AJP - Regu Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 289: R478-R485, 2005. First published April 21, 2005; doi:10.1152/ajpregu.00081.2005
0363-6119/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/2/R478    most recent
00081.2005v1
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 (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tao, W.
Right arrow Articles by Sherwood, E. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tao, W.
Right arrow Articles by Sherwood, E. R.

NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Cardiovascular dysfunction caused by cecal ligation and puncture is attenuated in CD8 knockout mice treated with anti-asialoGM1

Weike Tao,4 Victor T. Enoh,1 Cheng Y. Lin,1 William E. Johnston,4 Peng Li,4 and Edward R. Sherwood1,2,3

Departments of 1Anesthesiology and 2Microbiology and Immunology, The University of Texas Medical Branch and 3The Shriners Hospital for Children, Galveston; 4Department of Anesthesiology and Pain Management, The University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 4 February 2005 ; accepted in final form 20 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study was designed to assess hemodynamics and myocardial function at 18 h after injury caused by cecal ligation and puncture (CLP) in CD8-knockout mice treated with anti-asialoGM1 (CD8KO/{alpha}AsGM1 mice). Arterial pressure was measured by carotid artery cannulation, and left ventricular pressure-volume measurements were obtained by use of a 1.4-Fr conductance catheter. Blood acid-base balance and indexes of hepatic, renal, and pulmonary injury were also measured. CD8KO/{alpha}AsGM1 mice exhibited higher mean arterial pressure and increased systemic vascular resistance compared with wild-type mice. Cardiac output was significantly decreased in wild-type, but not CD8KO/{alpha}AsGM1, mice compared with sham controls. Myocardial function was better preserved in CD8KO/{alpha}AsGM1 mice as indicated by less impairment of left ventricular pressure development over time, time varying maximum elastance, end-systolic pressure-volume relationship, and preload recruitable stroke work. The impairment in myocardial function was associated with induction of proinflammatory cytokine mRNAs in the hearts of wild-type mice. The hemodynamic derangements in wild-type mice were coupled with significant metabolic acidosis and elevated serum creatinine levels. Overall, this study shows that cardiovascular collapse and shock characterized by hypotension, myocardial depression, low systemic vascular resistance, and metabolic acidosis occurs after CLP in wild-type mice but is attenuated in CD8KO/{alpha}AsGM1 mice. These observations likely explain, in part, the previously observed survival advantage of CD8KO/{alpha}AsGM1 mice following CLP.

blood pressure; vascular resistance; cardiac output; left ventricular function; perfusion

{beta}2-MICROGLOBULIN KNOCKOUT mice treated with anti-asialoGM1 ({beta}2M/{alpha}AsGM1 mice) exhibit improved survival after cecal ligation and puncture (CLP) compared with wild-type mice (19). This survival benefit is associated with less hypothermia, decreased metabolic acidosis, attenuated systemic inflammation, and preserved cardiovascular function (19, 23). However, {beta}2MKO/{alpha}AsGM1 mice have multiple immunological defects including an absence of CD8+ T, natural killer (NK), and natural killer T cells as well as deficient expression of the class I major histocompatability complex and CD1 molecules (2, 15, 17). One of our goals has been to determine which of these immunological alterations confers resistance to CLP-induced injury. Our recent studies indicate that depletion of CD8+ T and NK cells is a major factor in this regard. Adoptive transfer of CD8+ T and NK cells into {beta}2MKO/{alpha}AsGM1 mice will reestablish CLP-induced mortality (19). More recently, we showed that mice specifically depleted of CD8+ T and NK cells exhibit improved survival and decreased systemic inflammation after CLP compared with wild-type controls (18). The survival benefit observed in CD8+ T and NK cell-deficient mice was comparable to that observed in {beta}2MKO/{alpha}AsGM1 mice. However, the physiological factors associated with improved survival in mice specifically depleted of CD8+ T and NK cells remain to be determined.

