AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 279: H1922-H1930, 2000;
0363-6135/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (16)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sielenkämper, A. W.
Right arrow Articles by Sibbald, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sielenkämper, A. W.
Right arrow Articles by Sibbald, W. J.
Vol. 279, Issue 4, H1922-H1930, October 2000

Diaspirin cross-linked Hb and norepinephrine prevent the sepsis-induced increase in critical O2 delivery

Andreas W. Sielenkämper, Pei Yu, Otto Eichelbrönner, Tammy MacDonald, Claudio M. Martin, Ian H. Chin-Yee, and William J. Sibbald

The A. C. Burton Vascular Biology Laboratory, Victoria Hospital Research Institute, and The University of Western Ontario, London, Ontario, Canada N6A 4G5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that support of arterial perfusion pressure with diaspirin cross-linked Hb (DCLHb) would prevent the sepsis-induced attenuation in the systemic O2 delivery-O2 uptake relationship. Awake septic rats were treated with a chronic infusion of DCLHb or a reference treatment [norepinephrine (NE)] to increase mean arterial pressure by 10-20% over 18 h. Septic and sham control groups received normal saline. Isovolemic hemodilution to create anemic hypoxia was then performed in a metabolic box during continuous measurement of systemic O2 uptake. O2 delivery was calculated from hemodynamic variables, and the critical point of O2 delivery (DO2 crit) was determined using piecewise regression analysis of the O2 delivery-O2 uptake relationship. Sepsis increased DO2 crit from 4.99 ± 0.17 to 6.69 ± 0.42 ml · min-1 · 100 g-1 (P < 0.01), while O2 extraction capacity was decreased (P < 0.05). DCLHb and NE infusion prevented the sepsis-induced increase in DO2 crit [4.56 ± 0.42 ml · min-1 · 100 g-1 (P < 0.01) and 5.04 ± 0.56 ml · min-1 · 100 g-1 (P < 0.05), respectively]. This was explained by a 59% increase in O2 extraction capacity in the DCLHb group compared with septic controls (P < 0.05), whereas NE treatment decreased systemic O2 uptake in anemic hypoxia (1.51 ± 0.08 vs. 1.87 ± 0.1 ml · min-1 · 100 g-1 in septic controls, P < 0.05). We conclude that DCLHb ameliorated O2 extraction capacity in the septic microcirculation, whereas NE decreased the metabolic demands of the tissues.

blood substitute; anemic hypoxia; cardiovascular; rat


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SEPSIS IS A SYNDROME that jeopardizes the integrity of many physiological pathways. Besides an activation of inflammatory cascades and a dysfunction of the systemic, regional, and microregional circulations, diffusive and convective O2 transport are perturbed. Diffusive O2 transport may be compromised in the lung, for example, because of acute respiratory distress syndrome (6, 18) or in the microcirculation, where tissue edema may increase diffusion distances and therefore compromise uptake of the systemically provided O2 (16). Convective O2 delivery (DO2) may be impaired when a depression in myocardial contractility interferes with the ability to appropriately increase cardiac output (CO) (10, 25), when vasoplegia of resistance vessels maldistributes blood flow between organs (21), or when microvascular dysfunction causes inadequate capillary perfusion (9, 19). In addition, it has been postulated that mitochondrial dysfunction in sepsis restricts the optimal use of available O2 (34, 38).

As a consequence of these abnormalities, the normal relationship between systemic DO2 and O2 uptake (VO2) is altered in sepsis, and the maximal O2 extraction capacity of the tissues is thereby decreased (23, 27). Under experimental conditions, this phenomenon becomes manifest as an elevation of the critical DO2 (DO2 crit), the point where systemic VO2 becomes dependent on O2 supply (23). In a recent study to determine the efficacy of an O2-carrying, cell-free Hb solution, diaspirin cross-linked Hb (DCLHb) (30), we found that infusing DCLHb improved O2 extraction capacity in septic rats (30). One possible explanation for this effect was that DCLHb recruited capillaries previously not perfused with red blood cells (RBCs), since a subsequent study demonstrated an increase in the density of RBC-perfused capillaries in the gut mucosa of septic rats after DCLHb infusion (31).

In addition to increasing microvascular perfusion, there are other explanations for the activity of Hb solutions to increase O2 extraction capacity in sepsis. Because Hb solutions are effective O2 carriers, but much smaller than RBCs, Hb in solution may access capillaries unavailable to RBCs, because their lumens are narrowed by edema (29). Hb molecules may also facilitate tissue oxygenation, since they are uniformly distributed within the plasma phase and thus reduce diffusion resistance for O2 (24).

The present study was designed to determine the effect of DCLHb infusion on the systemic VO2-DO2 relationship and to identify why DCLHb infusion increases the microvascular O2 extraction in sepsis. We chose to administer DCLHb chronically, in doses that provided a moderate increase in mean arterial blood pressure (MAP). Because Hb solutions will increase vascular resistance because of their effect to bind nitric oxide (13, 14, 28), we added a control group (septic rats) in which norepinephrine (NE) was infused to also increase vascular resistance. By this approach, we hoped to isolate any effects of DCLHb infusion on the microcirculation per se, that is, excluding the influence of DCLHb on vascular resistance. The interventions were infused over an 18-h period to allow sufficient time for complete expression of potential effects on the systemic VO2-DO2 relationship, as well as to enhance the potential generalizability of findings to the clinical situation. When the effects of both treatments on DO2 crit were determined after completion of the treatment phase by use of acute progressive isovolemic hemodilution and on-line measurements of VO2, we found that DCLHb and NE were equally effective at preventing the sepsis-induced increase of the DO2 crit.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The protocol of this study was approved by the Council on Animal Care of the University of Western Ontario (London, ON, Canada).

