Introduction

Several investigators have suggested that a depressionin left ventricular contractility is an important causeof circulatory collapse in severe sepsis [1, 2]. Somehave attributed this cardiac dysfunction to a low molec-ular weight substance that is present in septic plasma,possibly a cytokine released into the circulation as partof the inflammatory reaction induced by the infect-ing organism [2±4]. A number of investigators haveadvocated continuous arteriovenous hemofiltration

(CAVH) as a treatment to remove circulating factorsin sepsis [4±6]. In CAVH, the subject's blood is passedthrough a hemofilter to remove inflammatory media-tors in sepsis [4, 5]. In our laboratory, we previouslyprovided further evidence for the existence of a myo-cardial depressant factor in sepsis. Gomez et al. [4]showed that myocardial depression during experimentalsepsis in dogs could be reversed by the removal of acirculating substance of between 10000 and 30000 Dafrom plasma by CAVH in which myocardial depres-sant activity [filterable cardiodepressant substance

S. N. MinkX. LiD. BoseM. GuG. LiuH. JacobsR. B. Light

Early but not delayed continuousarteriovenous hemofiltration improvescardiovascular function in sepsis in dogs

Received: 12 November 1998Final revision received: 6 April 1999Accepted: 23 April 1999

Supported by the Heart and StrokeFoundation of Manitoba. Mai Gu was aFellow of the Medical Research Councilof Canada

S.N. Mink ()) × X. Li × D. Bose ×M. Gu × G. Liu × H. Jacobs × R. B. LightSection of Respiratory Medicine,Departments of Anesthesiology,Pharmacology and Therapeutics,Biochemistry and Molecular Biology,University of Manitoba, Winnipeg,Manitoba R3E-OZ3, Canada

Mailing address:Respiratory Investigation Unit, GF-221700 William Ave., Health Sciences Centre,Winnipeg, Manitoba R3E OZ3, CanadaTel.: + 1 (2 04) 787-2684Fax: + 1 (2 04) 787-48 26email: minksn@cc.umanitoba.ca

Abstract Objective: Continuous ar-teriovenous hemofiltration (CAVH)has been advocated as treatment toremove inflammatory mediatorsand thereby to improve hemody-namic parameters in sepsis. How-ever, the results obtained withCAVH have been inconsistent. In acanine model of bacteremic Pseu-domonas aeruginosa pneumonia, wetested the hypothesis that the timecourse of the institution of CAVHmay be important in obtaining abeneficial treatment effect.Methods: Two protocols were per-formed in phenobarbital-anesthe-tized dogs. In the early hemofiltra-tion study (EHS), CAVH for 3 h wasinitiated 2 h post-pneumonia beforemean arterial pressure (MAP) fell.In the late hemofiltration study(LHS), CAVH for 3 h was initiatedat 5 h post-pneomonia when a de-crease in MAP had already occur-red. Hemodynamic measurementsincluded cardiac output (CO),

stroke volume (SV), and strokework (SW). Myocardial depressantactivity [filterable cardiodepressantsubstance (FCS)] found in plasmawas assessed by bioassay at eachmeasurement interval.Results: In EHS, after 5 h of sepsis,SW, CO, and SV in the hemofilteredpneumonia group were higher ascompared with the nonhemofilteredpneumonia group. In contrast, inLHS, no differences in hemody-namic parameters were found be-tween the two pneumonia groups. Inboth EHS and LHS, plasma FCSactivity was decreased to similar ex-tents by CAVH.Conclusion: These results suggestthe time course of institution ofCAVH may be important in obtain-ing a beneficial treatment effect insepsis.

Key words Cardiac depression ×Myocardial depressant factor × Dog

Intensive Care Med (1999) 25: 733±743Ó Springer-Verlag 1999 EXPERIMENTAL

(FCS)] was detected by a right ventricular trabecularbioassay.

Nevertheless, the reported effects of CAVH on im-proving cardiovascular function in different models ofsepsis have been variable. For instance, in swine modelsof Staphylococcus aureus septicemia, and endotoxicshock, investigators have shown an improvement in car-diovascular function following hemofiltration [6, 7]. Onthe other hand, Freeman et al. [8] found that CAVHdid not improve survival or hemodynamics in a caninemodel of peritoneal sepsis.

Although the variable effects of CAVH may be dueto the different animal models used, it is likely that theseverity of sepsis examined would also be important, asmight the timing of the CAVH intervention. Early insti-tution of CAVH, before hemodynamic abnormalitiesare observed, may result in findings different than thoseobserved when hemodynamic abnormalities are alreadypresent. In a canine model of Pseudomonas aeruginosapneumonia, we have found that the physiological chan-ges produced over a 5-h period are rather predictable[9]. At 2-h post-inoculation, there is a slight fall inmean arterial pressure (MAP) as compared with pre-sepsis values (baseline), while at 5-h post-sepsis, MAPfalls to about two-thirds that of baseline.

