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Systemic response to low-dose endotoxin infusion in cats
Veterinary Immunology and Immunopathology 132 (2009) 167–174
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Research paper
Systemic response to low-dose endotoxin infusion in cats§ Amy E. DeClue a,*, Kurt J. Williams b, Claire Sharp a, Carol Haak a,1, Elizabeth Lechner a,2, Carol R. Reinero a a
Department of Veterinary Medicine and Surgery, University of Missouri, College of Veterinary Medicine, 900 E. Campus Drive, Columbia, MO 65211, United States Department of Pathobiology and Diagnostic Investigation, G380 Veterinary Medical Center, Michigan State University, East Lansing, MI 48824, United States
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2009 Received in revised form 26 May 2009 Accepted 3 June 2009
Sepsis is a common problem in feline patients and is associated with substantial morbidity and mortality. There has been little research investigating the physiologic response to bacterial infection in cats, in part because appropriate models have not been developed. The objective of this study was to characterize the response to low-dose LPS infusion in conscious, healthy cats. Measures of systemic inflammation, hemodynamic stability, coagulation, metabolic function, and organ damage were compared between placebo and low-dose LPS infusion (2 mcg/kg/h 4 h, IV) in cats, with each cat serving as its own control. Markers of systemic inflammation including temperature, plasma TNF activity, IL6, CXCL-8 and IL-10 concentrations were significantly increased and white blood cell counts were significantly decreased after LPS infusion. A biphasic hypotensive response was observed after initiation of LPS infusion without concurrent tachycardia. Additionally, LPS administration significantly increased blood glucose, lactate and creatinine concentrations. Patchy alveolar congestion, multifocal acute alveolar epithelial necrosis, and mild pulmonary edema were noted in the lungs along with acute centrilobular hepatocellular necrosis, and mild lymphocyte apoptosis in the spleen and/or intestinal Peyer’s patches. No biologically significant alterations in coagulation parameters developed after LPS infusion. Low-dose LPS infusion in cats induced systemic inflammation, hemodynamic derangement, metabolic alterations and mild organ damage. Low-dose endotoxin infusion is a viable pre-clinical model to study naturally developing sepsis in cats. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Animal model Cytokine Hypotension Inflammation Sepsis
1. Introduction Sepsis is a clinical syndrome that develops because of a systemic inflammatory response to infection (Anon., 1992). Although sepsis is recognized in cats, little is
§ This study was performed at the University of Missouri, College of Veterinary Medicine, Columbia, Missouri. * Corresponding author. Tel.: +1 573 882 7821; fax: +1 573 884 7563. E-mail address: [email protected] (A.E. DeClue). 1 Present address: Animal Emergency Center, 2100 W. Silver Spring Dr., Glendale, WI 53209, United States. 2 Present address: University of Florida, 2015 SW 16th Ave, Gainesville, FL 32610, United States.
0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.06.002
known about sepsis in this species. The few studies evaluating sepsis in this species were either retrospective clinical investigations or experimental investigations that frequently employed extensive instrumentation and overwhelming insult resulting in rapid death. While studies of naturally developing sepsis have provided useful clinical information, their retrospective nature has prevented meaningful evaluation of pathologic mechanisms. The overwhelming insult commonly used in experimental models is unlike the smoldering presentation of sepsis typically seen in clinical patients. Applying information from a rapidly fatal, severe insult to a clinical patient with more smoldering disease is difficult at best.
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Sepsis has been associated with many conditions including septic peritonitis, hepatic abscessation, pyothorax, bacteremia, pneumonia, endocarditis, pyelonephritis, and pyometra in the cat (Barrs et al., 2005; Brady et al., 2000; Costello et al., 2004; Sergeeff et al., 2004; Waddell et al., 2002). Mortality rates for feline patients with sepsis range from 22 to 79% (Barrs et al., 2005; Costello et al., 2004; Sergeeff et al., 2004; Waddell et al., 2002). Like other species, cats with naturally acquired sepsis often develop fever, leukocytosis, circulatory collapse, and multiple organ dysfunction (Brady et al., 2000; Segev et al., 2006; Sergeeff et al., 2004; Waddell et al., 2002). However, cats also develop relatively unique manifestations of sepsis including bradycardia, hypothermia and abdominal pain (Brady et al., 2000; Sergeeff et al., 2004; Waddell et al., 2002). Information about pathophysiology of these unique manifestations is lacking. In order to develop methods for early diagnosis and rational treatment strategies for sepsis in cats, a better understanding of the systemic response to infection in this species is necessary. Although studying the inflammatory response to infection would be ideally carried out in feline patients with the naturally developing syndrome, there are ethical limitations to this approach. An alternative method is the use of low-dose LPS infusion in research animals. Low-dose LPS infusion has been shown to more closely mimic the inflammatory response observed in naturally developing sepsis compared to high dose ‘‘intoxication’’ models of endotoxemia (Deitch, 1998). The objective of this study was to characterize the response to low-dose LPS infusion in conscious, healthy research cats. Measures of systemic inflammation, hemodynamic stability, coagulation and metabolic function were compared between placebo (saline) and low-dose LPS infusion.
2. Methods 2.1. Animals and experimental set up Six adult, purpose bred cats3 were used. Animals were cared for according to the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals and the study was approved by the Animal Care and Use Committee at the University of Missouri. The cats were maintained on commercial adult cat food and water ad libitum, but fasted prior to each day of procedures. On day 1, the cats were instrumented with 4 Fr single lumen jugular catheter4 and a 22 g cephalic catheter5 during a brief sedation (medetomidine6 15 mcg/kg and butorphanol tartrate7 0.2 mg/kg, IV). On day 2, placebo (saline, 2 ml/ kg/h 4 h, IV) was administered to the cats. On day 3, the cats received LPS derived from Escherichia coli 0127:B88 (2 mcg/kg/h diluted in saline 2 ml/kg/h 4 h, IV).
3 Liberty Research, Waverly, NY; Nutrition Colony, University of California, Davis, CA. 4 Cook Veterinary Products, Bloomington, IN. 5 Abbott, Abbott Park, IL. 6 Domitor1, Pfizer Animal Health, Exton, PA. 7 Torbugesic1, Fort Dodge, Fort Dodge, IA. 8 Sigma–Aldrich, St. Louis, MO.