Animal model. Forty-seven male Sprague-Dawley rats, weighing 320-380 g, were used after a 1-wk acclimatization period in our laboratory. Anesthesia was induced and maintained by halothane inhalation. Catheters were advanced into the left femoral vein, the superior vena cava, and the left carotid artery. A thermodilution CO probe (IT-21 thermocouple, Physiotemp Instruments, Clifton, NJ) was then positioned in the aortic arch via the carotid artery. After cannulation, rats were randomized to undergo sham laparotomy or laparotomy and cecal ligation and perforation (CLP), according to a previously standardized technique (9), to create sepsis. Fluid resuscitation with 0.9% saline (2 ml · 100 g-1 · h-1 iv) was started postoperatively. The carotid line was continuously flushed with heparin solution (45 IU/h) to maintain patency, and fentanyl (2 µg · 100 g-1 · h-1 iv) was provided to ensure adequate analgesia.

Experimental protocol. Figure 1 shows the experimental design of the study. Twenty-four hours after surgery, MAP and CO were determined, and blood samples were drawn to assess biochemistry, including blood gases. CLP-septic animals (n = 39) were then randomized to receive normal saline (NS) alone (n = 15) or a continuous infusion of DCLHb (n = 14) or NE (n = 10). With both DCLHb and NE, the goal was to administer a dose that increased MAP by 10-20% over the next 18 h. Sham rats (n = 8) received NS. Pilot experiments confirmed that this model of chronic infusion was technically possible and identified the general dose ranges required to achieve target pressures for DCLHb and NE. After 18 h of treatment, measurements were repeated, the animals were placed in a metabolic cage, and the arterial and venous lines were connected to withdrawal and perfusion pumps, respectively. Treatments were continued. After a 30-min acclimatization period, MAP, CO, arterial O2 content, and systemic VO2 were measured. Arterial blood (0.7 ml) was withdrawn to determine Hb concentration, arterial O2 saturation, and lactate concentration. Isovolemic hemodilution was then carried out (6 ml/h) to determine the systemic DO2-VO2 relationship. Systemic VO2 was measured semicontinuously (see below) while measurements of MAP and CO were repeated after every 2 ml of isovolemic hemodilution. Blood samples for arterial O2 content, Hb concentration, and lactate were simultaneously obtained. At all times, shed blood was replaced by identical volumes of warmed rat plasma obtained from donor rats.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1.   Experimental design. DO2 crit, critical O2 delivery; t, time; HD, hemodilution; CLP-NS, animals subjected to cecal ligation and perforation (CLP) and treated with normal saline (NS; i.e., septic controls); CLP-DC, septic rats treated with diaspirin cross-linked Hb (DCLHb) infusion; CLP-NE, septic rats treated with norepinephrine (NE) infusion.

Animals were excluded if technical failure (e.g., damage or blocking of arterial and venous catheters) occurred before the completion of the treatment phase. After completion of measurements, rats were euthanized with an overdose of pentobarbital sodium (65 mg), and postmortem examination was carried out.

Treatments and isovolemic hemodilution. Twenty-four hours after sepsis was induced, septic animals were randomized to receive a continuous infusion of DCLHb, NE, or placebo (NS). After a bolus infusion of 100 mg of DCLHb solution over 3 min to obtain effective plasma concentrations, DCLHb was infused at a rate of 70-300 mg · kg-1 · h-1. NE was adjusted to an effective dose within a few minutes and was then infused at a rate of 0.25-1.25 µg · kg-1 · min-1. Doses in the treatment groups were adjusted at 30 min and at 1, 2, 3, 6, and 12 h to maintain the increase in MAP at targeted levels. The femoral line was used for drug infusion, and adjustments for a constant infusion volume were made via the jugular line. CLP controls and sham rats received NS via both lines (CLP-NS group and sham group, respectively). Total infusion volumes were kept at a rate of 1.5 ml · 100 g-1 · h-1 in all groups. DCLHb was prepared by Baxter Healthcare (Round Lake, IL) as described previously (3, 22) and was formulated at a concentration of 100 g/l in a lactated electrolyte solution. NE was diluted in normal saline and administered at a concentration of 10 µg/ml.

For isovolemic hemodilution, rat plasma obtained from donor rats by use of a previously standardized protocol (30) and warmed to body temperature was filtered through a 40-µm transfusion filter. With the use of syringe pumps (Razel Scientific Instruments, Stamford, CT) set at a rate of 6 ml/h, blood was withdrawn via the arterial line and plasma was infused via the jugular line. In this way, DO2 was lowered in a stepwise manner to decrease it beyond the point of DO2 crit.

Measurements and calculations. Systemic VO2 was measured semicontinuously by means of an Oxymax system (Columbus Instruments, Columbus, OH). A constant flow of room air at a rate of 3.5 l/min was sampled by a paramagnetic O2 sensor for analysis of O2 content and then by an infrared CO2 analyzer. Reference measurements were made by sampling room air every five samples. Systemic VO2 was measured from the reduction of air O2 content within the closed system and displayed on-line. Five consecutive values obtained over a 60-s measurement period were averaged to determine VO2 at an individual time point.

