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Meeting Abstracts  |   October 2004
Local Anesthetics Reduce Mortality and Protect against Renal and Hepatic Dysfunction in Murine Septic Peritonitis
Author Affiliations & Notes
  • George Gallos, M.D.
    *
  • Dean R. Jones, M.D.
    *
  • Samih H. Nasr, M.D.
  • Charles W. Emala, M.D.
  • H Thomas Lee, M.D., Ph.D.
    §
  • * Resident, ‡ Associate Professor, § Assistant Professor, Department of Anesthesiology, Columbia University, New York, New York. † Clinical Fellow, Department of Pathology, Columbia University, and Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, New York.
Article Information
Meeting Abstracts   |   October 2004
Local Anesthetics Reduce Mortality and Protect against Renal and Hepatic Dysfunction in Murine Septic Peritonitis
Anesthesiology 10 2004, Vol.101, 902-911. doi:
Anesthesiology 10 2004, Vol.101, 902-911. doi:
THE incidence of sepsis and the associated mortality are increasing. On an annual basis, sepsis afflicts approximately 750,000 patients in the United States and accounts for nearly $16.7 billion in health care costs.1 When sepsis is associated with the development of acute multiorgan dysfunction, morbidity, mortality, and treatment expenditures greatly increase. For example, renal or hepatic dysfunction occurs in approximately 23% of all septic patients and is a significant clinical feature (38.2% and 54.3%, respectively) seen in patients who die from sepsis. Currently, no prophylactic therapy for patients in early sepsis has proven to avert progression to multiorgan dysfunction. Furthermore, no definitive treatment exists for those patients who progress to multiorgan failure.
Although the exact pathogenesis leading to multiorgan dysfunction remains to be elucidated, a complex inflammatory process seems to play a central role in initiating its development. An uncontrolled, hyperinflammatory response and associated uncontrolled, hyperexuberant cytokine production are known to be detrimental to organ function and are proposed to be the main cause precipitating multiorgan dysfunction in early sepsis. The murine cecal ligation and puncture (CLP) model resembles the human sepsis syndrome with respect to cytokine/chemokine generation and the development of fulminant multiorgan failure.2–4 Although various attempts at targeting particular mediators of inflammation have not proved universally successful in human sepsis trials, some studies have suggested a beneficial role from more global antiinflammatory modulation.5 
Local anesthetics have been shown to modulate inflammatory cascades and provide protection from ischemic reperfusion injury in the heart,6,7 lung,8,9 and liver.10 They are capable of exerting antiinflammatory effects on various cell types including monocytes, macrophages, and neutrophils.11 Therefore, we questioned whether infusion of local anesthetics after CLP would attenuate the expression of hyperinflammatory mediators, reduce organ injury and dysfunction, and provide a significant survival effect in this in vivo  murine model of septic peritonitis.
Materials and Methods
Mice
We used male C57BL/6 mice (approximately 20–25g) purchased from Harlan Laboratories (Indianapolis, IN). All animal protocols were approved by the Institutional Animal Care and Use Committee of Columbia University (New York, NY).
Delivery of Saline Vehicle or Local Anesthetics
Mice were anesthetized with intraperitoneal pentobarbital (50 mg/kg or to effect). A mini-osmotic pump (Model #2001; Durect Corporation, Cupertino, CA) was implanted into the subcutaneous space on the back of each animal. All pumps continuously delivered 1 μl/h of 10% lidocaine (∼5.0 mg·kg−1·h−1), 5% lidocaine (∼2.5 mg·kg−1·h−1), 2% bupivacaine (∼1.0 mg·kg−1·h−1), 1% bupivacaine (∼0.5 mg·kg−1·h−1), or saline vehicle for 7-day survival studies. Subsequent functional and molecular studies were then performed with 10% lidocaine and 1% bupivacaine at 24 h after CLP. These doses of local anesthetics were chosen to mimic clinically achieved plasma concentrations in patients receiving epidural local anesthetics for postoperative pain control. To confirm that clinically relevant local anesthetic plasma concentrations were achieved at 24 h with this delivery system, local anesthetic plasma concentrations from mice were confirmed by high performance liquid chromatography measurements at the Nathan Kline Institute (Orangeburg, NY) as described previously (table 1).12 
Table 1. Local Anesthetic Plasma Levels at 24 hours after Cecal Ligation and Puncture 
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Table 1. Local Anesthetic Plasma Levels at 24 hours after Cecal Ligation and Puncture 
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Induction of Sepsis
Six hours after pump implantation (the time interval is required for pump priming), the mice were reanesthetized with intraperitoneal pentobarbital and were allowed to spontaneously breathe room air on an electric heating pad under a warming light. CLP was performed as described by others.13 Briefly, the cecum was isolated through a 1-cm midline incision and the distal (0.5 cm) portion of the cecum below the ileocecal valve (to avoid bowel obstruction) was ligated with a 4–0 silk suture. The cecum was then punctured through and along its antimesenteric border (double puncture) with a 20-gauge needle and a small amount of stool was extruded through the puncture site. After instillation of 0.5 ml normal saline into the peritoneal cavity, the abdomen was closed in two layers. The mice remained on the heating pad until they recovered from the anesthetic. Twenty-four hours after CLP, mice were killed with an overdose of intraperitoneal pentobarbital, and specimens (plasma and kidneys) were collected. The left kidney was used for histologic evaluation and the right kidney was snap frozen in liquid nitrogen for all in vitro  studies described (e.g.  , DNA laddering, semiquantitative reverse transcriptase polymerase chain reaction, and immunoblotting).