In the present study, we examined the effect ofCAVH on cardiovascular function in P.aeruginosa -treat-ed dogs when CAVH was initiated at different timesafter the onset of sepsis. In one group, CAVH was initi-ated at 2-h post-inoculation (early hemofiltration study)before major hemodynamic changes were observed,while in the other group, CAVH was initiated at 5-hpost-sepsis (late hemofiltration study) when substantialpathophysiological changes were already present. In ad-dition, we followed FCS activity over the intervals to de-termine whether a reduction in FCS activity with CAVHtreatment was associated with an improvement in he-modynamics in the early and late studies [4, 10]. Wewere therefore able to determine whether the effect ofCAVH on cardiovascular parameters was dependentupon the time at which treatment was initiated.

Materials and methods

Experimental protocols

This study was approved by the Central Animal Care Committeeat the University of Manitoba. In the early hemodynamic study,two pneumonia groups were examined in which CAVH was initi-ated before major hemodynamic changes were observed. Thesetwo groups were termed pneumonia group with hemofiltrationand pneumonia group without hemofiltration, respectively. In thepneumonia group with hemofiltration (n = 12), after baseline mea-surements were performed (see below), right and left lower lobepneumonia was produced as previously described [9]. A broncho-scope was introduced into the airway, and 0.5 ml/kg of brain±heartinfusion broth mixed with 1010 colony forming units of P. aerugino-

sa /cc was instilled into each of the lower lobes. At 2-h post-inocu-lation, measurements were repeated and CAVH was begun (seedetails below). CAVH was continued for 3 h. Three hours of hemo-filtration was used in this protocol because we previously foundthat 2 to 3 h were necessary to restore contractility toward normalin canine sepsis [4]. A final set of measurements was obtained at5-h post-inoculation (5-h measurements) just after CAVH was dis-continued.

The above results were compared with those obtained in apneumonia group in which CAVH was not performed (pneumoniagroup without hemofiltration; n = 13). In this latter group, pneu-monia was produced immediately after baseline measurementswere obtained. No hemofiltration was performed, but CAVH vas-cular accesses were inserted. Otherwise, the protocol was thesame as that described in the pneumonia group with hemofiltra-tion.

In the early hemofiltration study, two control groups were alsoincluded. In the sham pneumonia group with hemofiltration(n = 4), 0.5 cc/kg of sterile brain±heart infusion broth was instilledduring bronchoscopy. CAVH was initiated at 2-h post-bronchial in-oculation. Measurements were obtained at baseline, 2 h and 5 h. Afinal control group included the sham pneumonia group withouthemofiltration (n = 5). In this latter group, after instillation of ster-ile inoculum was completed, hemofiltration was not performed, al-though wascular accesses for CAVH were inserted. Measurementswere obtained at identical intervals as for the other groups in thisstudy.

A second series of experiments was performed to examine theeffect of late CAVH on cardiovascular function in this model. Inthe late hemofiltration study, the groups were comparable to thoseused in the early hemofiltration study. These groups included apneumonia group with hemofiltration (n = 13), pneumonia groupwithout hemofiltration (n = 13), sham pneumonia group with he-mofiltration (n = 4), and sham pneumonia group without hemofil-tration (n = 4). In all groups, after baseline measurements were de-termined (see below), pneumonia or sham pneumonia was pro-duced and then measurements were repeated at the 5-h period(5 h). The animals were then randomized into CAVH or non-CAVH groups. A final full set of study measurements was obtainedat 8 h.

Animal preparation and hemodynamic measurements

On the day of the study, healthy mongrel dogs (20 to 30 kg) werebrought to the laboratory and were anesthetized with sodium pen-tobarbital (30 mg/kg intravenously). The trachea was intubatedwith an endotracheal tube and the lungs were mechanically venti-lated (Harvard Apparatus, S. Natick, Mass., USA). The ventilatorsettings were adjusted to maintain initial arterial partial pressureof carbon dioxide (PaCO2) at » 35 mm Hg and pH at » 7.35. The in-spired oxygen concentration was 100%. Supplemental pentobarbi-tal was administered according to the following protocol to main-tain relatively constant plasma concentrations of this anesthesiathroughout the experiment. Based on the kinetics of pentobarbitalelimination, the half-life of pentobarbital is approximately 8 h [11,12]. Additional doses of pentobarbital (approximately one-thirdthe initial anesthetizing dose in the early hemofiltration study,and one-half the initial anesthetizing dose in the late hemofiltra-tion study) were administered in three equally divided doses overthe time course of the experiment. These doses were infused intra-venously (over 10 to 15 min) and were administered at least 45 minbefore measurements were made in a given condition. This meth-od results in relatively stable plasma concentrations as determinedin a previous study [4].