MAP was measured with Uniflow disposable transducers (Baxter, Toronto, ON, Canada) and a monitor (model 78353B, Hewlett-Packard, Mississauga, ON, Canada). CO was measured by the thermodilution technique with use of 0.3 ml of NS at room temperature injected via the jugular catheter. The thermocouple output was analyzed with a Cardiotherm 500 AC-R CO computer (Columbus Instruments). Hb and arterial O2 saturation were assessed using a CO-oximeter (OSM2b hemoximeter, Radiometer, Copenhagen, Denmark), and lactate concentration was determined by means of a quantitative, enzymatic method (Paramax Analytical System, Baxter, Mississauga, ON, Canada). Arterial O2 content was measured directly using a Lex-O2-Con O2 analyzer (Lexington). Systemic DO2 was obtained by multiplying arterial O2 content by CO. Systemic vascular resistance (SVR) and systemic O2 extraction ratio were calculated using standard formulas. DO2 crit was determined using piecewise regression analysis of the VO2-DO2 relationship as described by Samsel and Schumacker (26). The whole body VO2-DO2 relationship is biphasic, with the point where systemic VO2 becomes dependent on O2 supply (the DO2 crit) defined at the point of transition from plateau to downslope (27). All possible pairs of regression lines were constructed over all points where DO2 and VO2 data had been obtained. The pairs of lines were then compared to find the pair with the lowest residual sum of squares of the perpendicular distances from the points to the lines. The DO2 crit was then determined by calculating the intersection point of this pair of lines.

Statistics. For statistical analysis, SigmaStat 2.03 software (Jandel, San Rafael, CA) was used. Mortality was analyzed using Fisher's exact test. ANOVA with post hoc tests and correction for multiple comparisons (Student-Newman-Keuls method) was performed to determine the effects of the treatments in the CLP-septic groups at 18 h and after hemodilution. To determine the effects of sepsis between the sham group and the CLP-septic control group, Student's t-test was used. The effects of sepsis and the effects of the treatments on blood pressure during hemodilution were analyzed using two-way ANOVA for repeated measurements with appropriate post hoc comparisons (Student-Newman-Keuls method). For all statistical tests, significance was assumed at P < 0.05. Values are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal model. Twenty-four hours after the surgical procedures, sham rats had recovered. All animals treated with CLP demonstrated reduced activity, piloerection, and exudation around the eyes and nose. The effects of CLP-sepsis on hemodynamic and biochemical markers are shown in Table 1. Septic rats presented with modest hypotension, an elevated CO, and a decreased SVR. CLP-sepsis was also characterized by leukopenia and thrombocytopenia, whereas the arterial lactate increased only slightly compared with the sham group. On postmortem examination, inspection of the abdominal contents revealed spillage of bowel contents and peritonitis in CLP-septic rats, whereas the aspect of the abdomen was normal in all sham rats.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Effects of CLP-sepsis on hemodynamic and biochemical markers at 24 h

Effects of DCLHb and NE infusion after 18 h of treatment. Our intention was to increase MAP with DCLHb or NE infusion in CLP-septic rats by 10-20% over 18 h. With either of the treatments, MAP, when averaged across all measurements of the treatment period, was kept in the desired range (Fig. 2, horizontal lines). Average blood pressure was 109 ± 2 mmHg for the sham group, 96 ± 5 mmHg for the CLP-NS rats, and 114 ± 3 and 109 ± 3 mmHg for the DCLHb- and NE-treated groups, respectively. Especially among the animals in the three septic groups, considerable variability in blood pressure was observed independent from treatment (Fig. 2, vertical lines).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2.   Goal-directed approach to increase mean arterial pressure (MAP) by 10-20% over 18 h in septic rats. Horizontal lines, MAP across all measured pressures during the 18-h treatment phase; vertical lines, range of pressures for each animal. See Fig. 1 legend for definition of abbreviations.

Mortality was determined for the treatment period including the time of isovolemic hemodilution before Do2 crit (e.g., O2 supply dependency) was reached. In the sham group, no mortality was observed. Mortality in the septic groups was 7 of 15 in the CLP-NS group (46.7%), 7 of 14 in the CLP-DC group (50%), and 2 of 10 in the CLP-NE group (20%). Differences in mortality among the treatment groups, and comparing the treatment group with the CLP-NS group, were not significant.

Table 2 summarizes the effects of DCLHb and NE infusion on CO, SVR, O2 transport, and biochemical markers after completion of the 18-h treatment phase. CO and systemic DO2 were decreased in the CLP-DC group compared with CLP-NS and CLP-NE groups, and O2 extraction ratio was higher in the CLP-DC than in the CLP-NE group. SVR was elevated in DCLHb-treated rats, but not in the CLP-NE group. There were no treatment effects on systemic VO2, arterial and venous O2 saturation, Hb concentration, white blood cell count, platelet count, and arterial lactate concentration.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Effects of 18 h of chronic infusion of diaspirin cross-linked Hb or NE in septic rats

Effects of DCLHb and NE infusion on MAP and O2 transport during isovolemic hemodilution. Figure 3 shows the changes in MAP during the isovolemic hemodilution procedure in all groups. Compared with baseline, there was a significant decrease in blood pressure during hemodilution in all except the DCLHb group. Compared with the sham group, CLP-septic rats were hypotensive during the hemodilution procedure (P < 0.05). Continuing the infusion of DCLHb or NE resulted in higher blood pressure than in untreated septic rats. Toward the end of the experiment, however, blood pressure in the NE-treated rats decreased to the level of the CLP-NS group (P < 0.05 vs. DCLHb group).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 3.   MAP during progressive hemodilution. DCLHb and NE treatment was continued using the effective doses at the end of the 18-h treatment phase. , sham; open circle , CLP-NS; black-down-triangle , CLP-DC; down-triangle, CLP-NE. Values are means ± SE. * P < 0.05 vs. baseline. dagger  P < 0.05, CLP vs. sham. # P < 0.05, DCLHb vs. CLP. § P < 0.05, NE vs. CLP. ** P < 0.05, NE vs. DCLHb.