Survival Studies
To determine the effect that local anesthetic infusions would have on mortality from CLP-induced septic peritonitis, continuous subcutaneous infusion of lidocaine and bupivacaine was administered to mice for 7 days until the pump reservoir was exhausted. Survival studies were performed with infusions of varying concentrations of lidocaine (5% and 10%) and bupivacaine (1% and 2%) to determine if dose dependency existed. All mice had free access to water and food and were frequently observed by dedicated research personnel to determine 7-day survival statistics. All severely moribund animals were euthanized with an overdose injection of anesthetic in adherence with our animal protocol. To examine if the manner of administration played an important role in survival, a separate small cohort of mice (n = 8) were bolused with lidocaine every 8 h totaling the dose they would have received with the continuous osmotic pump infusion. Specifically, a 0.8 mg subcutaneous lidocaine bolus every 8 h for 24 h was equivalent to an infusion of 10% lidocaine at 1 μl/h over 24 h.
Assessment of Renal and Hepatic Function after Sepsis
Renal function was assessed by measuring plasma creatinine 24 h after CLP by a colorimetric method based on the Jaffe reaction.14 Hepatic function 24 h after CLP was assessed by measuring plasma, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) concentrations using a commercially available colorimetric method (Sigma, St. Louis, MO).
Measurement of Systemic Cytokines by ELISA
Murine plasma tumor necrosis factor (TNF)-α and keratinocyte-derived chemokine (KC) concentrations taken 24 h after CLP were measured using commercially available ELISA kits according to manufacturer instructions (ALPCO, Windham, NH and R&D Systems, Minneapolis, MN, respectively).
Assessment of Renal Inflammation
Renal inflammation 24 h after CLP was determined by measurement of renal cortical myeloperoxidase activity (a marker of leukocyte infiltration) and immunoblotting for intracellular adhesion molecule (ICAM)-1 as described previously.12 In addition, measurements of mRNA encoding for the markers of inflammation (KC, macrophage inflammatory protein [MIP]-2, ICAM-1, monocyte chemoattractant protein [MCP]-1), TNF-α, regulated on activation normal T-cell expressed and secreted, interferon-induced protein 10, and glyceraldehyde 3-phosphate dehydrogenase (control for RNA quantity) in renal cortices with semiquantitative reverse transcriptase polymerase chain reaction were also performed as described previously.12 For each experiment, we also performed semiquantitative reverse transcriptase polymerase chain reaction under conditions yielding linear results for glyceraldehyde 3-phosphate dehydrogenase to confirm equal RNA input. Primers were designed based on published GenBank sequences (National Institutes of Health database, Bethesda, MD) for mice (table 2).
Table 2. Proinflammatory Sense and Anti-sense Primers Based on Published GenBank Sequences for Mice 
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Table 2. Proinflammatory Sense and Anti-sense Primers Based on Published GenBank Sequences for Mice 
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Assessment of Renal Necrosis and Apoptosis
Both necrosis and apoptosis was assessed by histologic evaluation. Renal apoptosis was also evaluated by DNA laddering as well as by in situ  Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling assay Assay (TUNEL).
Histologic Examinations
Explanted kidneys were bisected along the long axis and fixed in 10% formalin solution overnight. After automated dehydration through a graded alcohol series, transverse kidney slices were embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin. Morphological assessment was performed by an experienced renal pathologist (SHN) who was blinded to the treatments that each animal had received. An established grading scale (0–4) of necrotic injury to the proximal tubules was used for the histopathological assessment of damage as outlined by Jablonski et al.  15 For apoptotic assessment the pathologist examined hematoxylin and eosin stained mouse kidney sections under light microscopy for apoptotic bodies. As previously described, the degree of renal tubular apoptosis was quantified by counting the number of apoptotic bodies in proximal tubules in the corticomedullary area of the kidney.16 Twenty-five to fifty tubules were counted per field from each treatment group and at least six fields were examined per section. Results are representative of at least four separate experiments.
Detection of DNA Fragmentation by DNA Laddering
To assess potential differences in DNA laddering (indicative of apoptosis), extracted DNA (Wizard; Promega, Madison, WI) from dissected renal cortices taken at 24 h after CLP were subjected to electrophoresis for 6 h at 70 V in a 2.0% agarose gel in Tris-acetate-EDTA buffer. Gels were stained with ethidium bromide and photographed under ultraviolet illumination. DNA ladder markers (100 bp) were used as a reference for the analysis of internucleosomal DNA fragmentation.
Assessment of Renal Apoptosis by TUNEL Assay
To further evaluate potential differences in renal apoptosis, TUNEL assay was performed on formalin fixed mice kidney sections obtained at 24 h after CLP. Briefly, sections were deparaffinized and rehydrated. In situ  labeling of fragmented DNA was performed with TUNEL (green fluorescence) using a commercial in situ  cell death detection kit (Roche, Indianapolis, IN) according to manufacturer instructions. To visualize the total number of cells in the field, sections were also concomitantly stained with a red fluorescent monomeric cyanine nucleic acid stain (Molecular Probes, Eugene, OR).
Statistical Analysis
One-way analysis of variance was used to compare mean values across multiple treatment groups with a Dunnett post hoc  multiple comparison test. The ordinal values of the Jablonski scale were analyzed by the Kruskal-Wallis nonparametric test with Dunn posttest comparison between groups. Survival statistics were compared with a Kaplan-Meier curve and log-rank test. In all cases, a probability value less than 0.05 was taken to indicate significance.