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Vascular catheters were inserted under sterile conditions. Anarterial catheter was placed in the left femoral artery for obtainingblood to monitor arterial blood gases and for continuous recordingof MAP. Another catheter was placed into one jugular vein for theinfusion of fluids and supplemental anesthesia as required. Into theother jugular vein, a thermistor-tipped catheter was advanced intothe pulmonary artery to measure mean pulmonary arterial pres-sure (mPAP) mean pulmonary wedge pressure (mPWP), meanright atrial pressure (mRAP), and cardiac output (CO) by the ther-modilution technique (Columbus Instruments, Ohio, USA). Allthe fluid-filled catheters used for vascular pressure measurementswere connected to transducers (Cobe, Lakewood, Colo., USA)and were referenced to the level of the left atrium. Measurementswere obtained with the animal in the supine position and weretaken at end-expiration during which the ventilator was turned offfor approximately 3 to 5 s. All signals were displayed on an eight-channel recorder (Astra-Med, R. I., USA).

In the early and late hemofiltration studies, all groups were he-parinized (3 ml: 1000 units/ml) whether or not hemofiltration wasperformed, and heparinization was initiated at 2-h and 5-h post-in-oculation, respectively. In those groups in which hemofiltrationwas performed, the right femoral artery and vein were cannulatedwith polyethylene catheters for connection to a hemofilter (Gam-bro, CAVH Kit FH66, Hechingen, Germany) [4, 13]. The hemofil-ter permits all solutes up to a molecular mass of approximately30000 Da to be removed from the plasma. Approximately 2 to 3 lof filtrate was removed from the blood in the hemofiltered groups,and the average rate of removal for all of the dogs was (mean ± SE)33 ± 3 ml/kg per h. During hemofiltration, an arteriovenous flowrate of 2±3 l/min through the hemofilter was maintained with aroller pump (Cobe Perfusion System, Lakewood, Colo., USA).The pump was started slowly so that there would be little changein hemodynamics. The fluid removed was replaced with lactatedRinger's infusion given intravenously to maintain a zero net bal-ance. In the nonhemofiltered groups, arterial and venous accesseswere inserted as above, but hemofiltration was not performed.

In all groups, fluids were administered at an approximate rateof 5 ml/kg per h with the primary endpoint to keep mPWP 8 to10 mmHg throughout the experiment. The animal's core tempera-ture was maintained at 37�C by a warming blanket as needed.Moreover, since progressive metabolic acidosis develops in thispneumonia model [4, 13], the ventilator rate was adjusted as need-ed throughout the experiment to maintain pH » 7.25 to 7.35. Wepreferred to increase the ventilator rate rather than to give sodiumbicarbonate, since there are reports that sodium bicarbonate mayworsen cardiac function in metabolic acidosis [14].

In all groups, each full set of measurements consisted of arterialblood gas parameters and central hemodynamics. Central hemody-namics included mRAP, mPAP, mPWP, MAP, and CO. Calculatedvalues included stroke volume [SV = CO/heart rate (HR)], strokework (SW) = [SV (MAP � mPWP)], systemic vascular resistance[SVR = (MAP � mPWP)/CO], and pulmonary vascular resistance[PVR = (mPAP � mRAP)/CO]. Arterial blood gas analyses in-cluded oxygen tension (PaO2), PaCO2, pH, and hematocrit.

Following completion of all measurements, the animal wassacrificed. The lungs were excised and weighted as an index of thedegree of pneumonia produced.

Assessment of FCS activity in plasma

In a subset of each group of dogs (approximately five in eachgroup), FCS activity in plasma was determined at each measure-ment period by bioassay as previously described [4, 10]. Briefly,mongrel dogs (3 to 10 kg) were anesthetized with pentobarbitone.

Their hearts were removed and placed in ice-cold Krebs-Henseleitsolution (KH). The coronary vessels were flushed with 50 ml ofcold KH. Thin trabeculae (< 1 mm diameter) were obtained fromthe right ventricle and were tied at each end with 6-0 silk thread.Each thin muscle was suspended in a vertical constant temperaturebath (10 ml) containing KH (in mM: NaCl 118; KCl 4.7; CaCl2 2.5;MgSO4 1.2; KH2PO4 1.4; NaHCO3 25; dextrose 11). The solutionwas bubbled with O2 95 % and CO2 5% and maintained at 37 �C.Isometric contractions at optimum length were recorded with aforce transducer (Grass FTO3C) which was connected to a poly-graph (Grass Model 7). The muscle was stimulated electrically viaplatinum bipolar filed electrodes with rectangular pulses (1 msduration) at an intensity of 100% above threshold delivered at in-tervals of 2000 ms.

In each experiment, plasma samples (30 ml) were obtained ateach measurement period. The samples were sequentially passedthrough filters (Millipore, Canada) of different pore sizes (30000and 10000) to isolate the 10000 to 30000 Da fraction. The effectsof pre- vs post-sepsis plasma fraction (0.05 to 1 ml) on isometrictension were determined in separate trabeculae and were used asan index of FCS depressant activity [4, 10].

Supplemental pneumonia studies

Because hemofiltration appeared to be beneficial in the earlypneumonia study, another pneumonia group (n = 5) was studiedin which blood was passed through the hemofilter and circuit, butthe blood was not filtered. The objective for this group was to de-termine whether there may be cardiodepressant substances re-leased during sepsis that could be absorbed by the filter withoutnecessarily being filtered, or whether there was an unknown effectof blood passing through the circuit and hemofilter which causedthe improvement in hemodynamics observed in our model. In thisearly pneumonia sham hemofiltered group, the protocol and pre-paration was exactly the same as that described for the early pneu-monia group with hemofiltration, except that the port on the he-mofilter was capped, so that blood was not filtered.

