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10 g% DCLHb. The control group received shed blood diluted with Ringers to ahemoglobin content of 10 g%. Intravital microscopy was used for investigation of

the microcirculatory parameters and a multiwire platinum surface electrode formeasurement of local tissue pO2 in striated skin muscle in the dorsal skinfoldchamber of Syrian golden hamsters.

Resuscitation from hemorrhagic shock with 10 g% AUB revealed significantincrease of leukocytes rolling in postcapillary venules at 30 to 120 minutes afterresuscitation compared to baseline values. DCLHb turned out to reduce the num-ber of firmly adherent leukocytes after resuscitation compared to 10 g% AUB.Microvascular permeability as an indicator for functional endothelial integrityrevealed no significant differences between the groups. DCLHb and 10 g%AUB led to a significant increase in local tissue oxygenation after resuscitationfrom hemorrhagic shock. However, 10 g% AUB turned out to be most effectiveto restore the local tissue pO2compared to Dx-60.

Our findings indicate that DCLHb restores microvascular perfusion aftercritical hemorrhagic shock as efficient as Dx-60 and 10 g% AUB. The absenceof enhanced leukocyte-endothelium interaction after resuscitation with DCLHbimplies that this HBOC does not exacerbate formation of oxygen free radicalsduring reperfusion. DCLHb effectively increases local tissue pO2 after resusci-tation from hemorrhagic shock; however, not as effectively as 10 g% AUB.

Keywords: Blood substitutes; Hemoglobin based oxygen carriers; Leukocyte=endothelial cell interactions; Local tissue pO2; Microcirculation; Multiwiresurface electrode

INTRODUCTION

Hemoglobin based oxygen carriers (HBOCs [1]) are red blood cell (RBC)substitutes, which demonstrate therapeutic efficiency when used for

resuscitation from hemorrhagic shock [2,3]. However, to date, large scaleclinical application has been impeded due to unwanted side effects ofthese solutions consisting of nephrotoxicity [4], complement activation[5] and risk of transmission of infectious diseases, e.g. hepatitis, HIV orprion induced diseases [6]. Next to the refinement of the purification pro-cedure and the technical production process of these solutions [7], thechemical modification of the hemoglobin molecule was crucial to abolishthese adverse effects.

Diaspirin Crosslinked Hemoglobin (DCLHb, HemAssistTM) i s a

stromafree hemoglobin solution. The hemoglobin molecule is covalentlycrosslinked between the 99lysin of the aa-chains with a fumaryl bridge [8].

456 S. Hungerer et al.

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shock, which restores macrohemodynamic parameters such as the meanarterial pressure [10] and reverses a potential base deficit [11]. However,very limited data are available on the effects of DCLHb on microvascularparameters following resuscitation from hemorrhagic shock [12,13].

Preclinical investigations as well as clinical trials using DCLHb forresuscitation in hemorrhagic shock demonstrated promising results[2,14]. However, the recently published mortality analysis of the efficacytrial of DCLHb raises concerns about safety of these solutions [15].HBOCs are thought to enhance formation of free oxygen radicals,especially during reperfusion of postischemic tissues [16], e.g. afterresuscitation from hemorrhagic shock. One possible mechanism may bethe release of free iron ions from the prosthetic heme group, thus promot-ing the formation of hydroxyl radicals via the Haber-Weiss-reaction byacting as a Fenton reagent [17]. This mechanism might lead to anenhancement of lipid peroxidation and leukocyte=endothelial cell interac-tions with activation of the inflammation cascade, release of cytosolicenzymes and, ultimately destruction of postischemic tissues [18].

DCLHb has proven its efficacy in restoring microvascular para-meters in the microcirculation after ischemia and reperfusion [19] andhemorrhagic shock [20]. Aim of the current study was the investigationof the effects of 10g% DCLHb on the microcirculation, leukocyte-endothelial cell=cell interactions and local tissue oxygenation afterresuscitation from severe hemorrhagic shock. Special focus addressedresuscitation efficiency by comparing DCLHb (hemoglobin content10 g=dl) with autologous blood of identical hemoglobin concentrationie equal oxygen carrying capacity.

MATERIALS AND METHODS

Hemoglobin Solution

Diaspirin Crosslinked Hemoglobin [DCLHb, 99aa-3,5-bis(dibromosalicyl)fumararte hemoglobin, lot-no. HBXR-92-268-42793] was provided byBaxter, Healthcare Corp. (Deerfield, Illinois, USA). The characteristicsof the hemoglobin solution have been described previously [21]. Purifi-cation procedures include virus inactivation by heat pasteurization [22].