Reagents
Unless otherwise specified, all reagents were purchased from Sigma (St. Louis, MO). Protein content was determined with the Pierce Chemical (Rockford, IL) bicinchoninic acid protein assay reagent using bovine serum albumin as a standard.
Results
Local Anesthetic Infusion Protects against CLP-induced Mortality
We initially measured the effects of chronic local anesthetic infusion on mortality from CLP-induced septic peritonitis. As demonstrated in figure 1, infusion of lidocaine and bupivacaine significantly protected mice against CLP-induced mortality. Both lidocaine infusion groups (5% and 10%) significantly reduced mortality to 0% (n = 20) and 3% (n = 25), respectively, at 24 h compared with the saline vehicle group (24 h mortal-ity = 49%, n = 31; P  < 0.05). Log-rank analysis demonstrated that a significant level of protection was conferred by lidocaine throughout the study. Similar results were observed in the mice that received continuous subcutaneous infusions of bupivacaine. CLP-induced mortality with 1% and 2% bupivacaine infusion was 7% (n = 22) and 14% (n = 21, respectively) at 24 h, and a significant degree of protection compared with saline vehicle persisted throughout the study (P  < 0.05). The 2% bupivacaine infusion significantly improved survival versus  the 1% bupivacaine infusion (P  < 0.05). However, we chose to use the 10% lidocaine and 1% bupivacaine treatments in the remaining studies because these more accurately reflect clinically relevant doses. We also found the manner of administration had an impact on survival benefit. We observed no benefit in mortality with bolus administration (every 8 h for 24 h) of 10% lidocaine (data not shown).
Fig. 1. Kaplan-Meier survival curves were generated for C57 mice subjected to cecal ligation and double puncture with saline infusion (n = 35), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 5% (n = 20) or 10% (n = 35) lidocaine, and C57 mice subjected to cecal ligation and double puncture treated with infusion of 1% (n = 27) or 2% (n = 21) bupivacaine. Log-rank analysis demonstrated a significant improvement in survival for all local anesthetic infusion groups  versus  the saline vehicle group (  P  < 0.05). Log-rank analysis also demonstrated a significant improvement in survival in the 2% bupivacaine infusion group  versus  the 1% bupivacaine infusion group (  P  < 0.05). No statistical differences in mortality were observed between the lidocaine infusion groups.  BUPIV  = bupivacaine;  LIDO  = lidocaine  .
Fig. 1. Kaplan-Meier survival curves were generated for C57 mice subjected to cecal ligation and double puncture with saline infusion (n = 35), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 5% (n = 20) or 10% (n = 35) lidocaine, and C57 mice subjected to cecal ligation and double puncture treated with infusion of 1% (n = 27) or 2% (n = 21) bupivacaine. Log-rank analysis demonstrated a significant improvement in survival for all local anesthetic infusion groups  versus  the saline vehicle group (  P  < 0.05). Log-rank analysis also demonstrated a significant improvement in survival in the 2% bupivacaine infusion group  versus  the 1% bupivacaine infusion group (  P  < 0.05). No statistical differences in mortality were observed between the lidocaine infusion groups.  BUPIV  = bupivacaine;  LIDO  = lidocaine 
	.
Fig. 1. Kaplan-Meier survival curves were generated for C57 mice subjected to cecal ligation and double puncture with saline infusion (n = 35), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 5% (n = 20) or 10% (n = 35) lidocaine, and C57 mice subjected to cecal ligation and double puncture treated with infusion of 1% (n = 27) or 2% (n = 21) bupivacaine. Log-rank analysis demonstrated a significant improvement in survival for all local anesthetic infusion groups  versus  the saline vehicle group (  P  < 0.05). Log-rank analysis also demonstrated a significant improvement in survival in the 2% bupivacaine infusion group  versus  the 1% bupivacaine infusion group (  P  < 0.05). No statistical differences in mortality were observed between the lidocaine infusion groups.  BUPIV  = bupivacaine;  LIDO  = lidocaine  .
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Local Anesthetic Infusion Significantly Reduced CLP-induced Organ Dysfunction/Injury
The development of organ dysfunction and injury as a complication of sepsis is associated with increased mortality. We examined the degree of renal dysfunction by measuring plasma creatinine and the degree of hepatic dysfunction by measuring ALT and AST 24 h after CLP. In this model of sepsis, plasma creatinine was significantly increased in saline vehicle treated mice (1.2 ± 0.10 mg/dl, n = 13) compared with sham surgery controls (0.50 ± 0.10 mg/dl, n = 8) (table 3). Mice treated with 10% lidocaine or 1% bupivacaine infusion had significantly lower plasma creatinine at 24 h (0.50 ± 0.10 mg/dl, n = 10 and 0.50 ± 0.10 mg/dl, n = 5; P  < 0.001 respectively) compared with mice treated with saline vehicle alone Therefore, local anesthetic infusion alleviates CLP-induced renal injury. Similarly, 24 h after CLP operation AST and ALT concentrations were significantly increased in saline vehicle treated mice (AST, 349 ± 15 U/ml, n = 15 and ALT, 142 ± 10 U/ml, n = 12) compared with sham surgery controls (AST, 91 ± 11 U/ml, n = 8 and ALT, 20 ± 2 U/ml, n = 8; table 3). At 24 h after CLP, the lidocaine and bupivacaine treated groups also demonstrated lower AST (134 ± 13 U/ml, n = 7 and 169 ± 28 U/ml, n = 4 respectively; P  < 0.001) and ALT (78 ± 7 U/ml, n = 7 and 95 ± 23 U/ml, n = 4 respectively; P  < 0.001) compared with mice receiving vehicle alone. This data also demonstrates that local anesthetic infusion ameliorates CLP-induced hepatic injury in mice.