Statistical methods

The groups were studied in a randomized design (except for thesupplemental pneumonia studies) in which the ratio of pneumoniadogs to the control dogs was approximately 3 to 1. The power ofthe study was sufficient to detect a 25% difference in MAP, SW,SV, and CO between the hemofiltered and nonhemofilteredgroups. When data in a single group were compared, a one-way re-peated measures and Student-Neuman-Keuls' multiple compari-son test were used. When data were compared between groups, ei-ther a one-way nonrepeated measures analysis of variance (ANO-VA) or a two- or three-way ANOVA with the appropriate numberof repeated measures was used (Statpak, Northwest Analytical,Portland, Orl., USA). In the two- and three-way ANOVA, the in-teraction between factors was assessed in which significance in theinteraction term controls for experiment wise error and repeatedmeasurements [15]. If a significant interaction was present, thenthe treatments behaved differently over time. In that case, a Stu-dent-Neuman-Keuls was used to determine at which specific timeperiods a difference among the three treatments occurred. Resultsare recorded as mean ± 1SE.

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Results

Baseline parameters were not different between groupsin the early and late hemofiltration studies. In the earlyhemofiltration study, the changes in MAP measuredover the 5-h study are shown in Fig. 1 (upper panel). Inthe pneumonia group without hemofiltration, MAPwas unchanged at the 2-h period and then significantlydecreased compared with baseline at 5 h. In contrast, inthe pneumonia group with hemofiltration, MAP waswell maintained over the course of the experiment andat the 5-h period MAP was significantly higher than in

the nonhemofiltered pneumonia group. There were nochanges in MAP in the two control groups (Fig. 1 upperpanel).

The changes in SW found in the early hemofiltrationgroup are shown in Fig. 2 (upper panel). In the pneumo-nia group without hemofiltration, SW decreased at 5 has compared with baseline. In the pneumonia groupwith hemofiltration, SW measured at 5 h was signifi-cantly higher as compared with the nonhemofilteredgroup. There were no changes in SW observed in thetwo control groups over the course of the experiment(see Fig. 2, upper panel).

For the early hemofiltration study, the changes ob-served in CO and SV are shown in Table 1. At the 5-hmeasurement, CO and SV measured in the pneumoniagroup with hemofiltration were higher than correspond-ing values obtained in the pneumonia group without he-mofiltration. In Table 1, mPWP was unchanged betweenmeasurement intervals within groups, and mPWP andSVR were not different between pneumonia groups.

736

Fig.1 Mean arterial pressure is shown for the early upper paneland late hemofiltration studies lower panel at the different mea-surement intervals. By one-way repeated measures ANOVA andStudent-Neuman-Keuls * p < 0.05 vs baseline and + p < 0.05 pre-ceding condition. By two-way split-plot ANOVA, ! p < 0.05 hemo-filtered vs nonhemofiltered pneumonia groups

In contrast to the findings in the early hemofiltrationstudy, cardiovascular parameters obtained in the latehemofiltration study were not improved by hemofiltra-tion. In Fig. 1 (lower panel), MAP decreased to a similarextent in both hemofiltered and nonhemofiltered pneu-monia groups at 5 h and then decreased to the same ex-tent at the 8-h period. Moreover, SW showed similarfindings and decreased to comparable values over the8-h period in both pneumonia groups (Fig.2, bottom).Other hemodynamic parameters measured, such asCO, SV, and SVR, were also not different between thetwo pneumonia groups (see Table 2). There were no ef-fects of CAVH on hemodynamics in the sham pneumo-nia groups in either the 5-h or 8-h studies.

The arterial blood gas parameters obtained in theearly and late hemofiltration studies are shown in Ta-bles 3 and 4, respectively. In the pneumonia groups,

PaO2, PaCO2, and arterial pH decreased to comparableextents in the early and late pneumonia groups overthe course of study. The findings were not different inthe hemofiltered and nonhemofiltered groups. Therewere no changes in the latter parameters in the controlgroups in either study.

Lung weights obtained in the two studies are given inTable 5. Consistent with the longer time of sepsis, lungweights in the pneumonia groups in the late hemofiltra-tion study were higher than those measured in the earlyhemofiltration study, but were not different betweenthe hemofiltered and nonhemofiltered groups in eitherstudy.

Unlike the findings observed in the early pneumoniagroup with hemofiltration, in the early pneumoniasham hemofiltered group, hemodynamics significantlydecreased at 5 h when blood was passed through the he-mofilter and circuit, but not filtered. These results areshown in Table 6.