Animal Model

Microcirculation and Local Tissue pO2 Upon DCLHb Administration 457

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the Care and Use of Laboratory Animals (NIH Publication No. 80-23,revised 1985) and with the permission of the local ethic committee.The dorsal skin fold chamber in awake Syrian golden hamsters [23]was used for investigation of microcirculatory parameters and localtissue oxygen tension. The chamber was implanted 2448 h prior to theexperiment under ketamine=xylazine anesthesia (130=20mg=kg BWi.p.). Polyethylene catheters (Portex, Hythe, UK) were implanted in the

jugular vein for infusion of resuscitation fluids and carotid artery formonitoring of mean arterial pressure, heart rate, arterial blood gasesand hematocrit.

Experimental Protocol

Animals were randomly assigned to three different treatment groupsas described later. After implantation of the dorsal skin fold chambera recovery period of at least 48 hrs was followed before assessment ofbaseline values of microcirculatory parameters and tpO2. Shock wasinduced by bleeding the animals at a rate of 33 ml=kg BW maintaininga mean arterial pressure of 30 2 mmHg over a period of 45 minutes

by bleeding or reperfusion of the shed blood. Intravital microscopyand tissue oxygen measurements were performed at 30 min after onsetof hemorrhagic shock and at 10, 30, 60 and 120 minutes followingresuscitation.

Animals were resuscitated with either 33 ml=kg BW 6% Dextran60.000 (Dx-60; Mr 60.000; Schiwa, Germany) as an isooncotic controlsolution, 10 g% DCLHb or with shed blood diluted to a hemoglobincontent of 10.0 0.5g% from 14.2 0.4g% with lactated ringer.

Intravital Microscopy

The microcirculation of the striated skin muscle was assessed using acomputer controlled microscope (Zeiss, Jena, Germany), connected toa stepping motor [20] which allowed for exact repeated measurementsof the same vessel segments. In vivo staining of leukocytes was accom-plished by intravenous injection of 0.05% rhodamine 6G (0.15 mg=kgBW; Sigma Chemicals, Deisenhofen, Germany). This method enablesthe quantitative assessment of leukocyte=endothelial interactions [24].

Macromolecular leakage was assessed by calculation of the ratioextravascular versus intravascular leakage of the fluorescence marker


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computer assisted microcirculation analysis system [25] (CapImage1,Zeintl, Heidelberg, Germany). The following parameters were assessed:

1. Functional capillary density (FCD) defined as red cell perfused capil-laries of the striated skin muscle per area (cm=cm2) [26].

2. Leukocyte=endothelial cell interaction, determined as the number ofnon-adherent leukocytes (NAL=min), number of rolling leukocytes(cells=min) and firmly sticking leukocytes (cells=mm2). Rolling leuko-cytes are defined as slowly passing leukocytes along the endotheliallining, whereas sticking leukocytes did not detach from the endotheliallining during 30 seconds.

3. RBC velocity in capillaries and postcapillary venules (mm=s) andvenular shear rate using the following formula:

shear rate ~vvmean

vessel diameter 8:

Tissue Oxygen Measurement

A platinum multi-wire surface electrode (Clark-type MDO-electrode,

Eschweiler, Kiel, Germany) connected to a computer assisted amplifier(MID, Steindorf, Germany) was used for measurement of local tissue oxy-gen tension in the striated skin muscle [27,28]. At the preset time points ofinvestigation, the cover slips of the chamber window was gently removedand the tissue superfused with isotonic saline solution (B. Braun,Melsungen, Germany) at room temperature. The MDO-electrode, con-taining 8 platinum electrodes with each channel measuring the tpO2 ofapproximately a spherical area of 25mm2, was placed on the exposed tissue.An integrated temperature probe allowed for continuous measurement of

the local tissue temperature for online correction of the tpO2-values. Forthe collection of 100150 individual measurements the electrode wasmoved via a step-motor every 10 sec as described earlier [29].

Statistics

Because of the limited number of animals per treatment group non-parametric tests were used. For analysis between the groups, data weretested using the Kruskal-Wallis-test followed by the Mann Whitney U-

Test for analysis between groups or the Friedmann- and Wilcoxon-Testfor comparison within the groups. All significant values were corrected


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error of mean values for more rapid interpretation and comparabilityof the data with data from other studies.