Table 3. Markers of Organ Dysfunction, Markers of Systemic Inflammation, and Markers of Renal Inflammation 24 hours after the induction of sepsis 
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Table 3. Markers of Organ Dysfunction, Markers of Systemic Inflammation, and Markers of Renal Inflammation 24 hours after the induction of sepsis 
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Local Anesthetic Infusion Significantly Attenuated Proinflammatory KC and TNF-α Plasma Concentrations after CLP
TNF-α concentrations were undetectable in most sham surgery control mice (n = 5), indicating that baseline concentrations were less than 18.2 pg/ml (the sensitivity of our ELISA). In contrast, plasma TNF-α concentrations in mice undergoing CLP with saline vehicle was significantly increased (1626 ± 553 pg/ml, n = 6) in comparison with the sham and the local anesthetic groups. Infusion of lidocaine or bupivacaine resulted in undetectable plasma TNF-α concentrations, indicating that local anesthetics reduced TNF-α concentrations after CLP-induced sepsis to concentrations indistinguishable from sham controls (table 3). KC plasma concentrations at 24 h after operation were significantly increased in mice undergoing CLP with saline vehicle (21375 ± 5353 pg/ml, n = 5) compared with concentrations observed in the sham group (173 ± 19 pg/ml, n = 4). Lidocaine and bupivacaine significantly reduced KC plasma concentrations to 1346 ± 150 pg/ml, n = 7 and 3292 ± 770 pg/ml, n = 4, respectively (table 3).
Renal Myeloperoxidase Activity 24 h after CLP Is Reduced by Local Anesthetic Infusion
Myeloperoxidase is an enzyme present in leukocytes and is an index of tissue leukocyte infiltration.17 CLP increased myeloperoxidase activity (2.10 ± 0.24 ΔOD/min/mg protein, n = 9) in the renal cortex of mice compared with sham controls (0.02 ± 0.02 ΔOD/min/mg protein, n = 7). However, mice receiving 10% lidocaine or 1% bupivacaine infusion demonstrated significantly reduced myeloperoxidase concentrations (0.57 ± 0.11 ΔOD/min/mg protein, n = 3 and 0.31 ± 0.21 ΔOD/min/mg protein, n = 5, respectively) as compared with mice treated with vehicle alone (table 3).
Renal Protein and mRNA Expression of Proinflammatory Markers after CLP Is Reduced by Local Anesthetic Infusion
Immunoblotting for ICAM-1 protein from dissected renal cortices taken 24 h after CLP revealed that ICAM-1 protein expression was significantly higher in the CLP group receiving saline vehicle (263 ± 49% of sham, n = 6) versus  the lidocaine and bupivacaine treated groups (91 ± 16% of sham, n = 6 and 98 ± 55% of sham, n = 5; respectively) (table 3). In a separate set of experiments, saline-treated mice subjected to CLP demonstrated increased gene expression of KC, MIP-2, ICAM-1, MCP-1, TNF-α, regulated upon activation normal T-cell expressed and secreted, and interferon-induced protein 10 compared with sham controls. However, infusion of 10% lidocaine or 1% bupivacaine resulted in reduced mRNA quantities of KC, MIP-2, ICAM-1, and MCP-1 (fig. 2). mRNA quantity of TNF-α, regulated upon activation normal T-cell expressed and secreted, and interferon-induced protein 10 were not significantly different between treatment groups (data not shown). This occurred despite a decrease in systemic plasma concentrations of TNF-α with local anesthetic infusion as described above. This is consistent with other published findings that suggest suppression of TNF-α can occur at a posttranscriptional level.18 
Fig. 2. Semiquantitative reverse transcriptase polymerase chain reaction of proinflammatory markers keratinocyte derived chemokine (KC) (  A  ), macrophage inflammatory protein 2 (MIP-2) (  B  ), intracellular adhesion molecule-1 (ICAM-1) (  C  ), and monocyte chemoattractant protein 1 (MCP-1) (  D  ) from renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture (CLP) with saline infusion, C57 mice subjected to CLP while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Bar graphs represent densitometric quantifications of relative band intensities from reverse transcriptase polymerase chain reactions for each indicated mRNA, and are compilations of at least three separate experiments. A representative image of the semiquantitative reverse transcriptase polymerase chain reaction gels for each gene with sham, saline CLP, and 10% lidocaine with CLP is also shown.  bp  = basepair;  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine. #  P  < 0.05  versus  sham;  *P  < 0.05  versus  saline vehicle  .