In the early hemofiltration study, the changes ob-served in FCS activity are shown in Fig. 3. In both pneu-

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Fig.2 Stroke work is plotted for the early and late hemofiltrationstudies at the different intervals. See Fig.1 for statistics

monia groups, an increase in FCS activity was observedafter 2 h, which reached statistical significance as com-pared with baseline in the nonfiltered group. In the non-filtered group, FCS activity continued to increase at the5-h period as compared with the 2-h measurement. Incontrast, FCS activity decreased at the 5-h period in thehemofiltered group after 3 h of CAVH. The decrease inFCS activity observed at the 5-h period in the hemofil-tered group was not due to a nonspecific effect of hemo-filtration, since FCS activity remained unchanged in thesham pneumonia group in which hemofiltration wasperformed. There was also no change in depressant ac-tivity in the control group without hemofiltration.

In the late hemofiltration study, the changes in FCSobserved over the course of the study are shown inFig. 4. In both pneumonia groups, FCS increased at 5 has compared with baseline. In the pneumonia groupwithout hemofiltration, FCS activity remained at thishigh level between the 5- and 8-h period. In the pneu-monia group with hemofiltration, treatment caused adecrease in FCS activity as compared with the 5-h peri-od and as compared with the results found in the non-hemofiltered pneumonia group. In the two controlgroups (Fig. 4, lower panel), FCS activity did not changeover the course of the experiment.

In the early pneumonia sham hemofiltered group,there was no removal of FCS when blood was passedthrough the hemofilter and circuit, but not filtered (seeFig. 5).

Discussion

The present study shows that in the early hemofiltrationstudy, CAVH caused an improvement in cardiovascularfunction in this pneumonia model. At the 5-h measure-ment, MAP, SW, CO, and SV determined in the hemo-filtered group were higher than parameters measuredin the nonhemofiltered group. In the nonhemofilteredgroup, whereas cardiac function decreased between 2 hand 5 h, this was not observed in the hemofiltered pneu-monia group. This was not do a nonspecific effect of he-mofiltration, or related to blood passing through the he-mofilter and circuit without being filtered as assessed bythe appropriate control groups in the early hemofiltra-tion study.

In a previous study, we showed that myocardial de-pression in sepsis could be ascribed to a circulating sub-stance of < 30000 Da, and that removal of this sub-stance by CAVH was associated with an improvementin contractility in experimental sepsis [4]. In that study,we also found that depressant activity could be identi-fied in the ultrafiltrate to support the contention thatthis substance was actually filtered and not simply ab-sorbed onto the hemofilter membrane. In a subsequent

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Table 1 Hemodynamic parameters in the early hemofiltrationstudy. Values are mean ± SE (CO cardiac output, SV stroke vol-ume, mPWP mean pulmonary wedge pressure, SVR systemic vas-cular resistance)

Baseline 2 h 5 h

Pneumonia with hemofiltration(n = 12) (n = 5) (n = 12)

CO (l/min) 5.6 ± 0.5 5.0 ± 0.5 5.9 ± 0.5!SV (ml) 44 ± 6 39 ± 7 48 ± 4!mPWP (mm Hg) 9 ± 1.5 11 ± 7 9 ± 3SVR (mmHg/l × min) 29.8 ± 2.6 28.4 ± 4.5 23 ± 2

Pneumonia without hemofiltration(n = 13) (n = 5) (n = 13)

CO (l/min) 5.9 ± 0.5 5.4 ± 1* 4.4 ± 0.4*+SV (ml) 40 ± 3 39 ± 7 30 ± 2*mPWP (mm Hg) 8.5 ± 2 9 ± 1 9 ± 2SVR (mmHg/l × min) 27 ± 0.5 27 ± 3.3 25.5 ± 2

Sham pneumonia with hemofiltration (n = 4)CO (l/min) 6.3 ± 0.4 5.7 ± 0.6 4.3 ± 0.3SV (ml) 49 ± 8 49 ± 7 38 ± 10mPWP (mm Hg) 9.5 ± 1 10 ± 2 10 ± 2SVR (mmHg/l × min) 28 ± 2 28 ± 2 30 ± 1

Sham pneumonia without hemofiltration (n = 5)CO (l/min) 5.1 ± 0.45 5.1 ± 0.5 4.6 ± 0.5SV (ml) 40 ± 7 38 ± 7 37 ± 9mPWP (mm Hg) 8 ± 1 8 ± 1 9.4 ± 1SVR (mmHg/l × min) 32 ± 0.02 35 ± 1 29 ± 1.5

By ANOVA + Student-Neuman-Keuls * p < 0.05 vs baseline;+ p < 0.05 vs 2 h within a group. By two-way ANOVA & Student-Neuman-Keuls ! p < 0.05 hemofiltration vs nonhemofiltrationgroup

Table 2 Hemodynamic parameters in the late hemofiltrationstudy. Values are mean ± SE

Baseline 5 h 8 h

Pneumonia with hemofiltration (n = 13)CO (l/min) 5.7 ± 0.4 4.8 ± 0.5 5.2 ± 0.6SV (ml) 34 ± 3 33 ± 4 35 ± 5mPWP (mm Hg) 8 ± 5 8.5 ± 0.4 8 ± 5SVR (mmHg/l × min) 25 ± 6 21 ± 6* 17 ± 6*