In the CLP-NS group, DO2 crit was increased compared with the sham group (from 4.99 ± 0.17 to 6.69 ± 0.42 ml · min-1 · 100 g-1, P < 0.01; Fig. 4). At the DO2 crit, all the following were also changed compared with the sham rats: 1) O2 extraction capacity was depressed (20%, P < 0.05); 2) Hb concentration was greater (67 ± 5 vs. 44 ± 2 g/l, P < 0.001); and 3) systemic VO2 was greater (18.5 ± 1 vs. 15.3 ± 0.9 ml · min-1 · 100 g-1, P < 0.05). CO, however, was not different between the sham and CLP septic rats at the DO2 crit (299 ± 24 and 314 ± 22 ml/min, respectively).


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 4.   Effects of DCLHb and NE infusion on DO2 crit. Values are means ± SE; n = 7 sham, 8 CLP-NS, 7 CLP-DC, and 7 CLP-NE. # P < 0.05 vs. sham. * P < 0.05 vs. CLP-NS. ** P < 0.01 vs. CLP-NS.

In the DCLHb- and NE-infused septic rats, the sepsis-induced increase in DO2 crit was prevented (P < 0.01 and P < 0.05 vs. CLP-NS; Fig. 4). At the DO2 crit, systemic VO2 was decreased in the CLP-NE group (-19% vs. CLP-NS, P < 0.05; Fig. 5A) but not in the CLP-DC group. The Hb concentration was lower in DCLHb- and NE-treated rats (P < 0.05 and P < 0.001, respectively; Fig. 5B). The O2 extraction ratio was increased by 59% in DCLHb rats compared with the CLP-NS group (P < 0.05), whereas it was the same in the CLP-NE group (Fig. 5C). CO at DO2 crit tended to decrease with DCLHb infusion compared with the CLP-NE group (P = 0.05; Fig. 5D). Body temperature was not different between groups (37.8 ± 0.4, 38 ± 0.4, and 37.7 ± 0.5°C in CLP-NS, CLP-DC, and CLP-NE, respectively).


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of DCLHb and NE infusion on key variables of O2 transport at DO2 crit. Values are means ± SE; n = 8 CLP-NS, 7 CLP-DC, and 7 CLP-NE. A: systemic O2 uptake (VO2). * P < 0.05 vs. CLP-NS; dagger P < 0.05 vs. CLP-DC. B: Hb concentration. * P < 0.05 vs. CLP-NS; ** P < 0.001 vs. CLP-NS. C: systemic O2 extraction ratio. * P < 0.05 vs. CLP-NS; ** P < 0.05 vs. CLP-NE. D: cardiac output (CO). P = 0.05 (not significant), CLP-NE vs. CLP-DC.

When the CO-SVR relationship was examined at the completion of the 18-h treatment phase, the CLP-NS group was clearly characterized by a high CO-low SVR ("hyperdynamic") profile compared with the sham group. DCLHb-treated rats, compared with the CLP-NS group, presented with a low CO-high SVR profile. A similar effect was also seen in the CLP-NE group but to a much lesser degree (Fig. 6). Isovolemic hemodilution caused a shift to higher CO and lower SVR in all groups. However, the CLP-DC group, but not the CLP-NE group, maintained a low CO-high SVR profile.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6.   CO vs. systemic vascular resistance (SVR). , sham; open circle , CLP-NS; black-down-triangle , CLP-DC; down-triangle, CLP-NE. Arrows, changes in the CO-SVR relationship from the time when the 18-h treatment phase was completed to the point when DO2 crit was reached. Anemic hypoxia caused a shift to higher CO and lower vascular resistances in all 4 groups. DCLHb treatment produced a low CO-high SVR profile compared with the CLP-NS group. Values are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This experiment explored the effects of a chronic infusion of Hb solution, DCLHb, on the systemic VO2-DO2 relationship. With a chronic, 18-h infusion of DCLHb, as well as with NE infusion in a control group, we prevented the usual adverse effect of a sepsis-induced increase in DO2 crit. This novel finding supports our conclusion that the disturbance in convective DO2 seen in sepsis, which depresses the host's ability to extract O2, is amenable to treatment.

Approach and animal model. Recent studies suggested that, in the presence of inadequate DO2, cell-free Hb solutions may increase the maximal O2 extraction capacity (24, 30, 32). Therefore, this study aimed at preventing sepsis-induced alterations in the systemic VO2-DO2 relationship by using the hemodynamic properties of the DCLHb, that is, to support arterial perfusion pressure (28) and to improve microvascular perfusion (31). We also chose this approach to mimic the clinical scenario, where support of blood pressure by chronic infusion of agents that increase vascular resistance and/or CO is a cornerstone of therapies to improve outcome from septic shock (33).