Fig. 2. Semiquantitative reverse transcriptase polymerase chain reaction of proinflammatory markers keratinocyte derived chemokine (KC) (  A  ), macrophage inflammatory protein 2 (MIP-2) (  B  ), intracellular adhesion molecule-1 (ICAM-1) (  C  ), and monocyte chemoattractant protein 1 (MCP-1) (  D  ) from renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture (CLP) with saline infusion, C57 mice subjected to CLP while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Bar graphs represent densitometric quantifications of relative band intensities from reverse transcriptase polymerase chain reactions for each indicated mRNA, and are compilations of at least three separate experiments. A representative image of the semiquantitative reverse transcriptase polymerase chain reaction gels for each gene with sham, saline CLP, and 10% lidocaine with CLP is also shown.  bp  = basepair;  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine. #  P  < 0.05  versus  sham; 
	*P  < 0.05  versus  saline vehicle 
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Fig. 2. Semiquantitative reverse transcriptase polymerase chain reaction of proinflammatory markers keratinocyte derived chemokine (KC) (  A  ), macrophage inflammatory protein 2 (MIP-2) (  B  ), intracellular adhesion molecule-1 (ICAM-1) (  C  ), and monocyte chemoattractant protein 1 (MCP-1) (  D  ) from renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture (CLP) with saline infusion, C57 mice subjected to CLP while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Bar graphs represent densitometric quantifications of relative band intensities from reverse transcriptase polymerase chain reactions for each indicated mRNA, and are compilations of at least three separate experiments. A representative image of the semiquantitative reverse transcriptase polymerase chain reaction gels for each gene with sham, saline CLP, and 10% lidocaine with CLP is also shown.  bp  = basepair;  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine. #  P  < 0.05  versus  sham;  *P  < 0.05  versus  saline vehicle  .
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Local Anesthetic Treatment Decreases CLP-induced Renal Apoptosis
Although no appreciable renal necrosis was observed 24 h after CLP-induced sepsis (Jablonski scores were 0 for all groups), CLP-induced sepsis in mice receiving saline vehicle resulted in a significant increase in renal cortical apoptosis. However, local anesthetic infusion decreased the magnitude of renal apoptosis after CLP, as indicated by quantification of apoptotic bodies, the appearance of DNA laddering, and TUNEL positive cells.
There was a minimal occurrence of renal apoptotic bodies seen in sham operated mice (0.023 ± 0.022 apoptotic bodies/tubule). CLP resulted in a significantly higher number of renal apoptotic bodies (0.320 ± 0.117 apoptotic bodies/tubule; P  < 0.05). In contrast, infusion of 10% lidocaine (0.018 ± 0.008 apoptotic bodies/tubule, n = 6) or 1% bupivacaine (0.006 ± 0.004 apoptotic bodies/tubule, n = 4) resulted in decreased renal apoptosis at 24 h after CLP (P  < 0.05).
Similar results were observed when renal cortices were assessed for DNA laddering (fig. 3). Sham-operated mice displayed little to no DNA laddering. Samples from mice subjected to CLP and saline infusion showed obvious DNA laddering composed of DNA fragments in multiples of 180 basepairs, whereas samples from mice receiving local anesthetic infusion demonstrated minimal to no evidence of DNA laddering. Figure 3is representative of three separate experiments.
Fig. 3. Representative image of DNA laddering in renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture with saline infusion, C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Image is representative of three separate experiments.  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine 
Fig. 3. Representative image of DNA laddering in renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture with saline infusion, C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Image is representative of three separate experiments.  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine 
Fig. 3. Representative image of DNA laddering in renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture with saline infusion, C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Image is representative of three separate experiments.  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine 
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Furthermore, we failed to detect TUNEL positive corticomedullary kidney cells in sections from sham-operated mice (fig. 4). Consistent with our above findings, mice treated with saline infusion undergoing CLP showed increased TUNEL positive staining in the corticomedullary junction. However, mice receiving local anesthetic infusion and subjected to CLP showed reduced TUNEL-positive cells in the corticomedullary junction after 24 h. Figure 4is representative of three separate experiments.
Fig. 4. Representative image of TUNEL staining of renal cortices of sham operated C57 control mice (  A  ), C57 mice subjected to cecal ligation and double puncture with saline infusion (  B  ), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine (  C  ), and C57 mice treated with infusion of 1% bupivacaine (  D  ). Image is representative of three separate experiments.  Bupiv  = bupivacaine;  Lido  = lidocaine  .
Fig. 4. Representative image of TUNEL staining of renal cortices of sham operated C57 control mice (  A  ), C57 mice subjected to cecal ligation and double puncture with saline infusion (  B  ), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine (  C  ), and C57 mice treated with infusion of 1% bupivacaine (  D  ). Image is representative of three separate experiments.  Bupiv  = bupivacaine;  Lido  = lidocaine 
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Fig. 4. Representative image of TUNEL staining of renal cortices of sham operated C57 control mice (  A  ), C57 mice subjected to cecal ligation and double puncture with saline infusion (  B  ), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine (  C  ), and C57 mice treated with infusion of 1% bupivacaine (  D  ). Image is representative of three separate experiments.  Bupiv  = bupivacaine;  Lido  = lidocaine  .
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Discussion
The major findings of the current study are that chronic infusions of lidocaine or bupivacaine improve mortality, provide significant protection from acute renal and hepatic dysfunction, and attenuate the hyperinflammatory response and renal apoptosis associated with murine septic peritonitis induced by CLP.
Sepsis remains a major health challenge facing contemporary medicine. Despite advances in antibiotic, hemodynamic, and ventilatory support, sepsis remains responsible for approximately 215,000 deaths in the United States each year.1 Given the high mortality rate associated with sepsis (which ranges between 30% and 70%), therapeutic interventions are primarily judged by their impact on survival.19 
To more accurately recapitulate the complex immunology of human sepsis, we adopted the cecal ligation and double puncture model that produces a clinical picture that more accurately reflects human sepsis than animal models that utilize endotoxin or bacteria.3,20 This model induces septic peritonitis and has been shown to resemble human sepsis with respect to cytokine generation and progression to multiorgan failure and response to certain therapeutic interventions.21 Another advantage of this model is the ability to manipulate the magnitude of sepsis. In preliminary studies, we compared survival responses to graded injuries from 18-gauge, 20-gauge, and 22-gauge CLP. We chose to employ a 20-gauge needle to induce a lethal degree of sepsis with a high predilection for fulminant multiple organ failure in the absence of any interventions. Our mortality rates correspond well with other published survival studies using this genetic strain in CLP.22 We deliberately chose this severe model of sepsis to elucidate whether local anesthetic infusion could improve survival, even in the face of a known lethal injury. By 48 h after CLP, we observed a profound level of mortality (78%) and morbidity in the majority of mice receiving saline vehicle alone. They uniformly exhibited piloerection and conjunctival secretions, were disheveled in appearance, remained sedentary, and abstained from food and drink. No mouse from this treatment group survived beyond 3 days. In contrast, mice receiving local anesthetic infusion were more robust, mobile, and resumed drinking and eating by 4 days, and a significant number survived past 7 days.
