was further subjected to a Newman–Keuls test forpost-hoc analysis. Significance obtained by two-way ANOVA was fur-ther subjected to a Bonferroni test for post-hoc analysis. Data is shownas mean ± SEM and was considered statistically significant when thedifference in mean values between groups had a P value less than 0.05.

Results

Mean arterial pressure and heart rate

In control sham animals, mean arterial pressure (MAP) and heartrate (HR) were maintained at a constant level ranging from 120 to140 mm Hg and 410 to 440 bpm for MAP and HR, respectively (Fig.1A, B). MAP and HR values for CASP animals receiving crystalloids(Ringer's lactate 16 ml/kg and 64 ml/kg) and different HES solutions(16 ml/kg 130/0.4 and 200/0.5 HES by different providers) were com-parable to those of sham animals.

Plasma extravasation

There was a significant increase in plasma extravasation in all CASPanimals compared to sham animals prior to receiving fluids (IVM1,Fig. 2A). All CASP groups maintained a significantly increased levelof plasma extravasation compared to sham animals post fluid

Please cite this article as: Wafa, K., et al., Short time impact of differentexperimental sepsis in rats, Microvasc. Res. (2014), http://dx.doi.org/10.1

ED P

RO

Oadministration (IVM2, Fig. 2A). Additionally, all groups demonstrat-ed an increasing trend in plasma extravasation post fluid administra-tion (IVM2) when compared to respective IVM1 control readings.However, the CASP 64 ring group demonstrated a significant in-crease in plasma extravasation post fluid administration (IVM2)compared to the CASP 64 ring IVM1 control reading.

Leukocyte–endothelial interactions

Administration of crystalloids or colloids in CASP animals did not sig-nificantly alter the number of temporary adherent leukocytes in themesenteric venules compared to sham animals (Fig. 2B). However, thenumber of temporary adherent leukocytes did significantly increase inthe sham animals post fluid administration (IVM2) compared to IVM1baseline control.

The number of firmly adherent leukocyteswas significantly elevatedin all CASP groups receiving fluids compared to sham animals (Fig. 2C).Firmly adherent leukocytes remained elevated after fluid administra-tion in all CASP groups compared to sham animals (IVM2, Fig. 2C).

Laboratory parameters

There were no significant differences in blood gas and hematocritanalyses for animals which underwent sham surgery and received16 ml/kg Ringer's solution (Sham) when compared with CASP animalswhich received either crystalloids or colloids (Table 1).

Discussion

In this study,we report a significant increase in plasmaextravasationdue to sepsis; however, there were no significant differences in plasmaextravasation related to the use of crystalloids versus hydroxyethylstarch colloids. Additionally, neither the use of crystalloids nor colloidshad an effect on leukocyte–endothelial interactions.

Crystalloids are composed of ions, which can easily pass through theendothelial wall and therefore have a short intravascular presence.Colloids are composed of larger molecular weight molecules andtherefore do not cross the endothelial barrier under normal condi-tions, allowing for a long intravascular persistence. Due to the com-position of crystalloids, the administration of a crystalloid solutionwill theoretically expand the plasma volume by 200 ml per liter in-fused, whereas the administration of a colloid solution provides aplasma volume expansion equal to or more than the volume infused(Vercueil et al., 2005; Vincent and Gottin, 2011). However duringsepsis, systemic inflammatory response causes an increase in vascu-lar permeability, which can lead to colloid extravasation into theinterstitial space (Lee and Slutsky, 2010).

hydroxyethyl starch solutions on the mesenteric microcirculation in016/j.mvr.2014.07.012

T

F

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

V-CASP16 T-CASP16 H-CASP16 HS-CASP16

Pre Post Pre Post Pre Post Pre Post

7.50 ± 0.04 7.50 ± 0.04 7.46 ± 0.04* 7.49 ± 0.05 7.49 ± 0.08 7.50 ± 0.12 7.46 ± 0.04* 7.48 ± 0.0393.03 ± 14.18 123.25 ± 22.44$# 83.45 ± 20.79 120.30 ± 40.59$# 90.69 ± 24.40 126.21 ± 28.46$%# 78.69 ± 14.89 104.03 ± 9.0231.42 ± 3.19 28.65 ± 3.00 33.72 ± 2.74 27.40 ± 7.31# 30.22 ± 6.32$ 26.53 ± 7.30 33.96 ± 5.11 28.41 ± 2.1026.23 ± 1.21 24.68 ± 1.04 24.76 ± 1.90 23.52 ± 5.31 25.20 ± 2.06 23.88 ± 1.97 24.61 ± 1.24* 23.64 ± 1.351.98 ± 1.46 −0.137 ± 1.71 0.590 ± 2.15 −1.57 ± 6.74 0.873 ± 2.19 −1.21 ± 1.98 0.510 ± 1.53 −0.856 ± 1.59

93.76 ± 2.60$% 96.41 ± 1.73 88.13 ± 6.57* 93.63 ± 3.65# 91.23 ± 4.65$% 95.3 ± 1.41 86.19 ± 6.07* 94.64 ± 0.81#