A primary objective of this study was to compare the effects of DCLHb infusion on the systemic VO2-DO2 relationship with a septic control group, which received only NS to adjust for the infused volume. A sham control group was added to demonstrate the effects of sepsis and to allow an estimation of possible effects of the DCLHb in relation to the insult. A third group that received NE infusion targeted to achieve the same effect on MAP during the 18-h treatment period was intended to control for the vascular effects of the DCLHb solution on arteriolar reactivity and tone. Specifically, our intention was to isolate the effects of the Hb solution on blood pressure from its properties to modify O2 transport capacity due to altered capillary convective and diffusive O2 transport (24, 31).

In this study, DO2 crit was the key parameter used to assess the effects of sepsis and the interventions on O2 transport capacity. The importance of the systemic DO2 crit is that this parameter defines the DO2 where O2 extraction is maximized DO2 critand the systemic VO2 becomes dependent on the systemic DO2 if the latter is reduced beyond this point. Thus the systemic DO2 crit is the ultimate physiological threshold to the manifestation of tissue hypoxia and shock (27). Two mechanisms have been discussed to explain the presence of a critical value of O2 supply: diffusion limitation in the microcirculation and physiological arterial-venous shunt (37). It is assumed that, at least on average in the whole body, arterial-venous shunting is more important to determine the maximal value of systemic O2 extraction (37).

To calculate the DO2 crit, we used a hemodilution model that was developed and standardized in our laboratory (11, 30). This model provides direct and on-line measurements of systemic VO2 from awake rats and thus allows the determination of DO2 crit from regression against a larger number of consecutive DO2 measurements, as originally described by Samsel and Schumacker (26).

When CLP-septic rats were compared with sham animals 24 h after CLP and before randomization to the treatment protocols, they had developed characteristic signs of sepsis as defined by a consensus conference (2): leukopenia, thrombocytopenia, and mild hypotension. Also, a modest increase in CO and loss of vascular resistance indicated a hyperdynamic cardiovascular response. When O2 extraction capacity was determined after 42 h, CLP-sepsis was associated with increased DO2 crit and an O2 extraction deficit compared with the sham group. Myocardial function appeared to be intact, since septic animals reached the same cardiac index at DO2 crit. Very similar sepsis-induced changes in O2 extraction capacity have been demonstrated in dogs by Nelson et al. (23), who proposed that microvascular injury might be the cause of the sepsis-induced attenuation in O2 transport. For the sepsis model as used in this experiment, alterations in arteriolar vascular reactivity (21), as well as reduced capillary perfusion and attenuation in microvascular blood flow in microvascular networks of different organs, have been demonstrated previously (5, 9, 19, 20).

It is important that chronic infusion of the catecholamine, NE, resulted in the same 10-20% increase in blood pressure that was achieved in Hb-treated septic rats throughout the 18-h treatment period. Therefore, the NE group may be regarded as an appropriate control for the blood pressure component of the effects of DCLHb infusion on the systemic VO2-DO2 relationship.

Mortality in this study was not significantly different between the septic groups, although the data for the NE group might suggest a decreased mortality compared with septic controls and DCLHb-treated rats. This study, however, was not designed to study effects of DCLHb and NE on mortality. An additional power analysis revealed that a larger sample size would have been required to determine treatment effects on mortality.

Also, it has to be considered that this study presents data from survivors of the septic insult. One cannot exclude that this introduced bias on some of the results. However, because the objective of this study was to determine the effects of chronic DCLHb or NE infusion in sepsis, which is a syndrome with high lethality under experimental and clinical conditions, this was unavoidable.

Effects of interventions. The typical, sepsis-induced alteration of the VO2-DO2 relationship in CLP-septic rats was prevented by DCLHb, as indicated by a decreased DO2 crit compared with placebo-treated septic rats. In rats treated with the Hb solution, this effect was associated with an increased ability to extract O2, suggesting improved diffusive and/or convective O2 transport in the microcirculation. In the NE group, the sepsis-related increase in DO2 crit was also prevented, but O2 extraction was not increased at the critical point. However, in this group, a tendency for a decrease in systemic VO2 indicated a modulation of the hyperdynamic metabolic response to sepsis.

One possibility is that the only pharmacological property common to DCLHb and NE, that is, to increase vascular resistance, explains the observed effects on DO2 crit. Indeed, loss of vascular resistance, also referred to as "septic vasoplegia," is a characteristic consequence of the inflammation process in sepsis as a result of nitric oxide overproduction (17, 21). Septic vasoplegia is followed by decreased perfusion pressures and inappropriate distribution of blood flows (17, 21), which may be the underlying cause for the within-organ, microregional O2 supply-demand imbalance in sepsis (4, 36). Therefore, improved blood flow distribution and increased perfusion pressure could explain the protective effect of DCLHb and NE infusion against sepsis-induced alterations of the VO2-DO2 relationship. Moreover, evidence for beneficial effects on the septic microcirculation have been demonstrated previously for DCLHb (31) and NE (40).

It is striking, however, that only DCLHb infusion increased the maximal O2 extraction capacity, whereas NE infusion, as indicated by a modest fall in VO2, preserved a normal DO2 crit via reduction of the metabolic needs. Despite comparable effects on blood pressure, this indicates that the effects of DCLHb and NE on the VO2-DO2 relationship could be explained, alternatively, by unique properties of each of the two agents.