It is well known that mortality in sepsis is strongly influenced by the development of organ injury and dysfunction.1 In concordance, our study showed chronic local anesthetic infusion not only decreased CLP-induced mortality, it significantly reduced the magnitude of CLP-induced renal and hepatic injury. Local anesthetics were able to decrease both creatinine and ALT/AST, suggesting that the protection conferred by local anesthetics was global and not restricted to a particular organ. Creatinine concentrations were not statistically different between sham and local anesthetic groups. Taken together, chronic local anesthetic infusion provided global protection from organ injury and dysfunction resulting from severe sepsis.
The fact that global antiinflammatory regimens have shown some degree of success in human sepsis lends credence to interventions predicated upon antiinflammatory modulation.5 These beneficial human sepsis studies utilizing insulin, low dose steroids, and activated protein C all share antiinflammatory characteristics.23–25 Similarly, there exists ample evidence of the potent antiinflammatory effects of local anesthetics. In a rabbit model of lung injury, Takao et al.  demonstrated that lidocaine infusion decreased various chemotactic factors and resulted in less neutrophil infiltration.26 In a separate study lidocaine and bupivacaine inhibited the release of inflammatory mediators leukotriene B4 and interleukin-1 from human neutrophils and monocytes.27 Several studies have also demonstrated that local anesthetics, in addition to possessing antithrombotic effects, can inhibit inflammatory cell migration and proinflammatory cytokine generation.28–30 On the basis of these studies, we hypothesized that modulation of initial inflammatory responses after septic insult with local anesthetics would lead to improved mortality rates as well as hepatic and renal protection, as inflammation is a major component of cell death associated with renal and hepatic injury in sepsis. In accordance with other published findings, significant inflammatory processes are elicited during CLP-induced sepsis in our saline vehicle group (shown by considerable elevations in proinflammatory plasma cytokines, renal mRNA expression, myeloperoxidase activity, and ICAM-1 protein expression). Although no mechanistic data are provided in this study, we show that local anesthetic treatment reduced inflammatory responses after CLP in mice. We speculate that the protective effects of local anesthetics occur by limiting inflammatory cell (macrophage, neutrophil, lymphocyte) activation or by attenuating inflammation on renal and hepatic parenchymal cells.
We first examined plasma concentrations of proinflammatory cytokines (TNF-α and KC) to assess the general state of inflammation in our mice. TNF-α is a prototypic example of a proinflammatory cytokine that plays an important role in propagating a host of secondary inflammatory cascades. As such, it has been shown in a variety of studies to mediate many of the immunopathologic features seen in sepsis. It also has been implicated to directly cause acute renal failure in sepsis by acting on the TNF-1 receptor31 and TNF-α has also been shown to cause multiorgan failure and lethality in sepsis.32 We demonstrated that local anesthetic infusion resulted in a dramatic decrease in circulating concentrations of TNF-α 24 h after CLP. Local anesthetics mitigated TNF-α plasma concentrations to undetectable concentrations (comparable to sham controls), and this may in part account for the decreased renal injury we observed in local anesthetic treated mice. Murine KC is a proinflammatory alpha chemokine that putatively represents the functional homologue of human interleukin-8. As such, KC not only serves as a potent leukocyte attractant and activator but its overexpression in experimental peritonitis has also been associated with liver enzyme abnormalities and increased early mortality.33 We also demonstrated a reduction in plasma KC concentrations with local anesthetic infusion, which may have served to prevent overamplification of proinflammatory responses mediated by KC. These results illustrate that local anesthetic infusion attenuates the overexuberant production of proinflammatory cytokines induced during severe sepsis.
To further examine our hypothesis that local anesthetics decrease inflammation, we then focused on gene expression in the kidney to examine if this phenomenon also occurred at the organ level. Previous studies have demonstrated that up-regulation of chemokines within the kidney accompanies the acute renal failure resulting from polymicrobial sepsis.34 We found reduced gene transcription of KC, MIP-2, ICAM-1, and MCP-1 in mice receiving local anesthetic infusion. In addition to the systemic plasma reductions in KC described above, we also observed renal KC gene expression was attenuated with local anesthetic infusions. Similar to KC, MIP-2 plays a pivotal role in leukocyte recruitment. Previous studies have shown a positive correlation with blunting MIP-2 overexpression and increased survival in sepsis models.35 Similarly, we observed substantial elevations in KC and MIP-2 mRNA concentrations with CLP, and a significant attenuation of this overexpression in KC and MIP-2 mRNA concentrations was seen in those mice treated with chronic infusion of lidocaine and bupivacaine. ICAM-1 is a proinflammatory adhesion molecule that participates in leukocyte recruitment and is up-regulated during acute inflammatory responses. MCP-1 is a chemokine also involved in the pathogenesis of sepsis that has been shown by other authors to indirectly contribute to renal injury.36 It plays an important role in the elicitation of both macrophages and neutrophils during sepsis. We also show that chronic local anesthetic infusion results in a decrease in MCP-1 gene expression in lidocaine and bupivacaine treated mice. We did not observe significant differences in gene expression of TNF-α, regulated on activation normal T-cell expressed and secreted, or interferon-induced protein 10. We did see a decrease in systemic plasma concentrations of TNF-α with local anesthetic infusion, suggesting that inhibition of TNF-α expression may occur at a posttranscriptional level.