Hemodynamic alterations and cardiac contractile dysfunction are key manifestations of injury caused by CLP. Our laboratory (22) reported studies on hemodynamic function in mice after CLP and showed that wild-type mice exhibit significant hypotension associated with myocardial depression and decreased systemic vascular resistance. In a subsequent study (23), our laboratory showed that {beta}2M/{alpha}AsGM1 mice exhibit improved myocardial function, increased systemic vascular resistance, and less hypotension than wild-type mice after CLP. On the basis of these observations, we hypothesized that mice specifically depleted of CD8+ T and NK cells would be resistant to cardiovascular dysfunction after CLP. We chose to examine mice depleted of both CD8+ T and NK cells because our previous studies have shown that depletion of NK cells alone does not improve survival after CLP and that isolated CD8+ T cell depletion provides only marginal benefit (18). Removal of both CD8+ T and NK cells causes decreased inflammation and improved survival that is far beyond that observed with depletion of either cell population alone. To test our hypothesis, we utilized the conductance catheter model of hemodynamic monitoring to assess cardiovascular function and myocardial contractility in CD8+ T and NK cell-depleted mice after CLP. In addition, CLP-induced myocardial inflammation, acid-base balance, and indexes of kidney, liver, and lung injury were measured.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Female, 6- to 8-wk-old C57BL/6J and CD8 knockout (CD8KO, strain B6.129S2-Cd8atm1Mac) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). NK cells were depleted by treatment of mice with anti-asialoGM1 (50 µg ip; Cedarlane Laboratories, Hornby, Ontario, Canada) 24 h before CLP. Treatment of mice with antibody against the surface glycolipid asialoGM1 causes targeted depletion of NK cells (19, 24). All studies were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and the University of Texas Southwestern Medical Center in Dallas. All studies conformed to National Institutes of Health Guidelines for the Care and Use of Experimental Animals.

CLP. Mice were anesthetized with 2% isoflurane in oxygen via facemask. A 1- to 2-cm midline incision was made through the abdominal wall; the cecum was identified and ligated with a 3-0 silk tie 1 cm from the tip. Care was taken not to cause bowel obstruction. A single puncture of the cecal wall was performed with a 20-gauge needle. The cecum was lightly squeezed to express a small amount of stool from the puncture site to ensure complete perforation. The cecum was returned to the abdominal cavity, and the incision was closed with a continuous suture. Mice received 2 ml of lactated Ringer's solution subcutaneously in the dorsal area for fluid resuscitation. Sham mice underwent anesthesia, laparotomy, and wound closure but were not exposed to the CLP procedure. Sham procedures were performed on both wild-type and CD8KO/{alpha}AsGM1 mice. Hemodynamic measurements were performed at 18 h after sham or CLP procedures in wild-type and CD8KO/{alpha}AsGM1 mice. This time point was chosen because wild-type mice exhibit significant inactivity and physical evidence of CLP-induced morbidity but do not exhibit mortality until 24–30 h after CLP.

Left ventricular pressure-volume and systemic blood pressure measurements. Mice were anesthetized with 2% inhaled isoflurane in oxygen as described above. Septic mice typically required a lower concentration of isoflurane compared with sham; thus adjustment was made on the basis of the overall appearance and responsiveness of mice during the surgical preparation, but all data were collected at a standardized inhaled isoflurane concentration of 1.5%. A temperature probe (YSI 400, Level 1, Rockland, MA) was inserted into the rectum for initial temperature measurement by a digital thermometer (TM 2000D, RSP, Irvine, CA) as soon as mice were anesthetized. Mice were then placed on the procedure bench with heating pads (T/Pump, Gaymar, Orchard Park, NY) and lamps to maintain the body temperature between 36 and 37.5°C.

The surgical procedure was performed under an operating microscope (Olympus SZ40, Leeds Instruments, Irving, TX). A small incision was made in the anterior neck to expose the trachea and the left carotid artery. The trachea was incised and intubated with an endotracheal tube modified from a 20-gauge intravenous catheter. The animal was then placed on positive-pressure ventilation using a rodent ventilator (model 683, Harvard, South Natick, MA) with a tidal volume of 400–450 µl and rate of 80–95 breaths per minute. These values were adjusted on the basis of the animal's carbon dioxide production guided by expired gas capnometry (Capnomac Ultima, Tewksbury, MA). The left carotid artery was exposed and cannulated with stretched PE-10 tubing and transduced to an analog pressure module (MP-100, Biopac Systems, Goleta, CA). The analog signal of the mean arterial pressure was interfaced with the data-acquisition system described below.

A clamshell incision was made over the anterior chest wall 2 mm above the level of the xyphoid process. The chest cavity was entered with small mosquito clamps that were then applied across the sternum. The sternum was transected with an electrocautery system (Valleylab, Force 2, Boulder, CO) and the mediastinum was exposed. A 4-0 silk tie was passed around the inferior vena cava (IVC) just above the diaphragm. The pericardium was excised at the left ventricular apex. The apex was then punctured with a 27-gauge needle occluded with bone wax. A 1.4-Fr catheter with pressure and conductance sensors (SPR 839, Millar Instruments, Houston, TX) was passed through the puncture along the longitudinal axis of the left ventricle. Left ventricular volume was estimated by the impedance-conductance technique. The catheter's angle and position were adjusted to obtain optimized flow-volume curves. Specifically, measurements were made at the position from which the maximum volume measurement was obtained.