2.2. Sample collection Rectal temperature, Doppler9 systolic arterial blood pressure and heart rate were evaluated at baseline and then every 30 min for 6 h after initiation of the infusion. Blood was harvested from the jugular catheter at a volume of no more than 6 ml/kg total for the duration of the experiment. For each volume of blood collected, an equal volume of 0.9% saline was administered. Blood was anticoagulated with either lithium heparin or sodium citrate, immediately placed on ice and centrifuged (1800 g 10 min, 4 8C). The plasma was harvested and frozen at 80 8C until analysis. Whole blood samples for complete blood count were anticoagulated with potassium EDTA. On day 3, at 8 h, the cats were euthanized via pentobarbital sodium10 overdose and tissues were collected immediately for histopathology. 2.3. Cytokine analyses TNF activity—Plasma TNF activity was evaluated at baseline, 0.5, 1, 1.5, 2, 3, 4 and 6 h after initiation of the infusion using a modification of a previously described cytotoxicity bioassay (Baarsch et al., 1991). Briefly, cells from mouse fibroblast cell line (L929) were cultured on 96 well plates. Diluted plasma samples were added to the wells in triplicate. After a 20 h incubation with MEM plus horse serum (1%) and actinomycin D8 (3 mg/ml), 3[4,5-dimethylthiazol-2-yl]-2,5,-di-phenyl tetrazolium bromide8 (MTT) was added and the cells incubated for an additional 2.5 h. The formazen crystals were solubilized in dimethylformanide11 (50%) and SDS11 (20%). Color development after 1 h was measured at 630 nm. Feline rTNF12 was used to construct a standard curve. The lower limit of detection for this assay is 1.0 ng/ml. Plasma IL-1b, IL-6, CXCL-8, IL-10—Plasma IL-1b, IL-6 and CXCL-8 were evaluated at baseline, 1, 2, 3, 4, and 6 h and IL-10 was evaluated at baseline, 4 and 8 h after the initiation of the infusion. For IL-1b, IL-6 and CXCL-8 ELISAs, plasma was diluted 1:2 with 1% BSA in PBS (IL-6, CXCL-8) or 25% FBS in PBS (IL-1b) prior to analysis. Samples for IL-10 were assayed undiluted. Assays were performed in duplicate using commercially available ELISA kits13 following the manufacturer’s directions. The lower limit of detection for these assays are 31.25, 400, 62.5, and 125 pg/ml for IL-1b, IL-6, CXCL-8, IL-10, respectively. 2.4. Blood and urine analyses Complete blood count (CBC)—A CBC was evaluated at baseline, 1, 2, 3, 4 and 6 h after initiation of the infusion
9 10
Parks Medical Electronics, Las Vegas, NV. Beuthanasia-D Special, Schering-Plough Animal Health Corp., Union,
NJ. 11 12 13
Fischer Scientific International, Pittsburgh, PA. Endogen, Rockford, IL. R&D systems, Minneapolis, MN.
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using a Coulter Counter.14 A manual 200 differential cell count was performed on Wright’s stained blood smears by an investigator blinded to the treatment group. Glucose concentrations—Whole blood glucose concentrations were evaluated at baseline and then hourly for 6 h after initiation of the infusion using an accu-check1 advantage15 glucometer. Lactate—Whole blood lactate concentrations were evaluated at baseline, 2, 4 and 6 h after initiation of the infusion using an AccutrendTM Lactate Analyzer.16 Plasma and urine biochemistry—Biochemical analysis of plasma urea nitrogen, creatinine, alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin were evaluated at 6 h using an Olympus AU400e automated wet chemistry analyzer.17 Urine specific gravity was determined using a refractometer. Coagulation—Prothrombin time (PT), partial thromboplastin time (PTT), antithrombin activity, fibrinogen and Ddimer concentrations were determined from citrated plasma at 8 h by a commercial laboratory.18 Histopathology—Following sacrifice of the animals, tissue samples were collected from the lung, liver, small intestine, pancreas, kidney, heart, skeletal muscle and spleen. The right cranial lung lobe was cannulated with a 14 g flexible canula, and inflated with 10% neutral buffered formalin prior to submersion. All tissues were allowed to remain in the fixative for at least 24 h prior to processing. Representative sections of all tissues were trimmed, embedded in paraffin, and routinely processed prior to staining with hematoxylin and eosin. The slides were evaluated by a single veterinary pathologist (KW) and compared to established histologic characteristics for each organ (Eurell, 2004; Foust and Getty, 1958; Rhodin, 1978; Samuelson, 2007). Observations were reported using descriptive statistics. Statistical analysis—The data were analyzed using commercially available software (SAS/STAT1).19 A mixed linear repeated measures model with a compound symmetry covariance structure was fitted to the data; variance parameters were estimated using the REML method. Numerical data concerning each of the treatments were analyzed using a Fisher’s least significant difference test. A P-value <0.05 was considered significant. For statistical purposes, the lower limit of the detection was used for samples below the limit of detection as this was the most conservative assumption. 3. Results 3.1. Physiologic parameters After LPS administration, rectal temperature steadily increased over the duration of the study with a peak at 5 h (Fig. 1A). Placebo administration, conversely, was associated with a steady decrease in rectal temperature. Rectal
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Fig. 1. Serial measurement of rectal temperature (A) and systolic arterial blood pressure (B) after placebo (&) and LPS infusion (&). Rectal temperature showed a significant increase after LPS administration and a gradual decrease after placebo administration. Endotoxin administration resulted in a significantly decreased blood pressure at 1.5–2.5 and 5–6 h. Group means were not significantly different at baseline between treatments for blood pressure or temperature. Data are expressed as group mean SE. ap < 0.05. bp 0.01. cp 0.001.