Renal apoptosis is often an important component in the development of acute renal failure. In renal ischemia and reperfusion injury models, it has been demonstrated that increases in apoptosis were directly associated with increased renal inflammation.37 Daemen et al.  also demonstrated that blocking apoptosis prevented renal inflammation.38 Evidence is emerging that renal apoptosis also plays a significant role in the development of acute renal failure in sepsis.39,40 In the current study, we observed increased levels of apoptosis in renal tubule cells 24 h after CLP and a concomitant increase in renal dysfunction. In contrast, mice treated with local anesthetic infusion demonstrated decreased renal apoptosis and significantly improved renal function. Whether the decrease in renal apoptosis was mediated by local anesthetic suppressing proinflammatory mediators or by a direct effect on apoptotic pathways remains to be elucidated. We conclude from our data that in this sepsis model local anesthetic infusion is associated with decreased apoptosis and improved renal function after CLP.
A great deal of valuable work and attention has recently been dedicated to the immunosuppressive and hypoinflammatory phase that follows the initial hyperinflammatory phase of sepsis.20 There is a growing body of evidence to suggest that uncontrolled, hyperexuberant cytokine regulation, as occurs in sepsis, can lead to apoptosis in a variety of cell types and subsequently manifest as immunosuppression. The fact that immunosuppression may arise secondary to the uncontrolled hyperinflammatory phase leads to the question of whether attenuation of the hyperinflammatory phase would aid in preventing subsequent immunosuppression. We agree that once a generalized state of immunosuppression is achieved, it logically follows that further immunosuppressive or antiinflammatory modulation would be deleterious. However, as already mentioned, there are three clinical studies that have demonstrated a benefit from antiinflammatory modulation in sepsis. We believe the benefits we have demonstrated in this study likely follow similar antiinflammatory mechanisms.
In conclusion, we demonstrate that chronic infusion of local anesthetics provides protection from CLP-induced mortality and acute organ dysfunction. As the pathogenesis of organ dysfunction in sepsis is largely mediated by an imbalanced, uncontrolled inflammatory response, we believe that local anesthetics improve organ function after septic insult by attenuating this hyperinflammatory process. This attenuation is global in nature, exerting its effect on a variety of proinflammatory molecules, and culminates in a reduction in organ injury and thereby protects from progression to multiple organ dysfunction. Given the protective benefit on survival and organ dysfunction observed with chronic local anesthetic infusion, our findings could have important therapeutic implications for patients in early sepsis. Although it is premature to advocate intravenous anesthetics in septic patients, this intervention may someday prove beneficial for patients in early sepsis. Intense work is now being performed to elucidate the cell types involved in mediating the protective effects of local anesthetics and the cellular signaling mechanisms involved in protection.
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Fig. 1. Kaplan-Meier survival curves were generated for C57 mice subjected to cecal ligation and double puncture with saline infusion (n = 35), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 5% (n = 20) or 10% (n = 35) lidocaine, and C57 mice subjected to cecal ligation and double puncture treated with infusion of 1% (n = 27) or 2% (n = 21) bupivacaine. Log-rank analysis demonstrated a significant improvement in survival for all local anesthetic infusion groups  versus  the saline vehicle group (  P  < 0.05). Log-rank analysis also demonstrated a significant improvement in survival in the 2% bupivacaine infusion group  versus  the 1% bupivacaine infusion group (  P  < 0.05). No statistical differences in mortality were observed between the lidocaine infusion groups.  BUPIV  = bupivacaine;  LIDO  = lidocaine  .
Fig. 1. Kaplan-Meier survival curves were generated for C57 mice subjected to cecal ligation and double puncture with saline infusion (n = 35), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 5% (n = 20) or 10% (n = 35) lidocaine, and C57 mice subjected to cecal ligation and double puncture treated with infusion of 1% (n = 27) or 2% (n = 21) bupivacaine. Log-rank analysis demonstrated a significant improvement in survival for all local anesthetic infusion groups  versus  the saline vehicle group (  P  < 0.05). Log-rank analysis also demonstrated a significant improvement in survival in the 2% bupivacaine infusion group  versus  the 1% bupivacaine infusion group (  P  < 0.05). No statistical differences in mortality were observed between the lidocaine infusion groups.  BUPIV  = bupivacaine;  LIDO  = lidocaine 
	.
Fig. 1. Kaplan-Meier survival curves were generated for C57 mice subjected to cecal ligation and double puncture with saline infusion (n = 35), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 5% (n = 20) or 10% (n = 35) lidocaine, and C57 mice subjected to cecal ligation and double puncture treated with infusion of 1% (n = 27) or 2% (n = 21) bupivacaine. Log-rank analysis demonstrated a significant improvement in survival for all local anesthetic infusion groups  versus  the saline vehicle group (  P  < 0.05). Log-rank analysis also demonstrated a significant improvement in survival in the 2% bupivacaine infusion group  versus  the 1% bupivacaine infusion group (  P  < 0.05). No statistical differences in mortality were observed between the lidocaine infusion groups.  BUPIV  = bupivacaine;  LIDO  = lidocaine  .