RESULTS

Systemic Hemodynamic Parameters

Prior to induction of hemorrhagic shock, mean arterial pressure andheart rate were in the range of physiological values of hamsters [30].The mean arterial pressure during the shock period of 45 minutes was

found to not be significantly different between the experimental groups.The MAP remained between 30 6 and 32 4 mmHg (Table 1). A pre-vious study from our laboratory [20] had shown that, without resusci-tation, animals did not survive longer than 60 minutes when the MAPremained at 30 mmHg. In the present study, resuscitation with Dx-60failed to restore the MAP to pre-shock values, whereas DCLHb and10 g% AUB administration resulted in MAP comparable to thoseobserved prior to hemorrhagic shock.

The heart rate was monitored using arterial pressure curve and

revealed no significant differences between the groups. In animalsresuscitated with DCLHb the heart rate increased at 10 minutes afterresuscitation to 412 48 beats per minute compared to baseline valuesof 374 18 min1, whereas animals resuscitated with Dx-60 or 10 g%AUB did not experience heart rates above the pre-shock values (Table 1).

During shock period in almost 50% of the animals it was not poss-ible to draw blood from the intraarterial catheter for arterial blood gasand hematocrit analysis. Therefore these data were not considered forstatistical analysis. The arterial blood gas analysis showed a decrease of

Table 1. Heart rate (HR) and mean arterial pressure (MAP) before, shock,during shock and after resuscitation (mean SD, n 68 animals per treatmentgroup; p< 0.05 vs. NaCl 0.9% and Dx-60, Mann-Whitney U-Test)

Time after resuscitation

Time points Baseline Shock 10 min 30 min 60 min 120 min

Dx-60 HR 364 22 240 52 342 55 382 41 368 35 310 56

MAP 98 5 30 6 58 11 67 12 65 10 64 1410 g% AUB HR 370 28 266 48 327 60 380 57 397 56 420 30

MAP 106 4 31 5 89 16 96 9 90 11 101 7


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systemic oxygen tension during shock period in the animals. 120 minutesafter resuscitation oxygen tension reached baseline values in animalsresuscitated with DCLHb and AUB 10%.

Hematocrit was decreased after resuscitation with Dx-60 andDCLHb whereas 10% AUB caused no changes of systemic hematocritfrom baseline values of 39.0 1.4 to 38.2 2.4% after 120 min afterresuscitation.

Microcirculatory Parameters

The diameters of arterioles or venules in the striated skin muscle of thedorsal skin fold chamber did not differ significantly between the threegroups. A vasoconstriction of the arteriolar vessels, as described in theliterature [31], was not found in animals resuscitated with DCLHb(Fig. 1).

However, the leukocyte=endothelial cell interactions represented bythe number of rolling and sticking leukocytes revealed significant changes.In animals resuscitated with 10 g%AUB, a significant increase in the num-ber of rolling leukocytes was detected at 30, 60 and 120 minutes after

Figure 1 Diameter of arterioles (A) and postcapillary venules (B) in striated skin


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resuscitation (Fig. 2A). The baseline number of rolling leukocytes prior toinduction of shock was 16.2 2.1 cells=minutes. At 30 minutes a value of33.4 5.0 was observed. The maximum was reached at 120 minutes afterresuscitation with 37.6 6.2 cells=minutes (p< 0.05 Wilcoxon Test). Nosignificant differences were found for the number of rolling leukocytes inanimals treated either with Dx-60 or DCLHb.

The number of leukocytes sticking to the endothelium of postcapil-

lary venules turned out to be increased in animals receiving Dx-60 andautologous blood, respectively, when compared to the group treated withDCLHb. In the early time course after reperfusion (10 and 60 minutes),the number of sticking leukocytes was significantly reduced in animalsresuscitated with DCLHb compared to 10 g% AUB (Fig. 2B). No signifi-cant changes were noted in animals receiving Dx-60.

The macromolecular leakage of FITC-Dextran (MW 150.000Dalton), as an indicator for functional endothelial integrity, revealedno significant changes. In animals treated with Dx-60 and DCLHb, an

increase of macromolecular leakage during the shock period was notable,although these changes were not seen in the group resuscitated with auto-

Figure 2. Rolling leukocytes (A) and sticking leukocytes (B) in postcapillaryvenules after resuscitation from hemorrhagic shock (mean SEM, n 68 ani-mals per exp. group; x p< 0.05 vs. Baseline, Wilcoxon test; # p< 0.05 10g%

AUB vs. 10 g% DCLHb, Mann Whitney U-Test).