Aside from cardiovascular effects, DCLHb is characterized by 1) excellent O2-carrying properties (8), 2) a rightward-shifted O2 dissociation curve [PO2 at which Hb is half-saturated (P50) = 32.4 mmHg] compared with human blood (8, 30), and 3) a characteristic distribution in the plasma, outside the RBCs (24). In this study, total Hb concentration was not increased in DCLHb-treated rats, and systemic DO2 was decreased, excluding transfusion effects as a cause for increased O2 extraction capacity. Also, it is unlikely that differences in P50 explain the effects of DCLHb on tissue O2 extraction capabilities, since compared with rat blood, which is characterized by a higher P50 of 37-38 mmHg, the O2 dissociation curve of DCLHb is shifted leftward. Studies on the effects of a leftward-shifted O2 dissociation curve on the physiological adaptation to acute decreases in DO2 reported only unfavorable effects on tissue oxygenation (39). Eventually, the distribution of DCLHb within the plasma compartment is (alternative to the effects on the vasculature) the only other possible explanation for increased O2 extraction capacity after DCLHb infusion. In a situation where microcirculatory perfusion is impaired, as in sepsis (9, 19), a homogeneous intravascular distribution of DCLHb may increase diffusion capacity and thus improve the abilities of the tissue to extract O2. For example, DCLHb could serve as a carrier or intermediary vehicle for O2 released from RBCs. A recent study in which a geometrical model was used, in fact, reported that the presence of Hb molecules outside the RBC decreases the diffusion resistance for O2 (24).

For the CLP-NE group, the unexpected decrease in systemic VO2 may also provide an explanation for the preservation of a normal VO2-DO2 relationship. This decrease in VO2 suggests that NE exhibited anti-inflammatory effects, implying that suppression of the typical systemic inflammatory process in sepsis decreased the O2 needs of the tissues. This assumption is supported by two recent studies demonstrating that catecholamines modulate monocyte receptor status and cytokine expression during inflammation in a potentially beneficial manner as a result of beta 2-adrenorecepter activation (1, 12). In addition, others who studied the effect of NE infusion on O2 extraction capacity using an endotoxin model where DO2 crit was determined by a progressive decrease in CO also reported a decrease in DO2 crit (40). However, this work included no septic controls to relate this benefit of NE infusion to the extent of the lesion (40).

Alternatively, the decrease in tissue VO2 in our study could suggest some degree of tissue ischemia after NE infusion. However, arterial lactate, which has been used as a marker of tissue ischemia (15, 35), was not increased in the NE-treated rats at completion of the treatment phase. Also, it would be expected that, in the presence of baseline ischemia, DO2 crit would be reached at a higher value, opposite to the findings in this study. It therefore appears that no significant compromise of tissue oxygenation occurred during NE infusion.

Intervention effects on cardiac performance. In this study the improvement in the VO2-DO2 relationship with DCLHb and NE infusion did not only occur in the presence of different effects of the two agents on O2 transport but was also associated with differences in the hemodynamic profile. After 18 h of treatment, the DCLHb group presented with a decreased systemic DO2, most likely to be explained by a reflex fall in CO secondary to the increase in vascular resistance. In NE-treated rats, CO and systemic DO2 were not affected, probably since myocardial contractility was supported simultaneously with the increase in blood pressure. From this observation, one can conclude that in the DCLHb group no support of myocardial contractility and systemic DO2 was required to increase O2 extraction capacity. This conclusion is supported by the analysis of the relationship between CO and SVR (Fig. 6), since the low CO-high SVR profile in DCLHb-treated was maintained even when systemic DO2 had been diminished to the critical point. The latter observation may also confirm the assumption that the decrease in CO and systemic DO2 observed at 18 h of treatment did not reflect a decreased demand of O2 supply, because, otherwise, isovolemic hemodilution would have caused CO to rise (7).

In summary, this study provides clear evidence for an improved systemic VO2-DO2 relationship after goal-directed chronic infusion of DCLHb and NE to increase blood pressure in septic rats. The possibility exists that this beneficial effect of DCLHb and NE is the sole consequence of increased perfusion pressure and subsequently improved microvascular perfusion. However, our results show that DCLHb infusion primarily increased O2 extraction capacity, whereas NE infusion appeared to decrease tissue O2 demand. Therefore, the observed effects on the sepsis-induced anomalies of the VO2-DO2 relationship could also be explained by unique, but different, effects of each of the two studied agents: DCLHb may favor O2 transport in the microcirculation, whereas NE may modulate the inflammatory response to sepsis.


    ACKNOWLEDGEMENTS

This study was supported by Baxter Healthcare (Round Lake, IL), Medical Research Council of Canada Group Grant GR-12816 and Grant MT-13940, and Heart and Stroke Foundation of Ontario Grant NA3733. A. Sielenkämper was supported, in part, by a grant from the Department of Anesthesiology and Intensive Care, Westfälischen Wilhelms-Universität, Münster, Germany.


    FOOTNOTES

Address for reprint requests and other correspondence: W. J. Sibbald, The London Health Sciences Centre, Victoria Campus, 375 South St., London, ON, Canada N6A 4G5 (E-mail: wsibbald{at}julian.uwo.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 August 1999; accepted in final form 17 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, E, Kaneko DJ, and Shenkar R. Effects of endogenous and exogenous catecholamines on LPS-induced neutrophil trafficking and activation. Am J Physiol Lung Cell Mol Physiol 276: L1-L8, 1999[Abstract/Free Full Text].

2.   ACCP/SCCM. . Consensus Conference. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 101: 1644-1655, 1992[Abstract/Free Full Text].