temperature differed significantly between groups starting at 3.5 h after initiation of LPS infusion. None of the cats developed hypothermia after LPS infusion. Lipopolysaccharide administration induced a significant biphasic hypotensive response compared with placebo (Fig. 1B). An initial hypotensive response was seen with a nadir at 1.5 h. The second blood pressure decline began at approximately 5 h, but the nadir was not determined since data collection ended at 6 h. There was no significant difference in heart rate between treatment groups at any time point (data not shown). 3.2. Cytokine analyses Mean plasma TNF activity increased significantly after LPS administration compared with placebo (Fig. 2A). Plasma TNF activity increased starting at 1 h with peak activity at 1.5 h. Interleukin-1b was not detected at any time point after placebo infusion. After LPS infusion, IL-1b was detectable in the plasma of only 2 cats at 6 h (86 6 pg/ml). Plasma IL-6 and CXCL-8 concentrations were increased significantly for the duration of the experiment starting at 2 h (Fig. 2B and C). The peak plasma concentration for both inflammatory mediators was at 4 h. Plasma IL-10 was significantly higher in the LPS group at 4 h compared with placebo but was not detectable in either group at baseline or 8 h (Fig. 2D). 3.3. Blood and urine evaluation
14 15 16 17 18 19
Coulter Electronics, Hialeah, FL. Roche Diagnostics, Basel, Switzerland. Sports Resource Group, Hawthorne, NY. Olympus America, Irving, TX. Animal Health Diagnostic Center, Cornell University, Ithica, NY. SAS Institute Inc., Cary, NC.
Total white blood cell and neutrophil counts remained stable over time after placebo administration (Fig. 3A and B). The white blood cell count significantly decreased starting at 1 h with a nadir at 4 h and recovery by 6 h after LPS
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Fig. 3. Dynamic alterations in white blood cell count (A), neutrophil count (B) and lymphocyte count (C) after placebo (&) and LPS infusion (&). White blood cell, neutrophil and lymphocyte counts were significantly lower in the LPS treated cats at multiple time points. Group means were not significantly different at baseline between treatments for white blood cell, neutrophil or lymphocyte counts. Data are expressed as group mean SE. ap < 0.05. bp 0.01. cp 0.001.
Fig. 2. Dynamic change in plasma TNF activity (A), IL-6 (B), CXCL-8 (C) and IL-10 (D) after placebo (&) and LPS infusion (&). Plasma TNF activity significantly increased with a peak at 1.5 h after initiation of LPS infusion. Plasma IL-6 and CXCL-8 concentrations significantly increased with a peak at 4 h after initiation of LPS infusion. Plasma IL-10 concentration was significantly greater after LPS infusion but was not detectable after either treatment at 8 h. Group means were not significantly different at baseline between treatments for plasma TNF activity, IL-6, CXCL-8 or IL-10 concentrations. Data are expressed as group mean SE. ap < 0.05. b p 0.01. cp 0.001.
administration. The neutrophil count behaved similarly to the WBC count, but with maximal decrease at 2 h (Fig. 3B). Conversely, the lymphocyte count peaked at 2 h and then steadily declined regardless of treatment (Fig. 3C). However, lymphocyte counts at 4 and 6 h were significantly lower after LPS administration compared with placebo. There was
no statistical difference in hematocrit between the groups at any time point (data not shown). Due to platelet clumping, an accurate platelet count was not obtained from many samples and therefore data are not reported. Blood lactate concentrations were significantly higher at 4 and 6 h after LPS administration compared to placebo (Fig. 4A). Lipopolysaccharide administration also significantly increased blood glucose concentrations at 3, 5 and 6 h (Fig. 4B). Plasma creatinine was significantly higher and urine specific gravity was significantly lower 6 h after initiation of the LPS infusion compared with placebo (Table 1). There was no significant difference in BUN, ALP, ALT or albumin, between LPS and placebo administration at 6 h (Table 1). The PT was significantly prolonged after LPS administration at 8 h compared with placebo (Table 2). Partial thromboplastin time, antithrombin activity, fibrinogen and D-dimer concentrations were not significantly different between treatments (Table 2).
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Table 1 Blood and urine analytes 6 h after LPS and placebo treatment. Blood urea nitrogen (BUN), alanine aminotransferase (ALT), alkaline phosphatase (ALP), specific gravity (SG). Data are expressed as group mean SE.
Creatinine (mg/dl) BUN (mg/dl) Albumin (g/dl) ALT (U/l) ALP (U/l) Urine SG
LPS
Placebo
P-Value
1.3 0.01 23 2.5 2.9 0.1 65.6 11.1 19 3.5 1.015 0.0032
1.11 0.01 17.2 2.7 3.1 0.1 58.5 11.1 23.6 3.5 1.050 0.0032
0.028 0.065 0.084 0.185 0.059 <0.001
Table 2 Prothrombin time (PT), partial thromboplastin time (PTT), antithrombin, fibrinogen and D-dimer concentrations 8 h after LPS or placebo treatment. Data are expressed as group mean SE. All cats had a Ddimer concentration less than 250 ng/ml.
PT (s) PTT (s) Antithrombin (%) Fibrinogen (mg/dl) D-dimer (ng/ml)
LPS
Placebo
P-Value
20.9 0.5 20.6 2.7 90.2 4.3 125.2 11.1 <250
19.1 0.4 20.2 3.2 91.8 3 113.7 10 <250
0.033 0.926 0.561 0.438 1.0
3.4. Histopathology The lungs were the most consistently affected tissue following LPS infusion (Table 3; Fig. 5A and B). The lungs of all cats developed patchy alveolar congestion and multifocal acute alveolar epithelial necrosis. There were small foci of neutrophils within the alveoli, as well as mild edema
Fig. 5. Lung histopathology after LPS infusion. (A) Multiple foci of acute alveolar congestion and inflammation are present within the lungs (between arrows). Bar = 100 mm. (B) Higher magnification of acute alveolar injury; there is acute necrosis of alveolar walls (arrow), with small amounts of adjacent alveolar hemorrhage and infiltration by inflammatory cells. Bar = 50 mm.
Table 3 Histopathologic findings after LPS infusion. Total number of cats = 6.
Fig. 4. Dynamic change in blood lactate (A) and glucose (B) concentrations after administration of placebo (&) and LPS infusion (&). Blood lactate significantly increased at 4 h and remained increased for the duration of the study in the LPS treated cats. Similarly, blood glucose was significantly higher with LPS administration at 3, 5 and 6 h. Group means were not significantly different at baseline between treatments for blood lactate or glucose concentrations. Data are expressed as group mean SE. ap < 0.05. b p 0.01. cp 0.001.