2.26 ± 0.80 3.20 ± 0.45 2.81 ± 0.91 4.79 ± 5.37*$ 2.57 ± 0.91 3.26 ± 2.35 2.63 ± 0.86 3.14 ± 0.766.70 ± 0.56 6.33 ± 0.45 6.19 ± 0.68 5.43 ± 1.57 6.12 ± 0.74 5.58 ± 0.95 6.19 ± 0.67 5.71 ± 0.60

47.23 ± 3.54 41.78 ± 4.84# 47.46 ± 3.16 42.00 ± 10.10# 47.40 ± 2.95 41.71 ± 2.85# 47.09 ± 2.26 42.29 ± 2.80

5K. Wafa et al. / Microvascular Research xxx (2014) xxx–xxx

UNCO

RREC

Our results indicate that while there is a generalized increase in fluidextravasation in early sepsis as a result of inflammation, the use of thetested colloids and crystalloids did not result in increased fluidextravasation into the interstitium. However, administration of ahigher volume of crystalloid (64 ml/kg), a ratio of 1:4 between col-loid and crystalloid, to account for the higher acute volume effectresulted in a significant increase in plasma extravasation.

CASP-induced sepsis is a model of postoperative abdominal sep-sis and as such resembles human conditions more closely thanendotoxemia models that are often used in experimental sepsis re-search. Endotoxemia models do not take into account the invasionand replication of live bacteria. In our study, the selection of a 16Gstent was based on previously completed work to generate a suble-thal model with stable macrocirculation (Lustig et al., 2007; Zantl et al.,1998). Animals were hemodynamically stable with no macrocirculatorychanges up until sacrifice after CASP surgery (Lustig et al., 2007). Addi-tionally, there were no changes in functional capillary density reportedup to 18 h after surgery (Lustig et al., 2007). This provided us with anidealmodel to study the effects of crystalloid versus colloid fluid replace-ment in early sepsis.

To our knowledge, there are no studies, which investigate plasmaextravasation following fluid resuscitation in mesenteric postcapillaryvenules during sepsis; however, there are various studies that investi-gated the use of colloids on pulmonary leakage and albumin escaperate in sepsis. Results reported by two different groups demonstratedsome differences as to the role of colloids on fluid extravasation andcapillary leakage. Feng et al. report a dose dependent attenuation ofthe increase in pulmonary capillary leakwith the use of HES for fluid re-suscitation in a cecal ligation model (CLP) of sepsis in rat (Feng et al.,2007).Marx et al. in a porcinemodel of septic shock induced by abdom-inal feces introduction, report an increased but equal albumin escaperate with both the use of HES and Ringer's solution (Marx et al., 2002).Interestingly, despite an increased albumin escape rate the use of HESmaintained plasma volume and colloid osmotic pressure leading theauthors of the study to conclude that HES demonstrated intravascularpersistency even in the presence of albumin leakage.

While our plasma extravasation data conflicts with data reported byFeng et al. they are consistent with results obtained byMarx et al. (Fenget al., 2007; Marx et al., 2002). Variation in fluid extravasation could beas a result of different experimental models, CLP versus CASP, pul-monary venules versus mesenteric venules or due to the differentconcentration of HES used. Furthermore, Feng et al. tested increasingconcentrations of HES where the most pronounced effect wasachieved at 30 ml/kg, as compared to the 16 ml/kg concentrationof HES (Feng et al., 2007) that we tested.

In a hamster endotoxemia model the use of HES compared toisotonic saline improved functional capillary density, decreased li-popolysaccharide (LPS) induced venular leakage and decreased

Please cite this article as: Wafa, K., et al., Short time impact of differentexperimental sepsis in rats, Microvasc. Res. (2014), http://dx.doi.org/10.1

ED P

RO

Oleukocyte adhesion (Hoffmann et al., 2002). Hoffmann et al. attributedthese protective effects of HES on endotoxin-induced microcirculatorydefects on anti-inflammatory effects of HES. Specifically the reductionof granulocyte chemotaxis associatedwith down regulation of adhesionmolecules and possibly the attenuation of inflammatory cytokines(Hoffmann et al., 2002). In our model we did not appreciate a HES me-diated modulation of leukocyte rolling or adhesion in mesenteric post-capillary venules.

Data reported by Hoffmann et al. provide promising evidence for thepositive effect of HES on attenuating endotoxin-mediated inflamma-tion. Interestingly, Hoffmann et al. discuss the down regulation of IL-6levels as one of the possiblemechanisms responsible for decreasing leu-kocyte activation, citing an earlier study (Schmand et al., 1995). In ourmodel we have previously demonstrated the early up regulation andpersistence of IL-6. However, we did not observe any attenuation to el-evated leukocyte adherence post CASP surgery in the presence of HEScolloids of varying ratios (130/0.4 and 200/0.5).Whilewe did not inves-tigate the effects of HES volume replacement on IL-6 levels, the discrep-ancy in the reported leukocyte adhesion data could perhaps be due tomodel difference. As the CASP model presents a continuous source ofbacteria which are able to replicate and invade tissues, it more closelyresembles clinical sepsis. Live bacteria, bacterial toxins and/or virulencefactors trigger a different immunological responsewhen comparedwitha single endotoxin administration (Zantl et al., 1998). Furthermore,while the modulation of IL-6 may prevent leukocyte adhesion in abolus sepsis model, it may not be enough to have the same effect in aCASP model of sepsis.