The conductance catheter was interfaced with a pressure-volume analog signal amplifier (Aria 1, Millar Instruments). Real-time conductance and mean arterial pressure data were collected with an analog-to-digital converter (Power Lab 4SP, ADInstruments, Castle Hill, Australia). All variables were displayed and recorded (Chart ver. 4.12, ADInstruments). Initial pressure-volume curves were assessed. To standardize the preload, 50–100 µl of normal saline were slowly given via the arterial line to reach a left ventricular end-diastolic volume (LVEDV) between 15 and 18 relative volume units (RVU), which corresponded to 40–50 µl. The arbitrary RVU number was converted to true volume by using calibration wells with known diameters of 2–6 mm (Millar Instruments) containing heparinized blood from mice of each group heated to 37°C. Volume measurements were performed with blood from three mice in each experimental group. No significant differences in RVU measurements were observed between groups at any of the well diameters. These determinations were made to ensure that differences in blood resistivity did not exist between groups. The target range of the LVEDV of 40–50 µl was chosen as the end point of resuscitation and was used as the standardized condition for hemodynamic measurements because it was found to be typical of healthy animals undergoing the same procedure with minimal or no blood loss in our pilot experiments.

Left ventricular pressure and volume were measured first at the steady state without IVC occlusion. Heart rate, stroke volume, and cardiac output, as well as pressure development during isovolumic contraction (dp/dtmax) and relaxation (dp/dtmin), were also obtained. After the steady-state measurements were performed, IVC occlusion was performed by gently lifting the tie around the IVC while briefly suspending mechanical ventilation. Left ventricular end-systolic pressure-volume relationship (ESPVR) was obtained for each individual animal at the varying preload status produced by IVC occlusion. Time-varying maximum elastance (Emax) was obtained from the series of pressure-volume relationship regression curves at various preloads. Likewise, preload-recruitable stroke work (PRSW) data were generated with the varying LVEDVs. The slopes of the curves for all animals in the group were numerically averaged. To eliminate parallel conductance and resultant contribution of volume from the myocardium, a calibration with 10 µl of 15% hypertonic saline was conducted as described by Yang et al. (25).

RNAse protection assay. Hearts were harvested and flash frozen in liquid nitrogen. Samples were stored at –80°C until used. Total RNA was isolated using Tri-Reagent (Molecular Research Center, Cincinnati, OH). The RNAse protection assay was performed using the Riboquant system (B-D Pharmingen) per the manufacturer's instructions. Briefly, radiolabeled RNA probes were synthesized from DNA template sets by using T7 RNA polymerase, [32P]UTP, and pooled nonradiolabeled nucleotides. Isolated total RNAs (20 µg/sample) were hybridized with the purified riboprobes and subjected to RNAse digestion. Protected RNA species were separated on 5% polyacrylamide sequencing gels by use of 0.5x Tris-borate-EDTA running buffer. Gels were run at 50-W constant power for 70 min and dried under vacuum, and the protected fragments were visualized by use of autoradiography.

Assessment of hepatic, renal, and pulmonary injury. Lung injury was determined by measuring wet-to-dry weight and PO2-to-inspired oxygen fraction (FIO2) ratios. Wet-to-dry weight ratios were determined by harvesting lung tissue from euthanized mice. All extraneous tissue was removed; lungs were blotted dry and weighed. Lungs were then desiccated by heating (55°C) in a drying oven for 5 days and weighed. The wet-to-dry weight ratio was calculated as an index of increased lung water. PO2-to-FIO2 ratios were calculated from arterial PO2 measurements obtained from mice breathing 100% oxygen. Blood was harvested by use of heparinized syringes, and blood gas measurements were performed using iStat cartridges (iStat, East Windsor, NJ). Plasma alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and blood urea nitrogen (BUN)/creatinine levels were measured as indexes of acute liver and renal injuries, respectively. Blood was obtained by carotid artery laceration. ALT, AST, BUN, and creatinine levels were measured in the clinical chemistry laboratory at the Shriners Burns Hospital, Galveston.