Tissue
Finding
Number of cats
Lung
Alveolar congestion Alveolar epithelial cell necrosis Lymphocytic/eosinophilic vasculitis Perivascular edema Intra-capillary megakaryocytosis
6 6 6 6 6
Liver
Centrilobular necrosis/ acute coagulation Sinusoidal congestion
5
Small intestine
Spleen
Lymphocyte apoptosis in peyer’s patches
a
2 3
Sinusoidal neutrophilia 4 Lymphocyte apoptosisa 5 in white pulp a Characteristic features of lymphocyte apoptosis included shrunken and hyperchromatic cells with nuclear fragmentation with or without adjacent tingible body macrophages (Janeway, 2005).
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and moderate numbers of increased intra-capillary megakaryocytes. Additionally, 5/6 cats developed acute centrilobular hepatocellular necrosis, and mild lymphocyte apoptosis was observed in the spleen and/or intestinal Peyer’s patch in 5/6 of the cats (Table 3). No abnormalities were observed within the pancreas, kidney, heart, or skeletal muscle. 4. Discussion This study documents the systemic response to lowdose LPS infusion in cats. Endotoxin infusion resulted in the development of systemic inflammation demonstrated by fever; TNF, IL-6, CXCL-8 and IL-10 production; leucopenia, and neutropenia; hemodynamic derangement such as hypotension and relative bradycardia; metabolic dysfunction including hyperglycemia, hyperlactatemia; and early organ dysfunction including azotemia and histopathologic evidence of pulmonary and hepatic damage. Low-dose LPS infusion was chosen for this study to avoid several pitfalls found in high-dose bolus endotoxemia models, including: (1) acute overwhelming inflammation, (2) induction of an immediate severe hypodynamic circulatory response, (3) atypical massive cytokine production, (4) highly invasive instrumentation and (5) rapid mortality (Deitch, 1998). Additionally, the cats were not anesthetized to allow maintenance of the neuro-endocrine axis and unaltered assessment of cardiovascular function. Unlike models of overwhelming sepsis resulting in rapid mortality, this model is more likely to provide clinically useful data for the evaluation of novel biomarkers and therapeutics prior to evaluation in pet cats. Altered thermoregulation, hypothermia or fever, is a common finding in cats with naturally developing sepsis (Barrs et al., 2005; Brady et al., 2000; Costello et al., 2004; Sergeeff et al., 2004; Waddell et al., 2002). Cats in our study developed fever 3.5 h after the initiation of the LPS infusion, with a peak at 5 h; none developed hypothermia. Peak body temperature after intravenous bolus injection of LPS was reported to be at 4 h previously (McCann et al., 2005). The differences in timing of the peak febrile response between these two studies likely relates to the different methods of LPS administration (i.e. bolus injection versus infusion). The cats in this study had a biphasic hypotensive response to LPS infusion. Humans have an almost identical biphasic hypotensive response to LPS administration (Sufferedini et al., 1989). However, cats are unique in that there was no significant difference in heart rate after LPS administration; a finding that was especially interesting in light of the presence of hypotension. One would expect a hyperdynamic response to LPS characterized, in part, by tachycardia (Sufferedini et al., 1989). The findings of this study mimic those in cats with naturally developing sepsis (Brady et al., 2000). Low-dose LPS infusion resulted in significant increases in plasma TNF activity, IL-6, CXCL-8 and IL-10 concentrations. The peak plasma TNF activity at 1.5 h was at a similar time point as has been described previously in cats with endotoxemia (Otto and Rawlings, 1995). To the author’s knowledge, production of IL-6, CXCL-8 and IL-10 in
response to LPS in the blood have not been studied in cats previously. However, IL-6 has been documented to increase in the cerebrospinal fluid of cats after endotoxin challenge (Akarsu et al., 1998). In this study, the plasma IL6, CXCL-8 and IL-10 response exhibited a similar peak and duration to that in other species (DeClue et al., 2008; Kemna et al., 2005; LeMay et al., 1990; Reichenberg et al., 2002; Taniguchi et al., 2008; van Deventer et al., 1990). Interestingly, there was a limited plasma IL-1b response after LPS infusion in this study. There are several possible reasons for this. First, the assay utilized to measure IL-1b may not have been sensitive enough to detect the change in plasma IL-1b. Second, our sampling may have been too early in the course of inflammation to detect marked changes in plasma IL-1b. For the 2 cats that had detectable IL-1b concentrations, it was at 6 h after initiation of LPS. Finally, anti-inflammatory mediators like IL-10 suppress IL-1b production. The IL-1b response may have been blunted by the observed IL-10 expression or another unmeasured anti-inflammatory mediator. A similar phenomenon has been recognized in humans after LPS injection (Kemna et al., 2005). Cats with naturally developing sepsis may have a leucopenia, neutropenia and lymphopenia as was observed in this study; although leukocytosis is more commonly recognized (Brady et al., 2000; Costello et al., 2004; King, 1994; Waddell et al., 2002). Since laboratory evaluation of cats with naturally developing sepsis is rarely accomplished within the first few hours of the inflammatory process, it is not surprising that an acute leucopenic response, as demonstrated here, is often missed. Low-dose LPS infusion did not produce significant coagulation abnormalities. There was a significant prolongation of plasma PT, however this mild prolongation was not considered biologically important. These findings are consistent with a previous study where high doses of LPS (3–10 mg/kg, IV) were administered to cats with no acute changes in coagulation parameters (Lucas and Kitzmiller, 1972). However, in this same study, cats did develop moderate thrombocytopenia after high dose LPS administration (Lucas and Kitzmiller, 1972). Because many cats in our study had clumping of their platelets, we were unable to accurately determine if low-dose LPS infusion results in thrombocytopenia as well. Metabolic dysfunction, namely hyperglycemia and hyperlactatemia, was observed after LPS infusion. These changes have been previously documented in cats with experimentally induced endotoxemia and are also seen in cats and humans with naturally developing sepsis (Costello et al., 2004; Hughes et al., 1981) The proposed pathogenesis of hyperglycemia during sepsis relates to a change in the balance between insulin and counter regulatory hormones, notably catecholamines (Nelson, 2002; Rand et al., 2002). The sepsis-induced hyperlactatemia observed in this study may be due to documented hypotension; or other mechanisms not specifically evaluated in this study such as microcirculatory derangement and mitochondrial dysfunction. Based on plasma biochemical analysis, there was little evidence of organ damage after LPS infusion. Urine specific gravity was significantly lower and serum creatinine was