Pneumonia without hemofiltration (n = 13)CO (l/min) 6.1 ± 0.5 4.7 ± 4* 4.9 ± 0.5*SV (ml) 41 ± 2 30 ± 2* 31 ± 3*mPWP (mm Hg) 9 ± 5 10 ± 1 10 ± 0.7SVR (mmHg/l × min) 24 ± 7 18 ± 6* 17 ± 6*

Sham pneumonia with hemofiltration (n = 4)CO (l/min) 5.7 ± 0.5 4.7 ± 4.0 4.0 ± 0.3SV (ml) 38 ± 4 39 ± 10 34 ± 9mPWP (mm Hg) 10 ± 1 12 ± 1 12 ± 1SVR (mmHg/l × min) 23 ± 1 31 ± 2 25 ± 2

Sham pneumonia without hemofiltration (n = 4)CO (l/min) 5.1 ± 2 5.0 ± 0.2 5.3 ± 0.2SV (ml) 36 ± 3 31 ± 2 35 ± 3mPWP (mm Hg) 9 ± 0.5 10 ± 1 10 ± 0.7SVR (mmHg/l × min) 30 ± 2 35 ± 1 29 ± 8

By ANOVA and Student-Neuman-Keuls * p < 0.05 vs baseline;+ p < 0.05 vs 5 h within a group

study, the results showed that FCS was a protein of mo-lecular weight between 10 000 and 30000 Da and that ithad the properties of a protein [10]. Its activity washeat labile and could be eliminated by the enzymatic di-gestion with proteinase K. Our further studies indicatedthat FCS caused a depression in cardiac function by ei-ther inhibition of calcium release from the sarcoplasmicreticulum (SR) or by inhibition of the transfer of cal-cium from the SR uptake site to the SR release site[10]. FCS requires an intact SR membrane system tocause dysfunction, since it does not impair myofilamentcontraction in a skinned trabecular preparation [16].

Moreover, we have also shown [4, 10] that there is adecrease in isometric tension caused by nonseptic plas-ma due to background depressant activity, althoughthis effect is less than that found with FCS (see controlgroups in Fig. 3, 4 and Jha et al. [10]). BDS (back-ground depressant substance) has different chemicaland physical properties from FCS [10]. BDS is heatstable and its activity is not abolished by proteinase K.BDS appears to be of lipid moiety based on partition-

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Table 3 Arterial blood gas parameters in the early hemofiltrationstudy. Values are mean ± SE

Baseline 2 h 5 h

Pneumonia with hemofiltration(n = 12) (n = 5) (n = 12)

PaO2 (mm Hg) 440 ± 20 389 ± 20 298 ± 50*PaCO2 (mm Hg) 36 ± 1 33 ± 1 32 ± 1*pH 7.39 ± 0.01 7.36 ± 0.01 7.32 ± 0.01*Hematocrit (%) 34 ± 8 34 ± 2 28 ± 10*

Pneumonia without hemofiltration(n = 13) (n = 5) (n = 13)

PaO2 (mm Hg) 540 ± 42 433 ± 15 230 ± 115*PaCO2 (mm Hg) 37 ± 1 37 ± 1 33 ± 5pH 7.38 ± 0.01 7.35 ± 0.01 7.33 ± 0.04Hematocrit (%) 38 ± 10 38 ± 10 30 ± 15

Sham pneumonia with hemofiltration (n = 4)PaO2 (mm Hg) 485 ± 6 476 ± 3.5 468 ± 11PaCO2 (mm Hg) 364 ± 2 35 ± 3 34 ± 2pH 7.35 ± 0.02 7.33 ± 0.01 7.36 ± 0.01Hematocrit (%) 38 ± 1 39 ± 3 30 ± 6

Sham pneumonia without hemofiltration (n = 5)PaO2 (mm Hg) 467 ± 8 462 ± 11 475 ± 10PaCO2 (mm Hg) 33 ± 2 34 ± 2 31 ± 0.5pH 7.40 ± 0.01 7.36 ± 0.01 7.37 ± 0.01Hematocrit (%) 34 ± 1 36 ± 2 33 ± 3

By ANOVA and Student-Neuman-Keuls * p < 0.05 vs baseline

Table 4 Arterial blood gas parameters in the late hemofiltrationstudy. Values are mean ± SE

Baseline 5 h 8 h

Pneumonia with hemofiltration (n = 13)PaO2 (mm Hg) 516 ± 10 212 ± 30* 135 ± 30* +PaCO2 (mm Hg) 34 ± 4 32 ± 6 30 ± 4pH 7.4 ± 0.01 7.34 ± 0.06* 7.31 ± 0.01*Hematocrit (%) 37 ± 1 34 ± 8 32 ± 2*

Pneumonia without hemofiltration (n = 13)PaO2 (mm Hg) 523 ± 10 212 ± 140* 101 ± 10* +PaCO2 (mm Hg) 35 ± 1 32 ± 1 33 ± 1pH 7.39 ± 0.01 7.34 ± 0.01* 7.32 ± 0.01*Hematocrit (%) 40 ± 2 36 ± 4 33 ± 3*