Statistics. Data are expressed as means ± SE. Calculation of dp/dtmax, dp/dtmin, Emax, ESPVR, and PRSW were performed by use of pressure-volume analysis software (PVAN version 3.1, Millar Instruments). Comparisons among groups were performed by one-way analysis of variance followed by a post hoc Tukey's or Dunnett's test. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hemodynamic response of CD8KO/{alpha}AsGM1 mice after CLP. Hemodynamic measurements were made after placement of the left ventricular conductance catheter and resuscitation of mice to LVEDVs of 40–50 µl (Fig. 1). Wild-type mice had significantly lower mean arterial pressure despite adequate fluid resuscitation compared with sham mice at 18 h after CLP. Mean arterial blood pressure was not significantly different between CD8KO/{alpha}AsGM1 and sham mice and was significantly higher in CD8KO/{alpha}AsGM1 mice compared with wild-type mice (Fig. 1). Heart rate and stroke volume were not significantly different between sham mice and wild-type or CD8KO/{alpha}AsGM1 mice (Fig. 1). Cardiac output was significantly decreased in wild-type mice at 18 h after CLP compared with sham mice but was not significantly different than CD8KO/{alpha}AsGM1 mice. Systemic vascular resistance was significantly decreased in wild-type mice compared with sham and CD8KO/{alpha}AsGM1 mice (Fig. 1). CD8KO/{alpha}AsGM1 mice did not exhibit a significant change in systemic vascular resistance after CLP compared with sham mice.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 1. Systemic hemodynamics in mice after cecal ligation and puncture (CLP). Wild-type mice and CD8-knockout mice treated with anti-asialoGM1 (CD8KO/{alpha}AsGM1 mice) underwent tracheal intubation, carotid artery cannulation, and left ventricular conductance catheter placement 18 h after sham or CLP procedures. Mice were resuscitated to left ventricular end-diastolic volumes of 40–50 µl before measurement of hemodynamic variables. *P < 0.05 compared with sham mice. {blacklozenge}P < 0.05 compared with wild-type mice. Values are means ± SE; n = 5–16 mice per group. SVR, systemic vascular resistance.

 
Cardiac contractile function in CD8KO/{alpha}AsGM1 mice after CLP. Cardiac contractile function was determined in sham and post-CLP mice both during the steady state and after transient occlusion of the IVC. Assessment of pressure development over time during contraction and relaxation (dp/dtmax and dp/dtmin) of the left ventricle was initially measured during the steady state (Fig. 2). Wild-type mice showed significant decreases in dp/dtmax and dp/dtmin at 18 h after CLP compared with sham mice (Fig. 2). Comparison of sham and CD8KO/{alpha}AsGM1 mice after CLP did not reveal significant differences in dp/dtmax and dp/dtmin, but these parameters were significantly improved in CD8KO/{alpha}AsGM1 mice compared with wild-type mice at 18 h after CLP. Because dp/dtmax is a relatively preload-sensitive marker of contractility, dp/dtmax was corrected for LVEDV (Fig. 2). Both wild-type and CD8KO/{alpha}AsGM1 mice exhibited significant decreases in (dp/dtmax/LVEDV) at 18 h after CLP compared with sham mice. However, dp/dtmax/LVEDV determinations were significantly higher in CD8KO/{alpha}AsGM1 mice compared with wild-type mice (Fig. 2).



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2. Changes in pressure development during isovolumic contraction (dp/dtmax) and relaxation (dp/dtmin) after CLP. Mice underwent sham or CLP procedures. The left ventricle was cannulated with a 1.4-Fr conductance catheter, and left ventricular pressure and volume measurements were performed. Top: CD8KO/{alpha}AsGM1 mice exhibit improved dp/dtmax and dp/dtmin compared with wild-type mice after CLP. These variables were determined in sham and post-CLP mice in the steady state. Bottom: relationship of dp/dtmax to left ventricular end-diastolic volume (EDV) in sham and septic mice during brief occlusion of the inferior vena cava. *P < 0.05 compared with sham. {blacklozenge}P < 0.05 compared with wild-type mice. Values are means ± SE; n = 5–16 mice per group.

 
Load-insensitive indicators of left ventricular contractility such as the ESPVR, Emax, and PRSW were determined by measuring the left ventricular pressure-volume relationship during transient IVC occlusion (Fig. 3). ESPVR and Emax were significantly lower in wild-type and CD8KO/{alpha}AsGM1 mice after CLP compared with the sham group (Fig. 3). CD8KO/{alpha}AsGM1 mice exhibited a significantly higher ESPVR and Emax at 18 h after CLP compared with wild-type mice. PRSW was significantly lower in wild-type mice after CLP compared with sham controls (Fig. 3). PRSW in CD8KO/{alpha}AsGM1 mice was not significantly different compared with sham mice but was significantly higher than wild-type mice after CLP (Fig. 3).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. CD8KO/{alpha}AsGM1 mice exhibit improved myocardial contractility compared with wild-type mice after CLP. Left ventricular pressures and volumes were measured in mice using a 1.4-Fr conductance catheter during occlusion of the inferior cava at 18 h after CLP. The end-systolic pressure-volume relationship (ESPVR), maximal elastance (Emax), and preload-recruitable stroke work (PRSW) were determined. *P < 0.05 compared with sham. {blacklozenge}P < 0.05 compared with wild-type mice. Values are means ± SE; n = 5–16 mice per group.