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significantly higher in the LPS group, however this change is more likely due to decreased vasopressin sensitivity of the renal medulla and pre-renal azotemia as opposed to early renal failure (Grinevich et al., 2004). Additionally, the creatinine concentration remained within the reference interval for this laboratory. The lungs of all cats developed patchy alveolar congestion, multifocal acute alveolar epithelial necrosis, small foci of neutrophils within the alveoli, mild edema and moderate numbers of increased intra-capillary megakaryocytes. Respiratory failure is the most rapid type of organ failure in humans with sepsis (Bernard et al., 1997). In a retrospective study of severe sepsis in cats, 13/22 cats had necrosis or inflammation in the lung despite having a non-pulmonary source of infection (Brady et al., 2000). Additionally, Lucas described similar pulmonary pathology after administration of 3 and 10 mg/kg of E. coli LPS into the vena cava of anesthetized cats (Lucas and Kitzmiller, 1972). Given the findings of this study, those of Brady and Lucas, and the high incidence of sepsis-induced lung injury in humans, further study evaluating the importance of lung injury during sepsis and other forms of critical illness in cats is needed (Brady et al., 2000; Lucas and Kitzmiller, 1972). Centrilobular hepatic necrosis was evident in 5/6 cats after LPS infusion and ALT was mildly increased but not significantly so. In other species, LPS induced hepatocellular injury is accompanied by neutrophilic inflammation which was not a characteristic finding in this study (Kaur et al., 2006). Because of the lack of neutrophilic inflammation, the acute hepatocellular necrosis likely represents acute ischemic injury related rather than direct injury from LPS. There are several limitations to this study that should be mentioned. A cross-over design was employed in the current study to limit the number of cats utilized; with this design, the placebo was administered prior to LPS to avoid residual inflammatory effects. Therefore, we were unable to randomize the cross-over design. Baseline values for each variable measured were not statistically different between the two groups, but a time effect cannot be completely excluded. Additionally, there was no placebo control for the histologic evaluation of tissues. It is possible that the histologic changes noted in this study resulted from a process unrelated to LPS administration and thus these data should be interpreted with caution. In conclusion, administration of a low-dose infusion of LPS to cats results in systemic inflammation, hemodynamic derangement, metabolic dysfunction and mild organ damage that mimics many of the clinical alterations seen during naturally developing sepsis in cats. Although the inflammatory mediator response is similar to that of other species, cats have a unique cardiovascular response to LPS. In the future, this model may be used for evaluation of early diagnostics and novel therapeutics for sepsis prior to administration to pet cats. Conflict of interest The authors certify that they do not have any conflicts of interest related to this study.
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Acknowledgements The authors would like to acknowledge Dr. Leona Rubin for her generous donation of mouse fibroblast cells; Dr. Keith Branson for kindly providing infusion pumps; Matt Haight for his technical expertise; and Drs. Heather Markway, Kelley Thiemann and Rebecca Plamondon, and Rachael Cohen for their laboratory assistance. A portion of the funding for this study was from the University of Missouri Pi Chapter of Phi Zeta.
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novel cyclooxygenase-2 inhibitor in cats with lipopolysaccharideinduced pyrexia. Am. J. Vet. Res. 66, 1278–1284. Nelson, R., 2002. Stress hyperglycemia and diabetes mellitus in cats. J. Vet. Intern. Med. 16, 121–122. Otto, C., Rawlings, C., 1995. Tumor necrosis factor production in cats in response to lipopolysaccharide: an in vivo and in vitro study. Vet. Immunol. Immunopathol. 29, 183–188. Rand, J., Kinnaird, E., Baglioni, A., Blackshaw, J., Priest, J., 2002. Acute stress hyperglycemia in cats is associated with struggling and increased concentrations of lactate and norepinephrine. J. Vet. Intern. Med. 16, 123–132. Reichenberg, A., Kraus, T., Haack, M., Schuld, A., Pollmacher, T., Yirmiya, R., 2002. Endotoxin-induced changes in food consumption in healthy volunteers are associated with TNF-alpha and IL-6 secretion. Psychoneuroendocrinology 27, 945–956. Rhodin, J.A., 1978. Microscopic anatomy of the pulmonary vascular bed in the cat lung. Microvasc. Res. 15, 169–193. Samuelson, D.A., 2007. Textbook of Veterinary Histology, vol. xii. Saunders–Elsevier, St. Louis, 546 pp.
Segev, G., Klement, E., Aroch, I., 2006. Toxic neutrophils in cats: clinical and clinicopathologic features, and disease prevalence and outcome— a retrospective case control study. JVIM 20, 20–31. Sergeeff, J.F, Armstrong, P.J., Bunch, S.E., 2004. Hepatic abscesses in cats: 14 cases (1985–2002). JVIM 18, 295–300. Sufferedini, A., Fromm, R., Parker, M., Brenner, M., Kovacs, J., Wesley, R., Parrillo, J., 1989. The cardiovascular response of normal humans to the administration of endotoxin. N. Engl. J. Med. 321, 280–287. Taniguchi, T., Kurita, A., Kobayashi, K., Yamamoto, K., Inaba, H., 2008. Dose- and time-related effects of dexmedetomidine on mortality and inflammatory responses to endotoxin-induced shock in rats. J. Anesth. 22, 221–228. van Deventer, S., Buller, H., ten Cate, J., Aarden, L., Hack, C., Sturk, A., 1990. Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood 76, 2520–2526. Waddell, L., Brady, C., Drobatz, K., 2002. Risk factors, prognostic indicators, and outcome of pyothorax in cats: 80 cases (1986–1999). JAVMA 221, 819–824.