3.   Azari, M, Rohn K, and Picken J. Diaspirin crosslinked hemoglobin (DCLHb): characterization of the process and the product manufactured under GMP requirements for clinical studies. Artif Cells Blood Substit Immobil Biotechnol 22: 701-708, 1994[ISI][Medline].

4.   Bersten, A, and Sibbald WJ. Circulatory disturbances in multiple systems organ failure. Crit Care Clin 5: 233-254, 1992.

5.   Boczkowski, J, Vicaut E, and Aubier M. In vivo effects of Escherichia coli endotoxemia on diaphragmatic microcirculation in rats. J Appl Physiol 72: 2219-2224, 1992[Abstract/Free Full Text].

6.   Bone, RC. Sepsis and its complications: the clinical problem. Crit Care Med 22: S8-S11, 1994[ISI][Medline].

7.   Bowens, C, Spahn DR, Frasco PE, Smith LR, McRae RL, and Leone BJ. Hemodilution induces stable changes in global cardiovascular and regional myocardial function. Anesth Analg 76: 1027-1032, 1993[Abstract/Free Full Text].

8.   Chatterjee, R, Welty LV, Walder RY, Pruitt SL, Rogers PH, Arnone A, and Walder JA. Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha  chains (lysine 99alpha 1, lysine 99alpha 2). J Biol Chem 21: 9929-9937, 1986.

9.   Farquhar, I, Martin CM, Lam C, Potter R, Ellis CG, and Sibbald WJ. Decreased capillary density in vivo in bowel mucosa of rats with normotensive sepsis. J Surg Res 61: 190-196, 1996[ISI][Medline].

10.   Feltes, TF, Pignatelli R, Kleinert S, and Marisalco MM. Quantitated left ventricular systolic mechanics in children with septic shock utilizing noninvasive wall-stress analysis. Crit Care Med 22: 1647-1658, 1994[ISI][Medline].

11.   Fitzgerald, RD, Martin CM, Dietz GE, Doig GS, Potter RF, and Sibbald WJ. Transfusing RBCs stored in CPDA-1 for 28 days fails to improve tissue oxygenation in rats. Crit Care Med 25: 726-732, 1997[ISI][Medline].

12.   Guirao, X, Kumar A, Katz J, Smith M, Lin E, Keogh C, Calvano SE, and Lowry SF. Catecholamines increase monocyte TNF receptors and inhibit TNF through beta 2-adrenoreceptor activation. Am J Physiol Endocrinol Metab 273: E1203-E1208, 1997[Abstract/Free Full Text].

13.   Gulati, A, Sen AP, Sharma AC, and Singh G. Role of ET and NO in resuscitative effect of diaspirin cross-linked hemoglobin after hemorrhage in rat. Am J Physiol Heart Circ Physiol 273: H827-H836, 1997[Abstract/Free Full Text].

14.   Hart, JL, Ledvina M, and Muldoon M. Actions of diaspirin cross-linked hemoglobin on isolated rat and dog vessels. J Lab Clin Med 129: 356-363, 1997[ISI][Medline].

15.   Haupt, MT, Gilbert EM, and Carlson RW. Fluid loading increases oxygen consumption in septic patients with lactate acidosis. Am Rev Respir Dis 131: 912-916, 1985[ISI][Medline].

16.   Hersch, M, Gnidec AA, Bersten AD, Troster M, Rutledge FS, and Sibbald WJ. Histologic and ultrastructural changes in nonpulmonary organs during early hyperdynamic sepsis. Surgery 107: 397-410, 1990[ISI][Medline].

17.   Julou-Schaeffer, G, Gray GA, Fleming I, Schott C, Parratt JR, and Stoclet JC. Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway. Am J Physiol Heart Circ Physiol 259: H1038-H1043, 1990[Abstract/Free Full Text].

18.   Kollef, MH, and Schuster DP. The acute respiratory distress syndrome. N Engl J Med 332: 27-37, 1995[Free Full Text].

19.   Lam, C, Tyml K, Martin C, and Sibbald W. Microvascular perfusion is impaired in a rat model of normotensive sepsis. J Clin Invest 94: 2077-2083, 1994.

20.   Madorin, SM, Martin CM, and Sibbald WJ. Dopexamine attenuates flow motion in ileal mucosal arterioles in normotensive sepsis. Crit Care Med 27: 394-400, 1999[ISI][Medline].

21.   Martin, CM, Yaghi A, Sibbald WJ, McCormack D, and Paterson NAM Differential impairment of vascular reactivity of small pulmonary and systemic arteries in hyperdynamic sepsis. Am Rev Respir Dis 148: 164-172, 1993[ISI][Medline].

22.   Nelson, D, Azari M, Brown R, Burhop K, Bush S, Catarello J, Chuang H, Downing C, Estep T, Loewen A, McClure K, McDaniel A, Michalek E, Mozier N, Rohn K, Spicuzza J, Zieske P, and Zimmerman G. Preparation and characterization of diaspirin cross-linked hemoglobin solutions for preclinical studies. Biomater Artif Cells Immobilization Biotechnol 20: 423-427, 1992[ISI][Medline].

23.   Nelson, DP, Samsel RW, Wood LDH, and Schumacker PT. Pathological supply dependence of systemic and intestinal O2 uptake during endotoxemia. J Appl Physiol 64: 2410-2419, 1988[Abstract/Free Full Text].