 
Cardiac proinflammatory cytokine expression in CD8KO/{alpha}AsGM1 mice after CLP. Proinflammatory cytokines have been shown to cause myocardial dysfunction in models of trauma and systemic injury. In these studies, expression of mRNAs for several proinflammatory cytokines and chemokines were measured in wild-type and CD8KO/{alpha}AsGM1 mice at 18 h after CLP (Fig. 4). Heart tissue from wild-type mice expressed mRNAs for the proinflammatory cytokines IL-1{alpha}, IL-1{beta}, IL-6, and TNF-{alpha}. In addition, expression of MIP-2 mRNA, the mouse homolog of the human chemokine IL-8, was observed in the hearts of wild-type mice at 18 h after CLP. Expression of all of these cytokines and chemokines was markedly decreased in the hearts of CD8KO/{alpha}AsGM1 mice compared with wild-type mice (Fig. 4).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4. Heart proinflammatory cytokine and chemokine mRNA expression is attenuated in CD8KO/{alpha}AsGM1 mice after CLP. Heart tissue was harvested from mice at 18 h after CLP. Cytokine and chemokine mRNA expression was determined by RNAse protection assay. The different panels represent results obtained with different riboprobe templates containing probes for the specific cytokines and chemokines shown. Results are typical of those obtained in at least 3 different experiments using each riboprobe template set. L32 served as a loading control.

 
Acid-base balance and organ injury in wild-type and CD8KO/{alpha}AsGM1 mice after CLP. Metabolic acidosis is a common ramification of tissue hypoperfusion and shock. Because of the altered hemodynamics observed in mice after CLP, studies were undertaken to assess acid-base balance in mice at 18 h after CLP (Fig. 5). Wild-type mice exhibited significant acidosis that was associated with decreased blood bicarbonate levels and increased base deficit. These parameters were not significantly altered in CD8KO/{alpha}AsGM1 mice compared with sham mice and were significantly improved in CD8KO/{alpha}AsGM1 mice compared with wild-type mice after CLP (Fig. 5). Plasma PCO2 concentrations were not significantly different between groups.



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 5. Acid-base balance in wild-type and CD8KO/{alpha}AsGM1 mice after CLP. Arterial blood gases were analyzed at 18 h after CLP. *Significantly (P < 0.05) different from sham. {blacklozenge}Significantly (P < 0.05) different from wild-type (WT) mice. Values are means ± SE; n = 5–7 mice per group.

 
Indexes of specific organ injury were also assessed in mice at 18 h after CLP (Fig. 6). The plasma creatinine concentration was less than 0.1 mg/dl in sham and CD8KO/{alpha}AsGM1 mice but was significantly increased in wild-type mice. BUN, AST, and ALT levels were increased in both wild-type and CD8KO/{alpha}AsGM1 mice after CLP compared with sham controls but comparison of wild-type and CD8KO/{alpha}AsGM1 mice did not show significant differences (Fig. 6). Lung wet-to-dry weight and PO2-to-FIO2 ratios were not significantly different between groups (Fig. 6).



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 6. Markers of organ injury in mice at 18 h after CLP. Plasma was harvested for measurement of creatinine, blood urea nitrogen (BUN), and liver enzymes. Blood PO2 was determined by blood-gas analysis in mice breathing 100% oxygen. Lungs were harvested for determination of wet-to-dry weight ratios as described in MATERIALS AND METHODS. *Significantly (P < 0.05) different from sham. {blacklozenge}Significantly (P < 0.05) different from wild-type mice. Values are means ± SE; n = 5–7 mice per group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; FIO2, inspired oxygen fraction.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study shows that CD8KO/{alpha}AsGM1 mice have less hemodynamic dysfunction compared with wild-type mice after CLP. Specifically, CD8KO/{alpha}AsGM1 mice exhibit less myocardial depression, higher systemic vascular resistance, preserved cardiac output, and sustained systemic arterial blood pressure compared with wild-type controls. The hemodynamic alterations observed in wild-type mice after CLP parallel those described in our laboratory's previous reports (22, 23) except that cardiac output was decreased in this study. One explanation for this finding is that wild-type mice may be at the transition from a hyperdynamic state to a state of cardiovascular depression at the 18-h time point. Numerous investigators have shown that a hyperdynamic state will develop during the early stages of sepsis in subjects that have been adequately resuscitated (1, 5). However, subjects in the late stages of septic shock often exhibit myocardial depression with concomitant deterioration of cardiac output and systemic perfusion (4). Our studies were performed at 18 h after CLP. At this time point, wild-type mice exhibit severe hypoactivity, cachexia, and shivering. Mortality occurs at 24–30 h after CLP in this model (18). Therefore, it is likely that the mice utilized in the present study were entering a state of hypodynamic shock. In support of this hypothesis, we previously showed a downward trend in stroke volume and cardiac output from 12 to 18 h after CLP (22). This is in agreement with other studies that have demonstrated a hyperdynamic state during the first 12 h after CLP but development of cardiovascular depression by 20 h post-CLP (12, 26). Another possible explanation for the lower cardiac output is that mice in this study were not adequately resuscitated. This is unlikely because all mice were resuscitated to LVEDVs of 40–50 µl before initiation of cardiovascular measurements. Resuscitation protocols used in this study were identical to those used in our previous studies.