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Systemic response to low-dose endotoxin infusion in cats§ Amy E. DeClue a,*, Kurt J. Williams b, Claire Sharp a, Carol Haak a,1, Elizabeth Lechner a,2, Carol R. Reinero a a
Department of Veterinary Medicine and Surgery, University of Missouri, College of Veterinary Medicine, 900 E. Campus Drive, Columbia, MO 65211, United States Department of Pathobiology and Diagnostic Investigation, G380 Veterinary Medical Center, Michigan State University, East Lansing, MI 48824, United States
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2009 Received in revised form 26 May 2009 Accepted 3 June 2009
Sepsis is a common problem in feline patients and is associated with substantial morbidity and mortality. There has been little research investigating the physiologic response to bacterial infection in cats, in part because appropriate models have not been developed. The objective of this study was to characterize the response to low-dose LPS infusion in conscious, healthy cats. Measures of systemic inflammation, hemodynamic stability, coagulation, metabolic function, and organ damage were compared between placebo and low-dose LPS infusion (2 mcg/kg/h 4 h, IV) in cats, with each cat serving as its own control. Markers of systemic inflammation including temperature, plasma TNF activity, IL6, CXCL-8 and IL-10 concentrations were significantly increased and white blood cell counts were significantly decreased after LPS infusion. A biphasic hypotensive response was observed after initiation of LPS infusion without concurrent tachycardia. Additionally, LPS administration significantly increased blood glucose, lactate and creatinine concentrations. Patchy alveolar congestion, multifocal acute alveolar epithelial necrosis, and mild pulmonary edema were noted in the lungs along with acute centrilobular hepatocellular necrosis, and mild lymphocyte apoptosis in the spleen and/or intestinal Peyer’s patches. No biologically significant alterations in coagulation parameters developed after LPS infusion. Low-dose LPS infusion in cats induced systemic inflammation, hemodynamic derangement, metabolic alterations and mild organ damage. Low-dose endotoxin infusion is a viable pre-clinical model to study naturally developing sepsis in cats. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Animal model Cytokine Hypotension Inflammation Sepsis
1. Introduction Sepsis is a clinical syndrome that develops because of a systemic inflammatory response to infection (Anon., 1992). Although sepsis is recognized in cats, little is
§ This study was performed at the University of Missouri, College of Veterinary Medicine, Columbia, Missouri. * Corresponding author. Tel.: +1 573 882 7821; fax: +1 573 884 7563. E-mail address: [email protected] (A.E. DeClue). 1 Present address: Animal Emergency Center, 2100 W. Silver Spring Dr., Glendale, WI 53209, United States. 2 Present address: University of Florida, 2015 SW 16th Ave, Gainesville, FL 32610, United States.
0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.06.002
known about sepsis in this species. The few studies evaluating sepsis in this species were either retrospective clinical investigations or experimental investigations that frequently employed extensive instrumentation and overwhelming insult resulting in rapid death. While studies of naturally developing sepsis have provided useful clinical information, their retrospective nature has prevented meaningful evaluation of pathologic mechanisms. The overwhelming insult commonly used in experimental models is unlike the smoldering presentation of sepsis typically seen in clinical patients. Applying information from a rapidly fatal, severe insult to a clinical patient with more smoldering disease is difficult at best.
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Sepsis has been associated with many conditions including septic peritonitis, hepatic abscessation, pyothorax, bacteremia, pneumonia, endocarditis, pyelonephritis, and pyometra in the cat (Barrs et al., 2005; Brady et al., 2000; Costello et al., 2004; Sergeeff et al., 2004; Waddell et al., 2002). Mortality rates for feline patients with sepsis range from 22 to 79% (Barrs et al., 2005; Costello et al., 2004; Sergeeff et al., 2004; Waddell et al., 2002). Like other species, cats with naturally acquired sepsis often develop fever, leukocytosis, circulatory collapse, and multiple organ dysfunction (Brady et al., 2000; Segev et al., 2006; Sergeeff et al., 2004; Waddell et al., 2002). However, cats also develop relatively unique manifestations of sepsis including bradycardia, hypothermia and abdominal pain (Brady et al., 2000; Sergeeff et al., 2004; Waddell et al., 2002). Information about pathophysiology of these unique manifestations is lacking. In order to develop methods for early diagnosis and rational treatment strategies for sepsis in cats, a better understanding of the systemic response to infection in this species is necessary. Although studying the inflammatory response to infection would be ideally carried out in feline patients with the naturally developing syndrome, there are ethical limitations to this approach. An alternative method is the use of low-dose LPS infusion in research animals. Low-dose LPS infusion has been shown to more closely mimic the inflammatory response observed in naturally developing sepsis compared to high dose ‘‘intoxication’’ models of endotoxemia (Deitch, 1998). The objective of this study was to characterize the response to low-dose LPS infusion in conscious, healthy research cats. Measures of systemic inflammation, hemodynamic stability, coagulation and metabolic function were compared between placebo (saline) and low-dose LPS infusion.
2. Methods 2.1. Animals and experimental set up Six adult, purpose bred cats3 were used. Animals were cared for according to the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals and the study was approved by the Animal Care and Use Committee at the University of Missouri. The cats were maintained on commercial adult cat food and water ad libitum, but fasted prior to each day of procedures. On day 1, the cats were instrumented with 4 Fr single lumen jugular catheter4 and a 22 g cephalic catheter5 during a brief sedation (medetomidine6 15 mcg/kg and butorphanol tartrate7 0.2 mg/kg, IV). On day 2, placebo (saline, 2 ml/ kg/h 4 h, IV) was administered to the cats. On day 3, the cats received LPS derived from Escherichia coli 0127:B88 (2 mcg/kg/h diluted in saline 2 ml/kg/h 4 h, IV).
3 Liberty Research, Waverly, NY; Nutrition Colony, University of California, Davis, CA. 4 Cook Veterinary Products, Bloomington, IN. 5 Abbott, Abbott Park, IL. 6 Domitor1, Pfizer Animal Health, Exton, PA. 7 Torbugesic1, Fort Dodge, Fort Dodge, IA. 8 Sigma–Aldrich, St. Louis, MO.