24.   Page, TC, Light WR, McKay CB, and Hellums JD. Oxygen transport by erythrocyte/hemoglobin solution mixtures in an in vitro capillary as a model of hemoglobin-based oxygen carrier performance. Microvasc Res 55: 54-64, 1998[ISI][Medline].

25.   Piper, RD, Li F-Y, Myers ML, and Sibbald WJ. Structure-function relationships in the septic rat heart. Am J Respir Crit Care Med 156: 1473-1482, 1997[Abstract/Free Full Text].

26.   Samsel, RW, and Schumacker PT. Determination of the critical O2 delivery from experimental data: sensitivity to error. J Appl Physiol 64: 2074-2082, 1988[Abstract/Free Full Text].

27.   Schumacker, PT, and Samsel RW. Oxygen delivery and uptake by peripheral tissues: physiology and pathophysiology. Crit Care Clin 5: 255-269, 1989[ISI][Medline].

28.   Sharma, AC, Singh G, and Gulati A. Role of NO mechanism in cardiovascular effects of diaspirin cross-linked hemoglobin in anesthetized rats. Am J Physiol Heart Circ Physiol 269: H1379-H1388, 1995[Abstract/Free Full Text].

29.   Sibbald, WJ, and Sielenkämper AW. Blood substitutes---effects on the microcirculation. In: Tissue Oxygenation in Acute Medicine, edited by Sibbald WJ, Messmer K, and Fink MP.. New York: Springer, 1998, p. 318-331.

30.   Sielenkämper, AW, Chin-Yee IH, Martin CM, and Sibbald WJ. Diaspirin crosslinked hemoglobin improves systemic oxygen uptake in oxygen supply-dependent septic rats. Am J Respir Crit Care Med 156: 1066-1072, 1997[Abstract/Free Full Text].

31.   Sielenkämper, AW, Eichelbrönner O, Martin CM, Madorin SW, Chin-Yee IH, and Sibbald WJ. Diaspirin crosslinked hemoglobin improves mucosal perfusion in the ileum of septic rats. Crit Care Med 28: 782-787, 2000[ISI][Medline].

32.   Standl, T, Horn P, Wilhelm S, Greim C, Freitag M, Sputtek A, Jacobs E, and Schulte am Esch J. Bovine haemoglobin is more potent than autologous red blood cells in restoring muscular tissue oxygenation after profound isovolemic haemodilution in dogs. Can J Anesth 43: 714-723, 1997[Abstract/Free Full Text].

33.   Task Force of the American College of Critical Care Medicine. Practice parameters for hemodynamic support of sepsis in adult patients in sepsis. Crit Care Med 27: 639-660, 1999[ISI][Medline].

34.   Unno, N, Wang H, Menconi MJ, Tytgat SH, Larkin V, Smith M, Morin MJ, Chavez A, Hodin RA, and Fink MP. Inhibition of inducible nitric oxide synthase ameliorates lipopolysaccharide-induced gut mucosal barrier dysfunction in rats. Gastroenterology 113: 1246-1257, 1997[ISI][Medline].

35.   Vincent, JL, Roman A, De Backer D, and Kahn RJ. Oxygen uptake/supply-dependency: effects of short-term dobutamine infusion. Am Rev Respir Dis 142: 2-8, 1990[ISI][Medline].

36.   Walley, KR. Heterogeneity of oxygen delivery impairs oxygen extraction by peripheral tissues: theory. J Appl Physiol 81: 885-894, 1996[Abstract/Free Full Text].

37.   Walley, KR. Hypoxic hypoxia. In: Tissue Oxygenation in Acute Medicine, edited by Sibbald WJ, Messmer K, and Fink MP.. New York: Springer, 1998, p. 81-97.

38.   Welty-Wolf, KE, Simonson SG, Huang YT, Fracica PJ, Patterson JW, and Piantadosi CA. Ultrastructural changes in skeletal muscle mitochondria in Gram-negative sepsis. Shock 5: 378-384, 1996[ISI][Medline].

39.   Woodson, RD. Physiological significance of oxygen dissociation curve shifts. Crit Care Med 7: 368-373, 1979[ISI][Medline].

40.   Zhang, H, Smail N, Cabral A, Rogiers P, and Vincent JL. Effects of norepinephrine on regional blood flow and oxygen extraction capabilities during endotoxic shock. Am J Respir Crit Care Med 155: 1965-1971, 1997[Abstract].

Am J Physiol Heart Circ Physiol 279(4):H1922-H1930
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Anesth. Analg.Home page
G. M. T. Hare, K. M. Hum, S. Y. Kim, A. Barr, A. J. Baker, and C. D. Mazer
Increased Cerebral Tissue Oxygen Tension After Extensive Hemodilution with a Hemoglobin-Based Oxygen Carrier
Anesth. Analg., August 1, 2004; 99(2): 528 - 535.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
Y. Morita, I. Chin-Yee, P. Yu, W. J. Sibbald, and C. M. Martin
Critical Oxygen Delivery in Conscious Septic Rats under Stagnant or Anemic Hypoxia
Am. J. Respir. Crit. Care Med., March 15, 2003; 167(6): 868 - 872.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
O. Eichelbronner, M. D'Almeida, A. Sielenkamper, W. J. Sibbald, and I. H. Chin-Yee
Increasing P50 does not improve DO2CRIT or systemic VO2 in severe anemia
Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H92 - H101.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (16)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sielenkämper, A. W.
Right arrow Articles by Sibbald, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sielenkämper, A. W.