Sham pneumonia with hemofiltration (n = 4)PaO2 (mm Hg) 472 ± 6 486 ± 4 466 ± 1PaCO2 (mm Hg) 34 ± 2 31 ± 0.05 29 ± 0.05pH 7.39 ± 0.01 7.37 ± 0.01 7.37 ± 0.01Hematocrit (%) 33 ± 2 35 ± 2 34 ± 2

Sham pneumonia without hemofiltration (n = 4)PaO2 (mm Hg) 463 ± 5 464 ± 8 443 ± 9PaCO2 (mm Hg) 32 ± 1 31 ± 1 30 ± 1pH 7.41 ± 0.01 7.37 ± 0.01 7.38 ± 0.01Hematocrit (%) 35 ± 2 32 ± 2 30 ± 3

By ANOVA and Student-Neuman-Keuls * p < 0.05 vs baseline;+ p < 0.05 vs 5 h

Table 5 Lung weights (g) in the early and late hemofiltration stu-dies. Values are mean ± SE

Early hemofiltration studyHemofiltered pneumonia group 431 ± 20*Nonhemofiltered 403 ± 10*Hemofiltered control group 236 ± 5Nonhemofiltered control group 300 ± 6

Late hemofiltration studyHemofiltered pneumonia group 651 ± 20* +Nonhemofiltered pneumonia group 715 ± 30* +Hemofiltered control group 309 ± 5Nonhemofiltered control group 296 ± 4

By ANOVA and Student-Neuman-Keuls * p < 0.05 vs nonpneu-monia groups within a study; + p < 0.05 late vs early study

Table 6 Variables in the early pneumonia sham-hemofilteredgroup. Values are mean ± SE (n = 8)

Baseline 2 h 5 h

MAP (mmHg) 164 ± 8 146 ± 10 106 ± 9* +!SW (mmHg × ml) 6645 ± 676 6089 ± 109 4529 ± 1160*CO (l/min) 7.0 ± 0.4 6.2 ± 0.8 5.3 ± 1*SV (ml) 42 ± 2 43 ± 6 42 ± 8mPWP (mmHg) 8 ± 2 9 ± 2 8 ± 2PaO2 (mmHg) 555 ± 11 356 ± 20* 205 ± 38*PaCO2 (mmHg) 32 ± 2 34 ± 2 34 ± 2pH 7.4 ± 0.02 7.35 ± 0.01* 7.28 ± 0.01*Hct (%) 38 ± 2 38 ± 3 36 ± 4Lung weight (g) ± ± 490 ± 32

MAP mean arterial pressure, SW stroke work, CO cardiac output,SV stroke volume, mPWP mean pulmonary wedge pressure, Hcthematocrit, by ANOVA + Student-Neuman-Keuls * p < 0.05 vsbaseline; + p < 0.05 vs 2 h. By two-way ANOVA ! p < 0.05 vs earlypneumonia group with hemofiltration

ing experiments of plasma into lipid soluble and non-soluble fractions.

It is important to note that although hemofiltration isassociated with an improvement in cardiac function insepsis, this effect cannot be ascribed to changes in theplasma concentrations of pentobarbital or ionized cal-cium during this treatment. These possibilities were ad-dressed in an earlier study [4]. Plasma concentrationsof pentobarbital and ionized calcium were unchangedto account for the improvement in hemodynamic para-meters observed with CAVH in our initial study [4]. In

another study, we showed that the plasma concentra-tions of pentobarbital that are attained by our anesthe-sia protocol do not cause a depression in left ventricularcontractility in either in vivo or in vitro preparations, al-though HR and MAP under pentobarbital anesthesiaare higher compared to the awake condition [12]. Thus,the findings observed in the early hemofiltration studysupport the contention that CAVH removes a cardio-depressant substance in experimental sepsis.

In the present study, whereas early hemofiltrationwas associated with an improvement in cardiovascularfunction in sepsis, there was no improvement in cardio-vascular function observed in the late hemofiltrationstudy. As shown in Fig. 1, MAP decreased to the sameextent in both hemofiltered and nonhemofiltered pneu-monia groups in the late hemofiltration study. In addi-tion, in Fig. 2, SW was not improved by hemofiltration,and this parameter as well as CO and SV decreased to

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Fig.3 Filterable cardiodepressant activity is shown for the differ-ent groups in the early hemofiltration study. By two-way repeatedmeasures ANOVA * p < 0.05 vs baseline; + p < 0.05 vs 2-h mea-surement. By three-way ANOVA, ! p < 0.05 hemofiltered vs non-hemofiltered pneumonia groups

comparable extents in the hemofiltered and nonhemo-filtered pneumonia groups in the late study.

Nevertheless, despite the lack of hemodynamic im-provement observed with CAVH in the late hemofiltra-tion study, the results shown in Fig. 4 indicate that FCSactivity could still be reduced by hemofiltration. Thepossibilities for the lack of hemodynamic improvementdespite removal of FCS activity from plasma could berelated to a number of factors. Since we previouslyshowed that myocardial depression in sepsis could beacutely reversed by CAVH [4, 13], the binding of theFCS to the myocardial cell must be somewhat revers-ible. However, the time course of events that occursonce FCS is bound to the myocardial cell is not clear.