2.2. Sample collection Rectal temperature, Doppler9 systolic arterial blood pressure and heart rate were evaluated at baseline and then every 30 min for 6 h after initiation of the infusion. Blood was harvested from the jugular catheter at a volume of no more than 6 ml/kg total for the duration of the experiment. For each volume of blood collected, an equal volume of 0.9% saline was administered. Blood was anticoagulated with either lithium heparin or sodium citrate, immediately placed on ice and centrifuged (1800 g 10 min, 4 8C). The plasma was harvested and frozen at 80 8C until analysis. Whole blood samples for complete blood count were anticoagulated with potassium EDTA. On day 3, at 8 h, the cats were euthanized via pentobarbital sodium10 overdose and tissues were collected immediately for histopathology. 2.3. Cytokine analyses TNF activity—Plasma TNF activity was evaluated at baseline, 0.5, 1, 1.5, 2, 3, 4 and 6 h after initiation of the infusion using a modification of a previously described cytotoxicity bioassay (Baarsch et al., 1991). Briefly, cells from mouse fibroblast cell line (L929) were cultured on 96 well plates. Diluted plasma samples were added to the wells in triplicate. After a 20 h incubation with MEM plus horse serum (1%) and actinomycin D8 (3 mg/ml), 3[4,5-dimethylthiazol-2-yl]-2,5,-di-phenyl tetrazolium bromide8 (MTT) was added and the cells incubated for an additional 2.5 h. The formazen crystals were solubilized in dimethylformanide11 (50%) and SDS11 (20%). Color development after 1 h was measured at 630 nm. Feline rTNF12 was used to construct a standard curve. The lower limit of detection for this assay is 1.0 ng/ml. Plasma IL-1b, IL-6, CXCL-8, IL-10—Plasma IL-1b, IL-6 and CXCL-8 were evaluated at baseline, 1, 2, 3, 4, and 6 h and IL-10 was evaluated at baseline, 4 and 8 h after the initiation of the infusion. For IL-1b, IL-6 and CXCL-8 ELISAs, plasma was diluted 1:2 with 1% BSA in PBS (IL-6, CXCL-8) or 25% FBS in PBS (IL-1b) prior to analysis. Samples for IL-10 were assayed undiluted. Assays were performed in duplicate using commercially available ELISA kits13 following the manufacturer’s directions. The lower limit of detection for these assays are 31.25, 400, 62.5, and 125 pg/ml for IL-1b, IL-6, CXCL-8, IL-10, respectively. 2.4. Blood and urine analyses Complete blood count (CBC)—A CBC was evaluated at baseline, 1, 2, 3, 4 and 6 h after initiation of the infusion
9 10
Parks Medical Electronics, Las Vegas, NV. Beuthanasia-D Special, Schering-Plough Animal Health Corp., Union,
NJ. 11 12 13
Fischer Scientific International, Pittsburgh, PA. Endogen, Rockford, IL. R&D systems, Minneapolis, MN.
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using a Coulter Counter.14 A manual 200 differential cell count was performed on Wright’s stained blood smears by an investigator blinded to the treatment group. Glucose concentrations—Whole blood glucose concentrations were evaluated at baseline and then hourly for 6 h after initiation of the infusion using an accu-check1 advantage15 glucometer. Lactate—Whole blood lactate concentrations were evaluated at baseline, 2, 4 and 6 h after initiation of the infusion using an AccutrendTM Lactate Analyzer.16 Plasma and urine biochemistry—Biochemical analysis of plasma urea nitrogen, creatinine, alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin were evaluated at 6 h using an Olympus AU400e automated wet chemistry analyzer.17 Urine specific gravity was determined using a refractometer. Coagulation—Prothrombin time (PT), partial thromboplastin time (PTT), antithrombin activity, fibrinogen and Ddimer concentrations were determined from citrated plasma at 8 h by a commercial laboratory.18 Histopathology—Following sacrifice of the animals, tissue samples were collected from the lung, liver, small intestine, pancreas, kidney, heart, skeletal muscle and spleen. The right cranial lung lobe was cannulated with a 14 g flexible canula, and inflated with 10% neutral buffered formalin prior to submersion. All tissues were allowed to remain in the fixative for at least 24 h prior to processing. Representative sections of all tissues were trimmed, embedded in paraffin, and routinely processed prior to staining with hematoxylin and eosin. The slides were evaluated by a single veterinary pathologist (KW) and compared to established histologic characteristics for each organ (Eurell, 2004; Foust and Getty, 1958; Rhodin, 1978; Samuelson, 2007). Observations were reported using descriptive statistics. Statistical analysis—The data were analyzed using commercially available software (SAS/STAT1).19 A mixed linear repeated measures model with a compound symmetry covariance structure was fitted to the data; variance parameters were estimated using the REML method. Numerical data concerning each of the treatments were analyzed using a Fisher’s least significant difference test. A P-value <0.05 was considered significant. For statistical purposes, the lower limit of the detection was used for samples below the limit of detection as this was the most conservative assumption. 3. Results 3.1. Physiologic parameters After LPS administration, rectal temperature steadily increased over the duration of the study with a peak at 5 h (Fig. 1A). Placebo administration, conversely, was associated with a steady decrease in rectal temperature. Rectal
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Fig. 1. Serial measurement of rectal temperature (A) and systolic arterial blood pressure (B) after placebo (&) and LPS infusion (&). Rectal temperature showed a significant increase after LPS administration and a gradual decrease after placebo administration. Endotoxin administration resulted in a significantly decreased blood pressure at 1.5–2.5 and 5–6 h. Group means were not significantly different at baseline between treatments for blood pressure or temperature. Data are expressed as group mean SE. ap < 0.05. bp 0.01. cp 0.001.
temperature differed significantly between groups starting at 3.5 h after initiation of LPS infusion. None of the cats developed hypothermia after LPS infusion. Lipopolysaccharide administration induced a significant biphasic hypotensive response compared with placebo (Fig. 1B). An initial hypotensive response was seen with a nadir at 1.5 h. The second blood pressure decline began at approximately 5 h, but the nadir was not determined since data collection ended at 6 h. There was no significant difference in heart rate between treatment groups at any time point (data not shown). 3.2. Cytokine analyses Mean plasma TNF activity increased significantly after LPS administration compared with placebo (Fig. 2A). Plasma TNF activity increased starting at 1 h with peak activity at 1.5 h. Interleukin-1b was not detected at any time point after placebo infusion. After LPS infusion, IL-1b was detectable in the plasma of only 2 cats at 6 h (86 6 pg/ml). Plasma IL-6 and CXCL-8 concentrations were increased significantly for the duration of the experiment starting at 2 h (Fig. 2B and C). The peak plasma concentration for both inflammatory mediators was at 4 h. Plasma IL-10 was significantly higher in the LPS group at 4 h compared with placebo but was not detectable in either group at baseline or 8 h (Fig. 2D). 3.3. Blood and urine evaluation
14 15 16 17 18 19
Coulter Electronics, Hialeah, FL. Roche Diagnostics, Basel, Switzerland. Sports Resource Group, Hawthorne, NY. Olympus America, Irving, TX. Animal Health Diagnostic Center, Cornell University, Ithica, NY. SAS Institute Inc., Cary, NC.