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baseline values of 148 33.1 cm=cm2 to 41.1 43.4 cm=cm2 duringthe shock period. Resuscitation with 10 g% AUB almost immediatelyled to an increase of FCD and almost restored FCD after 120 minutes.Less prominent was this effect after resuscitation with Dx-60, althoughno significant differences were measurable after 30 minutes. DCLHbwas less effective in restoring the FCD. Baseline levels in this group were

130.6 27.0 cm=cm2, 38.8 27.0 during shock, 62.9 39.7 at 10 minutesand 66.6 39.5 cm=cm2 at 30 min after resuscitation. These changeswere significantly different when compared to respective data in theDCLHb and 10 g% AUB group during the time course of resuscitationat 30 minutes after resuscitation (Fig. 3B).

A significant reduction of velocity of red blood cells was detectable inall experimental groups during the shock period with levels of reduced tono flow and pendular flow in postcapillary venules, respectively (Fig. 4A).Resuscitation restored the RBCV in all experimental groups, although

Dx-60 seemed to be less effective as compared to DCLHb or AUB.The same trend could be observed for shear rates in postcapillary venules

Figure 3. Macromolecular leakage (A) and functional capillary density (B) in thestriated skin muscle following hemorrhagic shock and resuscitation. Animalswere treated either with Dx-60, 10 g% AUB or 10g% DCLHb (mean SEM,

n 68 per exp. group;

p< 0.05 vs. Dx-60 Mann Whitney U-test; x p< 0.05vs. baseline Wilcoxon test).


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baseline levels in animals treated with 10% DCLHb or 10% AUB at 60minutes after resuscitation, but not with Dx-60. At 60 and 120 minutesafter resuscitation this rebound was still significantly greater in animalstreated with 10% AUB when compared to treatment with Dx-60.

Local Tissue pO2

The local tissue pO2 ranged from 20.3 0.3 to 21.0 0.9 mmHg priorto shock in investigated treatment groups. During the shock period asignificant decrease of the local tissue oxygenation was measured in allgroups ranging from 3.3 0.8 to 4.9 0.8 mmHg. In animals treatedwith 10g% AUB tissue pO2 recovered more rapidly after resuscitationthan in animals treated with Dx-60 or DCLHb and reached values of16.4 1.5 mmHg and was found in normal range 60 minutes afterresuscitation. The groups treated with Dx-60 demonstrated a prolongedreduction of local tissue pO2 at 120 minutes after resuscitation with a

mean of 8.2 1.3 mmHg (Fig. 5A). The distribution of local pO2 valuesindicated a marked shift to hypoxic tissue values. This shift is represented

Figure 4. Red blood cell velocity in percentage of baseline values (A) andshear rates (B) in postcapillary venules of animals resuscitated either with Dx-60,10 g% AUB or 10 g% DCLHb (mean SEM, n 68 per exp. group; p< 0.05vs. Dx-60 Mann Whitney U-test; x p< 0.05 vs. baseline Wilcoxon test).


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resuscitation the number of hypoxic tissue values initially increased inanimals treated with 10 g% DCLHb and Dx-60. In contrast, 60 minutesafter resuscitation no significant hypoxic tissue values were observed inanimals treated with DCLHb and only 17% of tissue pO2 levels had tobe classified as hypoxic 60 minutes after resuscitation. In Dx-60 treatedanimals tissue hypoxia persisted during the complete time course after

resuscitation (Fig. 5B).

DISCUSSION

The current study focused on the effects of DCLHb on the microcircula-tion and local tissue oxygenation in striated skin muscle after resuscitationfrom severe hemorrhagic shock. Special focus addressed resuscitationefficiency by comparing DCLHb (hemoglobin content 10 g=dl) with auto-logous blood of identical hemoglobin concentration, i.e., equal oxygen

carrying capacity.The main findings of this study on resuscitation with DCLHb following

Figure 5. Local tissue oxygenation (A) and frequency of tissue hypoxia (B) withvalues ranging from 05 mmHg in striated muscle prior to shock and followingresuscitation with Dextran, 10 g% AUB or DCLHb (mean SEM, n 6 per

exp. group; p< 0.05 vs. Dx-60 Mann Whitney U-test; x p< 0.05 vs. baselineWilcoxon test).