Systemic perfusion appears to be maintained in CD8KO/{alpha}AsGM1 mice, as indicated by the absence of metabolic acidosis. Wild-type mice had severe metabolic acidosis by 18 h after CLP. Several studies have shown metabolic acidosis and elevated base deficit to be good surrogate markers of global tissue hypoperfusion (3, 27). The presence of metabolic acidosis indicates the existence of tissue ischemia and anaerobic metabolism. This may be due to local cecal ischemia after the ligation procedure or systemic hypoperfusion secondary to CLP-induced hypotension and decreased cardiac output. The former explanation seems unlikely because CD8KO/{alpha}AsGM1 mice underwent the same cecal ligation procedure as wild-type mice but did not exhibit metabolic acidosis. Examination of ceca at 18 h after CLP in both groups showed identical levels of necrosis distal to the ligature. Our observation of increased serum creatinine concentration in wild-type, but not CD8KO/{alpha}AsGM1, mice provides further evidence of CLP-induced hypoperfusion and is likely an early sign of renal insufficiency. We were unable to identify differences in indexes of dysfunction of liver or lung when comparing wild-type and CD8KO/{alpha}AsGM1 mice in our model. Specifically, plasma ALT and AST concentrations were elevated in both wild-type and CD8KO/{alpha}AsGM1 mice after CLP. BUN was also increased in both groups. These findings likely represent the intra-abdominal response to acute gastrointestinal injury. CLP did not cause alterations in lung wet-to-dry weight or PO2-to-FIO2 ratios. Therefore, CLP appears to induce a state of cardiovascular instability and shock without evidence of specific injury to liver and lung in wild-type mice, whereas CD8KO/{alpha}AsGM1 mice are resistant to these alterations.

CD8KO/{alpha}AsGM1 mice clearly exhibit an attenuated proinflammatory response after CLP. Our laboratory previously reported that CD8KO/{alpha}AsGM1 mice have much lower levels of proinflammatory cytokines in peritoneal fluid and blood after CLP compared with wild-type mice (18). The present study extends this observation by showing increased expression of mRNAs for several proinflammatory cytokines and chemokines in the hearts of wild-type but not CD8KO/{alpha}AsGM1 mice. The heart has been shown to be a particularly rich source of proinflammatory cytokines during systemic inflammation caused by sepsis, burns, and trauma (6). Proinflammatory cytokines are produced by cardiac myocytes in response to circulating mediators present in serum from burned subjects as well as to bacterial products such as endotoxin (7, 10, 13). Proinflammatory cytokines directly contribute to myocardial depression during systemic inflammation caused by trauma or sepsis (4, 10, 13, 14, 21). The functional roles of TNF-{alpha}, IL-1{beta}, and IL-6 in causing myocardial dysfunction after burn injuries have been extensively described by Horton and colleagues (6, 7). However, it is unclear what relative impact circulating cytokines have compared with those produced locally by cardiac myocytes. Our study does not demonstrate a direct cause-and-effect relationship between elevated myocardial cytokine levels and left ventricular dysfunction. However, a strong correlation is demonstrated, and on the basis of the functional studies of previous investigators, it is likely that the elevated systemic and intracardiac cytokine levels observed in wild-type mice after CLP contribute to the observed myocardial depression. Nevertheless, other factors such as macrophage inhibitory factor, oxygen free radicals, and nitric oxide have also been implicated in inflammation-induced myocardial dysfunction and may play a role in our model (6). Furthermore, it is also possible that the worsening metabolic acidosis seen in wild-type mice contributes to the cardiovascular collapse observed in the post-CLP period. The association between systemic acidosis and cardiovascular dysfunction during sepsis has been recognized (9). In fact, some investigators have shown that metabolic acidosis potentiates the production of proinflammatory cytokines (8). Therefore, the coexistence of metabolic acidosis and systemic inflammation may serve as potentiating factors for cardiovascular dysfunction during sepsis.

Understanding of the mechanisms by which CD8+ T and NK cell depletion confers protection from CLP-induced injury is complicated by the complexity of the CLP model. It is likely that CLP causes both acute infection and gut ischemia. The high bacterial counts in blood, peritoneal fluid, and lung after CLP demonstrate the presence of bacterial dissemination and systemic infection (18). Intestinal ischemia is also likely to contribute significantly to CLP-induced morbidity. However, the ischemic component of CLP has not been widely studied. The blood supply to the rodent cecum arises primarily from the superior mesenteric artery and runs from the base to the distal tip of cecum (11). Therefore, ligation of the cecum at its base will disrupt blood flow to areas distal to the ligation site resulting in tissue ischemia. The functional importance of cecal ischemia in CLP-induced injury is supported by the studies of Singleton and Wischmeyer (20), in which the length of cecum ligated, rather than puncture size, was the major predictor of inflammation and mortality. Other studies have shown that resection of the ischemic cecum will reverse CLP-induced mortality (16). Nevertheless, the functional role of CD8+ T and NK cells in mediating ischemia-induced injury in this model needs to be fully examined.