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Fig. 2. Semiquantitative reverse transcriptase polymerase chain reaction of proinflammatory markers keratinocyte derived chemokine (KC) (  A  ), macrophage inflammatory protein 2 (MIP-2) (  B  ), intracellular adhesion molecule-1 (ICAM-1) (  C  ), and monocyte chemoattractant protein 1 (MCP-1) (  D  ) from renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture (CLP) with saline infusion, C57 mice subjected to CLP while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Bar graphs represent densitometric quantifications of relative band intensities from reverse transcriptase polymerase chain reactions for each indicated mRNA, and are compilations of at least three separate experiments. A representative image of the semiquantitative reverse transcriptase polymerase chain reaction gels for each gene with sham, saline CLP, and 10% lidocaine with CLP is also shown.  bp  = basepair;  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine. #  P  < 0.05  versus  sham;  *P  < 0.05  versus  saline vehicle  .
Fig. 2. Semiquantitative reverse transcriptase polymerase chain reaction of proinflammatory markers keratinocyte derived chemokine (KC) (  A  ), macrophage inflammatory protein 2 (MIP-2) (  B  ), intracellular adhesion molecule-1 (ICAM-1) (  C  ), and monocyte chemoattractant protein 1 (MCP-1) (  D  ) from renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture (CLP) with saline infusion, C57 mice subjected to CLP while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Bar graphs represent densitometric quantifications of relative band intensities from reverse transcriptase polymerase chain reactions for each indicated mRNA, and are compilations of at least three separate experiments. A representative image of the semiquantitative reverse transcriptase polymerase chain reaction gels for each gene with sham, saline CLP, and 10% lidocaine with CLP is also shown.  bp  = basepair;  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine. #  P  < 0.05  versus  sham; 
	*P  < 0.05  versus  saline vehicle 
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Fig. 2. Semiquantitative reverse transcriptase polymerase chain reaction of proinflammatory markers keratinocyte derived chemokine (KC) (  A  ), macrophage inflammatory protein 2 (MIP-2) (  B  ), intracellular adhesion molecule-1 (ICAM-1) (  C  ), and monocyte chemoattractant protein 1 (MCP-1) (  D  ) from renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture (CLP) with saline infusion, C57 mice subjected to CLP while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Bar graphs represent densitometric quantifications of relative band intensities from reverse transcriptase polymerase chain reactions for each indicated mRNA, and are compilations of at least three separate experiments. A representative image of the semiquantitative reverse transcriptase polymerase chain reaction gels for each gene with sham, saline CLP, and 10% lidocaine with CLP is also shown.  bp  = basepair;  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine. #  P  < 0.05  versus  sham;  *P  < 0.05  versus  saline vehicle  .
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Fig. 3. Representative image of DNA laddering in renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture with saline infusion, C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Image is representative of three separate experiments.  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine 
Fig. 3. Representative image of DNA laddering in renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture with saline infusion, C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Image is representative of three separate experiments.  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine 
Fig. 3. Representative image of DNA laddering in renal cortices of sham operated C57 control mice, C57 mice subjected to cecal ligation and double puncture with saline infusion, C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine, and C57 mice treated with infusion of 1% bupivacaine. Image is representative of three separate experiments.  M  = 100 basepair DNA marker;  Bupiv  = bupivacaine;  Lido  = lidocaine 
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Fig. 4. Representative image of TUNEL staining of renal cortices of sham operated C57 control mice (  A  ), C57 mice subjected to cecal ligation and double puncture with saline infusion (  B  ), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine (  C  ), and C57 mice treated with infusion of 1% bupivacaine (  D  ). Image is representative of three separate experiments.  Bupiv  = bupivacaine;  Lido  = lidocaine  .
Fig. 4. Representative image of TUNEL staining of renal cortices of sham operated C57 control mice (  A  ), C57 mice subjected to cecal ligation and double puncture with saline infusion (  B  ), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine (  C  ), and C57 mice treated with infusion of 1% bupivacaine (  D  ). Image is representative of three separate experiments.  Bupiv  = bupivacaine;  Lido  = lidocaine 
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Fig. 4. Representative image of TUNEL staining of renal cortices of sham operated C57 control mice (  A  ), C57 mice subjected to cecal ligation and double puncture with saline infusion (  B  ), C57 mice subjected to cecal ligation and double puncture while receiving infusion of 10% lidocaine (  C  ), and C57 mice treated with infusion of 1% bupivacaine (  D  ). Image is representative of three separate experiments.  Bupiv  = bupivacaine;  Lido  = lidocaine  .
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Table 1. Local Anesthetic Plasma Levels at 24 hours after Cecal Ligation and Puncture 
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Table 1. Local Anesthetic Plasma Levels at 24 hours after Cecal Ligation and Puncture 
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Table 2. Proinflammatory Sense and Anti-sense Primers Based on Published GenBank Sequences for Mice 
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Table 2. Proinflammatory Sense and Anti-sense Primers Based on Published GenBank Sequences for Mice 
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Table 3. Markers of Organ Dysfunction, Markers of Systemic Inflammation, and Markers of Renal Inflammation 24 hours after the induction of sepsis 
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Table 3. Markers of Organ Dysfunction, Markers of Systemic Inflammation, and Markers of Renal Inflammation 24 hours after the induction of sepsis 
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