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density after resuscitation, (4) DCLHb was more effective to restore localtissue oxygenation than Dx-60, however not superior to AUB.

The dorsal skin fold chamber is an animal model suitable forthe investigation of microvascular disturbances following ischemia=reperfusion as represented by systemic hemorrhagic shock [32]. Potentialadvantages of this model are the possibility to investigate changes in themicrocirculation independently of anesthesia during the observation per-iod. The changes due to manipulation of the local tissue are minimized byimplantation the chamber several days before investigation. The validityof local tissue oxygenation measurements in this experimental setup hasbeen demonstrated previously [33] and the experimental protocol ofsevere hemorrhagic shock has been validated before. Without resusci-tation the animals would die in hemorrhagic shock after a period ofapproximately 60 minutes [20].

The above mentioned studies performed in our laboratory suggested abenefit of DCLHb for ischemia=reperfusion injury and after resuscitationfrom hemorrhagic shock on the striated skin muscle, when compared totreatment with the colloidal solution Dx-60. These effects were attributedto a reduction of leukocyte=endothelial cell interactions and improvementof local tissue oxygenation. In the present experimental setup, isovolemicresuscitation with autologous blood led to an increase of leukocyte-endothelial cell=cell interactions, i.e., the number of rolling and stickingleukocytes. Since whole blood was reinfused, which was stored for 45 min-utes and diluted with lactated ringer solution, the contact of shed bloodwith artificial surfaces might have induced an inflammatory activationof white blood cells as described by experimental models of extracorporealcirculation [34]. As demonstrated in previous studies DCLHb led to adecrease of leukocyte-endothelial cell=cell interactions in animals resusci-tated from hemorrhagic shock [20]. The exact pathophysiological mechan-

isms that are responsible for the reduction of leukocyte-endothelialcell=cell interactions remain to be elucidated. DCLHb has been describednot to increase free radical formation under conditions of ischemia=reperfusion [35]. Data from studies using experimental models of hemor-rhagic shock indicate the potential role of DCLHb as a nitric oxidescavenger [36]. However, this would imply an activation of leukocyte-endothelial cell=cell interactions [18]. The complexity of these interactionsof modified stroma free hemoglobin such as DCLHb and leukocyte-endothelial interactions were enhanced by the findings that hemoglobin

delivers nitric oxide [37,38]. The paracrine and endocrine functions ofnitric oxide bound to hemoglobin are not fully understood and to a lesser


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the local tissue oxygenation and functional capillary density, the acti-vation of leukocytes was most prominent in this experimental group.The recovery of local tissue oxygenation and the tissue perfusion withred blood cells in the striated muscle is not the single pre-requisite forthe reduction of leukocyte-endothelial cell=cell interactions in postcapil-lary venules. Another interesting finding is that, although resuscitationwith 10 g% AUB led to an increase of the leukocyte-endothelial cell=cellinteractions, the macromolecular leakage of FITC-Dextran as anindicator of the local endothelial integrity showed no significant changes.Therefore it must be concluded, that a decrease of leukocyte=endothelialcell=cell interactions does not translate into a better biological function ofmicrovascular parameters such as described above.

Local tissue oxygenation was most effectively restored by resusci-tation with autologous blood in the striated skin muscle. However, theincrease of tissue pO2 seen after DCLHb was significantly higher thanfollowing resuscitation with Dextran. These findings are in good accord-ance with other experimental animal studies: DCLHb demonstrated asignificant improvement of local tissue oxygenation when compared toDextran, however, without reaching baseline values [39,40]. In extremehemodilution, DCLHb contributes to local tissue oxygenation and oxy-gen delivery, although tissue hypoxia is present in the skeletal muscle[41]. Furthermore, DCLHb is known to increase the local tissue oxygen-ation by inducing a more homogenous oxygen distribution and a reducedshift of the histogram profile to lower tissue oxygen levels [40]. The effi-cacy of autologous blood to restore local tissue oxygenation in the earlyreperfusion period might be explained by the hem diluting effect afterresuscitation. Comparable results are seen after acute normovolemichemodilution in the skeletal muscle of dogs using a polarographic plati-num surface electrode and the radioactive microspheres technique [42].