In conclusion, mortality of wild-type mice after acutely lethal CLP is likely due to cardiovascular collapse and shock. These cardiovascular aberrations are attenuated in CD8KO/{alpha}AsGM1 mice and are associated with diminished intracardiac and systemic inflammation. The underlying mechanisms responsible for these observations remain to be fully defined. However, it is likely that depletion of NK and CD8+ T cells causes attenuation of the CLP-induced proinflammatory response, resulting in preserved hemodynamics and cardiac contractile function with resultant resistance to mortality after CLP.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by research grants from the National Institute of General Medical Sciences (R01 GM66885) and the Shriners of North America (8650 and 8780).


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. R. Sherwood, Dept. of Anesthesiology, The Univ. of Texas Medical Branch, Galveston, TX 77555-0591 (e-mail: ersherwo{at}utmb.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ahmed AJ, Kruse JA, Haupt MT, Chandrasekar PH, and Carlson RW. Hemodynamic responses to gram-positive versus gram-negative sepsis in critically ill patients with and without circulatory shock. Crit Care Med 19: 1520–1525, 1991.[Web of Science][Medline]
  2. Brutkiewicz RR, Bennink JR, Yewdell JW, and Bendelac A. TAP-independent, beta 2-microglobulin-dependent surface expression of functional mouse CD1.1. J Exp Med 182: 1913–1919, 1995.[Abstract/Free Full Text]
  3. Chang MC, Rutherford EJ, and Morris JA Jr. Base deficit as a guide to injury severity and volume resuscitation. J Tenn Med Assoc 86: 59–61, 1993.[Medline]
  4. Court O, Kumar A, Parrillo JE, and Kumar A. Clinical review: Myocardial depression in sepsis and septic shock. Crit Care 6: 500–508, 2002.[CrossRef][Web of Science][Medline]
  5. Hollenberg SM, Dumasius A, Easington C, Colilla SA, Neumann A, and Parrillo JE. Characterization of a hyperdynamic murine model of resuscitated sepsis using echocardiography. Am J Respir Crit Care Med 164: 891–895, 2001.[Abstract/Free Full Text]
  6. Horton JW. Left ventricular contractile dysfunction as a complication of thermal injury. Shock 22: 495–507, 2004.[Web of Science][Medline]
  7. Horton JW, Maass DL, White DJ, Sanders B, and Murphy J. Effects of burn serum on myocardial inflammation and function. Shock 22: 438–445, 2004.[Medline]
  8. Jensen JC, Buresh C, and Norton JA. Lactic acidosis increases tumor necrosis factor secretion and transcription in vitro. J Surg Res 49: 350–353, 1990.[CrossRef][Web of Science][Medline]
  9. Kellum JA, Song M, and Venkataraman R. Effects of hyperchloremic acidosis on arterial pressure and circulating inflammatory molecules in experimental sepsis. Chest 125: 243–248, 2004.[Medline]
  10. Knuefermann P, Nemoto S, Misra A, Nozaki N, Defreitas G, Goyert SM, Carabello BA, Mann DL, and Vallejo JG. CD14-deficient mice are protected against lipopolysaccharide-induced cardiac inflammation and left ventricular dysfunction. Circulation 106: 2608–2615, 2002.[Abstract/Free Full Text]
  11. Leung FW, Su KC, Pique JM, Thiefin G, Passaro E Jr, and Guth PH. Superior mesenteric artery is more important than inferior mesenteric artery in maintaining colonic mucosal perfusion and integrity in rats. Dig Dis Sci 37: 1329–1335, 1992.[Medline]
  12. Merx MW, Liehn EA, Janssens U, Lutticken R, Schrader J, Hanrath P, and Weber C. HMG-CoA reductase inhibitor simvastatin profoundly improves survival in a murine model of sepsis. Circulation 109: 2560–2565, 2004.[Abstract/Free Full Text]
  13. Nemoto S, Vallejo JG, Knuefermann P, Misra A, Defreitas G, Carabello BA, and Mann DL. Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart. Am J Physiol Heart Circ Physiol 282: H2316–H2323, 2002.[Abstract/Free Full Text]
  14. Parker MM. Pathophysiology of cardiovascular dysfunction in septic shock. New Horiz 6: 130–138, 1998.[Web of Science][Medline]
  15. Quinn DG, Zajac AJ, Hioe CE, and Frelinger JA. Virus-specific, CD8+ major histocompatibility complex class I-restricted cytotoxic T lymphocytes in lymphocytic choriomeningitis virus-infected beta2-microglobulin-deficient mice. J Virol 71: 8392–8396, 1997.[Abstract]