Reversibility may be harder to achieve as sepsis pro-ceeds along its course. This could be due to greater af-finity of binding of FCS to the myocardial cell per se,or because FCS causes transcription of other cellularmessengers that, in turn, cause myocardial depressionin sepsis. Thus, institution of CAVH late in the courseof sepsis is associated with removal of FCS from theplasma, but not with attenuating the effect of FCS onthe myocardium.

Another possibility for the lack of hemodynamic im-provement in the late hemofiltration study is that a lar-ger amount of FCS may be produced as compared withthe early hemofiltration study. Although FCS activitymeasured at 8 h in the late hemofiltration study (seeFig. 4) was not clearly greater than that observed at 5 hin the early hemofiltration study (see Fig. 3), this findingdoes not exclude the possibility that a larger amount ofFCS was produced in the late hemofiltration study.

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Fig.4 Filterable cardiodepressant activity is shown for the differ-ent groups in the late hemofiltration study; see Fig.3 for statistics

There might be a limit in the degree to which isometrictension could be reduced in our ventricular preparationby FCS. There may be a plateau in the response, suchthat at high concentrations of FCS, any further reduc-tion in isometric tension with increasing concentrationsof FCS might be difficult to detect in this preparation.Accordingly, in the late hemofiltration study, the burdenof cytokines and/or other vasoactive substances mayhave been too great for removal by 3 h of CAVH. Thegreater pneumonia weights would be consistent withthis hypothesis. Thus, a longer period of CAVH couldhave resulted in cardiovascular improvement in thelate pneumonia study.

Finally, it is possible that other myocardial depres-sant substances with various molecular weights becomemore clinically relevant in sepsis as the inflammatorycascade proceeds over the course of infection. Thesesubstances may be less amenable to filtration byCAVH, possibly contributing to the lack of hemody-namic improvement observed in the late pneumoniastudy. Whereas these other questions could be addres-sed in future studies, our results clearly show that for agiven interval of CAVH, hemodynamic recovery wasobserved when hemofiltration was administered earlyin this pneumonia model.

In terms of our methodology, it is important to notethat the CAVH protocol differs in some respects fromCAVH performed in clinical medicine. The use of ablood pump and the achievement of extraordinarilyhigh blood flow rates are not features of the clinical pro-tocol. The flow rates of 2 to 3 l/min used in the currentstudy are an order of magnitude higher than would be

clinically obtained [17, 18]. These procedures wereused to achieve the mean ultrafiltration rate of approxi-mately 30 ml/kg per h, which is the upper limit of stan-dard CAVH rates used clinically for renal replacementtherapy. Once a better understanding of the nature andtime course of production of cardiodepressant substan-ces in sepsis is determined, protocols can be examinedwhich maximize the removal of specific cardiodepres-sant substances in sepsis.

Furthermore, because of the possibility that the im-provement in hemodynamic variables observed in the5-h study was due to absorption of cardiodepressantsubstances by the hemofilter membrane rather than toblood filtration, a supplemental study was performed(i. e., early pneumonia sham hemofiltered group) inwhich blood was passed through the hemofilter and cir-cuit, but not filtered. In contrast to the results found inthe early pneumonia group with hemofiltration, cardio-vascular parameters decreased at 5 h in the early pneu-monia group with sham hemofiltration. Nevertheless, itis possible that membrane absorption of cardiodepres-sant substances may play a role under other conditions.

In the design of this study, we included many controlgroups. We tried to keep supportive therapies such asfluid administration and ventilation similar betweenpneumonia groups in the early and late studies. Hema-tocrits and arterial blood gases were not different be-tween respective pneumonia groups in the 5-h and 8-hstudies to suggest that such factors played a role in theresults observed. We recognize that because of the com-plexity of the experimental design there may have beenunrecognized factors that were not taken into accountin the present study. Furthermore, when pneumoniawas produced, a large inoculum of bacteria was adminis-tered, and sepsis proceeded more quickly than is usuallyobserved in the clinical setting. Thus, our results mustbe cautiously applied to the human condition.

Previous investigators have suggested that hemofil-tration may be useful to improve organ function and sur-vival in sepsis because cytokines and ªtoxinsº thatwould otherwise cause tissue damage could be removedby this technique [4±7]. However, this issue has beencontroversial [5, 19]. Our results suggest that the timecourse of the institution of CAVH may be important indetermining the effectiveness of CAVH. Our results in-dicate that hemofiltration may be beneficial in sepsis ifadministered early rather than waiting until cardiovas-cular collapse has occurred.

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Fig.5 There was no effect of sham hemofiltration on cardio-depressant activity in the pneumonia sham filtered group. 2-h and5-h values were significantly different from those at baseline(* p < 0.05 by two-way ANOVA and Student-Neuman-Keuls)

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