The most likely explanation for the inferior efficacy of DCLHb torestore local tissue oxygenation in striated skin muscle when comparedto whole blood appears to be the reduction of functional capillary density(FCD). FCD is an indicator for local tissue perfusion [26] and survival fol-lowing hemorrhagic shock was found to correlate with the recovery ofFCD after resuscitation [43]. The FCD is defined as the length of RBC-perfused capillaries per observation area [26]. Since DCLHb and Dextranare non-corpuscular solution, a reduction of FCD would be expected.Surprisingly, DCLHb led to a significant decrease of FCD 10 and 30 min-

utes after resuscitation, whereas the local tissue oxygenation showed asignificant increase at 30 minutes following resuscitation. Acute toxic


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These findings suggest a higher number of capillaries perfused by plasmaand DCLHb but not by RBC. Obviously this is an impediment of themethodology to measure the perfusion of capillaries with non-corpuscularoxygen carrying solutions. Other experimental data demonstrated similareffects of DCLHb on FCD after isovolemic exchange transfusion [20,44].In these studies FCD was reduced up to 48% from baseline whereas localtissue oxygenation was not impaired. The reduced FCD might be theconsequence of a specific action of DCLHb since pre-clinical data on apolyethylene glycol-modified hemoglobin with a hemoglobin content of4 g=dL demonstrated an increase of the FCD after resuscitation fromhemorrhagic shock in the same animal model using a similar experimentalprotocol [45]. The heterogeneity of the microcirculation under physiologi-cal and pathophysiological conditions due to the spatial variance of localtissue perfusion may also contribute to varying results [46]. The mechan-isms regulating the local microcirculation are not fully understood, how-ever our data might suggest an interaction of DCLHb with organ-specificfactors modulating the tissues microcirculation.

The experimental setup used in our study did not access the outcomeof animals after resuscitation and therefore it remains unclear whetherthe reduction of leukocyte-endothelial cell=cell interactions in animalstreated with DCLHb would positively influence mortality and lethality.

Previous experimental studies described the induction of arterialvasoconstriction after administration of stroma free hemoglobin solu-tions, e.g. DCLHb [31]. DCLHb might therefore contribute to a decreasein the peripheral local tissue oxygen tension, an effect not seen afterresuscitation with whole blood. Although no vasoconstriction was foundin our studies, the vasoconstriction might take place in the largerupstream vessels not visible in the dorsal skin fold chamber preparation.Studies using radioactive labeled microspheres revealed an increased

microvascular blood flow to the heart after infusion of DCLHb, whereasthis phenomenon was absent after infusion of unmodified hemoglobin[47]. This may indicate an organ specific action of DCLHb on the micro-circulation. Intravital microscopy studies in the dorsal skin fold chamberof the hamster using DCLHb for isovolemic exchange transfusion andhypervolemic infusion demonstrated an increase of the local RBCVand a very short lasting arteriolar vasoconstriction for a maximum periodof 120 seconds after hypervolemic infusion [20]. However, this short last-ing arteriolar vasoconstriction did not explain the prolonged increase of

the RBCV at 30 minutes of hemorrhagic shock.The novel finding of the present study is the efficacy of diluted


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non-diluted whole blood in restoring macro- and microcirculatory para-meters [39]. Isovolemic hemodilution has been shown to be efficient toincrease oxygen delivery to tissues with a peak at 30% hematocrit [48].In the present study, the dilution of the reinfused autologous blood didnot decrease the systemic hematocrit at the end of the observation period.The hypothesis that hemodilution contributes to a faster recovery oftissue pO2 is also objected by the experimental group using colloidalinfusion solution. Although Dextran led to a decrease of systemichematocrit after resuscitation, Dextran was less effective in restoringthe local tissue pO2.

Despite the promising data reported from pre-clinical and clinicaltrials, there are still impediments for HBOCs, such as DCLHb, for alarge-scale clinical use. The published data on post hoc mortality analysisof the clinical trauma trial revealed a higher mortality in patients treatedwith DCLHb than expected by survival analysis [15]. These data led tospeculations about the safety of modified hemoglobin solutions. Thesesolutions do not represent simple oxygen carriers, but interact with awide variety of factors, such as nitric oxide and endothelin, that areinvolved in the regulation of the local microcirculation of ischemic andreperfused organs. This study demonstrates the capability of DCLHbto substitute red blood cells but not to replace them.

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