  16. Remick DG, Bolgos GR, Siddiqui J, Shin J, and Nemzek JA. Six at six: interleukin-6 measured 6 h after the initiation of sepsis predicts mortality over 3 days. Shock 17: 463–467, 2002.[CrossRef][Web of Science][Medline]
  17. Rolph MS, Raupach B, Kobernick HH, Collins HL, Perarnau B, Lemonnier FA, and Kaufmann SH. MHC class Ia-restricted T cells partially account for beta2-microglobulin-dependent resistance to Mycobacterium tuberculosis. Eur J Immunol 31: 1944–1949, 2001.[CrossRef][Web of Science][Medline]
  18. Sherwood ER, Enoh VT, Murphey ED, and Lin CY. Mice depleted of CD8+ T and NK cells are resistant to injury caused by cecal ligation and puncture. Lab Invest 84: 1655–1665, 2004.[CrossRef][Medline]
  19. Sherwood ER, Lin CY, Tao W, Hartmann CA, Dujon JE, French AJ, and Varma TK. Beta 2 microglobulin knockout mice are resistant to lethal intraabdominal sepsis. Am J Respir Crit Care Med 167: 1641–1649, 2003.[Abstract/Free Full Text]
  20. Singleton KD and Wischmeyer PE. Distance of cecum ligated influences mortality, tumor necrosis factor-alpha and interleukin-6 expression following cecal ligation and puncture in the rat. Eur Surg Res 35: 486–491, 2003.[CrossRef][Web of Science][Medline]
  21. Sun M, Dawood F, Wen WH, Chen M, Dixon I, Kirshenbaum LA, and Liu PP. Excessive tumor necrosis factor activation after infarction contributes to susceptibility of myocardial rupture and left ventricular dysfunction. Circulation 110: 3221–3228, 2004.[Abstract/Free Full Text]
  22. Tao W, Deyo DJ, Traber DL, Johnston WE, and Sherwood ER. Hemodynamic and cardiac contractile function during sepsis caused by cecal ligation and puncture in mice. Shock 21: 31–37, 2004.[CrossRef][Web of Science][Medline]
  23. Tao W and Sherwood ER. {beta}2-Microglobulin knockout mice treated with anti-asialoGM1 exhibit improved hemodynamics and cardiac contractile function during acute intra-abdominal sepsis. Am J Physiol Regul Integr Comp Physiol 286: R569–R575, 2004.[Abstract/Free Full Text]
  24. Wang M, Ellison CA, Gartner JG, and HayGlass KT. Natural killer cell depletion fails to influence initial CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses. J Immunol 160: 1098–1105, 1998.[Abstract/Free Full Text]
  25. Yang B, Larson DF, Beischel J, Kelly R, Shi J, and Watson RR. Validation of conductance catheter system for quantification of murine pressure-volume loops. J Invest Surg 14: 341–355, 2001.[CrossRef][Web of Science][Medline]
  26. Zhou M, Arthur AJ, Ba ZF, Chaudry IH, and Wang P. The small intestine plays an important role in upregulating CGRP during sepsis. Am J Physiol Regul Integr Comp Physiol 280: R382–R388, 2001.[Abstract/Free Full Text]
  27. Ziglar MK. Application of base deficit in resuscitation of trauma patients. Int J Trauma Nurs 6: 81–84, 2000.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Ha, F. Hua, D. Grant, Y. Xia, J. Ma, X. Gao, J. Kelley, D. L. Williams, J. Kalbfleisch, I. W. Browder, et al.
Glucan phosphate attenuates cardiac dysfunction and inhibits cardiac MIF expression and apoptosis in septic mice
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1910 - H1918.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
V. T. Enoh, C. D. Fairchild, C. Y. Lin, T. K. Varma, and E. R. Sherwood
Differential effect of imipenem treatment on wild-type and NK cell-deficient CD8 knockout mice during acute intra-abdominal injury
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2006; 290(3): R685 - R693.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
V. T. Enoh, C. Y. Lin, T. K. Varma, and E. R. Sherwood
Differential effect of imipenem treatment on injury caused by cecal ligation and puncture in wild-type and NK cell-deficient {beta}2-microgloblin knockout mice
Am J Physiol Gastrointest Liver Physiol, February 1, 2006; 290(2): G277 - G284.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/2/R478    most recent
00081.2005v1
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 (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tao, W.
Right arrow Articles by Sherwood, E. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tao, W.
Right arrow Articles by Sherwood, E. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.