Conclusion

In our current study, while we observed an increase in plasma ex-travasation in mesenteric postcapillary venules due to sepsis we didnot observe any differences in plasma extravasation based on the fluidadministered. Current recommendations by the Surviving Sepsis Cam-paign strongly recommends against the use of HES solutions in sepsisor in critically ill patients due to increased risk of kidney failure andmortality (Dellinger et al., 2013). In a recent Cochrane systematic re-view, authors concluded that there was no evidence based on random-ized controlled trials that resuscitation with colloids reduces the risk ofdeath in patients with trauma, burns or following surgery, compared toresuscitation with crystalloids (Perel et al., 2013). While our data sup-ports the notion that theuse of colloids does not provide any advantageswhen compared to the use of crystalloids in early sepsis, no harm wasattributed to the use of colloids within the tested parameters.

Competing interests

None.

hydroxyethyl starch solutions on the mesenteric microcirculation in016/j.mvr.2014.07.012

320

321

322

323

324

325326327328329330331332333334335336337338

339340341342343344345346347348349350351352353354355356357358359360

6 K. Wafa et al. / Microvascular Research xxx (2014) xxx–xxx

Acknowledgments

This studywas supported by funding provided by the Department ofAnesthesiology and Intensive CareMedicine, ErnstMoritz Arndt Univer-sity Greifswald, Germany.

References

Brunkhorst, F.M., et al., 2008. Intensive insulin therapy and pentastarch resuscitation insevere sepsis. N. Engl. J. Med. 358, 125–139.

Dellinger, R.P., et al., 2013. Surviving Sepsis Campaign: international guidelines for man-agement of severe sepsis and septic shock, 2012. Intensive Care Med. 39, 165–228.

Feng, X., et al., 2007. Hydroxyethyl starch, but not modified fluid gelatin, affects inflam-matory response in a rat model of polymicrobial sepsis with capillary leakage. Anesth.Analg. 104, 624–630.

Hoffmann, J.N., et al., 2002. Hydroxyethyl starch (130 kD), but not crystalloid volume sup-port, improves microcirculation during normotensive endotoxemia. Anesthesiology97, 460–470.

Lee, W.L., Slutsky, A.S., 2010. Sepsis and endothelial permeability. N. Engl. J. Med. 363,689–691.

Lustig, M.K., et al., 2007. Colon ascendens stent peritonitis—a model of sepsis adopted tothe rat: physiological, microcirculatory and laboratory changes. Shock 28, 59–64.

UNCO

RRECT

Please cite this article as: Wafa, K., et al., Short time impact of differentexperimental sepsis in rats, Microvasc. Res. (2014), http://dx.doi.org/10.1

OF

Marx, G., et al., 2002. Hydroxyethyl starch and modified fluid gelatin maintain plasmavolume in a porcine model of septic shock with capillary leakage. Intensive CareMed. 28, 629–635.

Myburgh, J.A.,Mythen, M.G., 2013. Resuscitation fluids. N. Engl. J. Med. 369, 1243–1251.Myburgh, J.A., et al., 2012. Hydroxyethyl starch or saline for fluid resuscitation in intensive

care. N. Engl. J. Med. 367, 1901–1911.Perel, P., et al., 2013. Colloids versus crystalloids for fluid resuscitation in critically ill

patients. Cochrane Database Syst. Rev. 2, CD000567.Perner, A., et al., 2011. Comparing the effect of hydroxyethyl starch 130/0.4 with balanced

crystalloid solution on mortality and kidney failure in patients with severe sepsis (6S—Scandinavian Starch for Severe Sepsis/Septic Shock trial): study protocol, designand rationale for a double-blinded, randomised clinical trial. Trials 12, 24.

Schmand, J.F., et al., 1995. Effects of hydroxyethyl starch after trauma-hemorrhagic shock:restoration of macrophage integrity and prevention of increased circulatinginterleukin-6 levels. Crit. Care Med. 23, 806–814.

Vercueil, A., et al., 2005. Physiology, pharmacology, and rationale for colloid administra-tion for the maintenance of effective hemodynamic stability in critically ill patients.Transfus. Med. Rev. 19, 93–109.

Vincent, J.L., Gottin, L., 2011. Type of fluid in severe sepsis and septic shock. MinervaAnestesiol. 77, 1190–1196.

Zantl, N., et al., 1998. Essential role of gamma interferon in survival of colon ascendensstent peritonitis, a novel murine model of abdominal sepsis. Infect. Immun. 66,2300–2309.

ED P

RO

hydroxyethyl starch solutions on the mesenteric microcirculation in016/j.mvr.2014.07.012