Total white blood cell and neutrophil counts remained stable over time after placebo administration (Fig. 3A and B). The white blood cell count significantly decreased starting at 1 h with a nadir at 4 h and recovery by 6 h after LPS
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Fig. 3. Dynamic alterations in white blood cell count (A), neutrophil count (B) and lymphocyte count (C) after placebo (&) and LPS infusion (&). White blood cell, neutrophil and lymphocyte counts were significantly lower in the LPS treated cats at multiple time points. Group means were not significantly different at baseline between treatments for white blood cell, neutrophil or lymphocyte counts. Data are expressed as group mean SE. ap < 0.05. bp 0.01. cp 0.001.
Fig. 2. Dynamic change in plasma TNF activity (A), IL-6 (B), CXCL-8 (C) and IL-10 (D) after placebo (&) and LPS infusion (&). Plasma TNF activity significantly increased with a peak at 1.5 h after initiation of LPS infusion. Plasma IL-6 and CXCL-8 concentrations significantly increased with a peak at 4 h after initiation of LPS infusion. Plasma IL-10 concentration was significantly greater after LPS infusion but was not detectable after either treatment at 8 h. Group means were not significantly different at baseline between treatments for plasma TNF activity, IL-6, CXCL-8 or IL-10 concentrations. Data are expressed as group mean SE. ap < 0.05. b p 0.01. cp 0.001.
administration. The neutrophil count behaved similarly to the WBC count, but with maximal decrease at 2 h (Fig. 3B). Conversely, the lymphocyte count peaked at 2 h and then steadily declined regardless of treatment (Fig. 3C). However, lymphocyte counts at 4 and 6 h were significantly lower after LPS administration compared with placebo. There was
no statistical difference in hematocrit between the groups at any time point (data not shown). Due to platelet clumping, an accurate platelet count was not obtained from many samples and therefore data are not reported. Blood lactate concentrations were significantly higher at 4 and 6 h after LPS administration compared to placebo (Fig. 4A). Lipopolysaccharide administration also significantly increased blood glucose concentrations at 3, 5 and 6 h (Fig. 4B). Plasma creatinine was significantly higher and urine specific gravity was significantly lower 6 h after initiation of the LPS infusion compared with placebo (Table 1). There was no significant difference in BUN, ALP, ALT or albumin, between LPS and placebo administration at 6 h (Table 1). The PT was significantly prolonged after LPS administration at 8 h compared with placebo (Table 2). Partial thromboplastin time, antithrombin activity, fibrinogen and D-dimer concentrations were not significantly different between treatments (Table 2).
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Table 1 Blood and urine analytes 6 h after LPS and placebo treatment. Blood urea nitrogen (BUN), alanine aminotransferase (ALT), alkaline phosphatase (ALP), specific gravity (SG). Data are expressed as group mean SE.
Creatinine (mg/dl) BUN (mg/dl) Albumin (g/dl) ALT (U/l) ALP (U/l) Urine SG
LPS
Placebo
P-Value
1.3 0.01 23 2.5 2.9 0.1 65.6 11.1 19 3.5 1.015 0.0032
1.11 0.01 17.2 2.7 3.1 0.1 58.5 11.1 23.6 3.5 1.050 0.0032
0.028 0.065 0.084 0.185 0.059 <0.001
Table 2 Prothrombin time (PT), partial thromboplastin time (PTT), antithrombin, fibrinogen and D-dimer concentrations 8 h after LPS or placebo treatment. Data are expressed as group mean SE. All cats had a Ddimer concentration less than 250 ng/ml.
PT (s) PTT (s) Antithrombin (%) Fibrinogen (mg/dl) D-dimer (ng/ml)
LPS
Placebo
P-Value
20.9 0.5 20.6 2.7 90.2 4.3 125.2 11.1 <250
19.1 0.4 20.2 3.2 91.8 3 113.7 10 <250
0.033 0.926 0.561 0.438 1.0
3.4. Histopathology The lungs were the most consistently affected tissue following LPS infusion (Table 3; Fig. 5A and B). The lungs of all cats developed patchy alveolar congestion and multifocal acute alveolar epithelial necrosis. There were small foci of neutrophils within the alveoli, as well as mild edema
Fig. 5. Lung histopathology after LPS infusion. (A) Multiple foci of acute alveolar congestion and inflammation are present within the lungs (between arrows). Bar = 100 mm. (B) Higher magnification of acute alveolar injury; there is acute necrosis of alveolar walls (arrow), with small amounts of adjacent alveolar hemorrhage and infiltration by inflammatory cells. Bar = 50 mm.
Table 3 Histopathologic findings after LPS infusion. Total number of cats = 6.
Fig. 4. Dynamic change in blood lactate (A) and glucose (B) concentrations after administration of placebo (&) and LPS infusion (&). Blood lactate significantly increased at 4 h and remained increased for the duration of the study in the LPS treated cats. Similarly, blood glucose was significantly higher with LPS administration at 3, 5 and 6 h. Group means were not significantly different at baseline between treatments for blood lactate or glucose concentrations. Data are expressed as group mean SE. ap < 0.05. b p 0.01. cp 0.001.
Tissue
Finding
Number of cats
Lung
Alveolar congestion Alveolar epithelial cell necrosis Lymphocytic/eosinophilic vasculitis Perivascular edema Intra-capillary megakaryocytosis
6 6 6 6 6
Liver
Centrilobular necrosis/ acute coagulation Sinusoidal congestion
5
Small intestine
Spleen
Lymphocyte apoptosis in peyer’s patches
a
2 3
Sinusoidal neutrophilia 4 Lymphocyte apoptosisa 5 in white pulp a Characteristic features of lymphocyte apoptosis included shrunken and hyperchromatic cells with nuclear fragmentation with or without adjacent tingible body macrophages (Janeway, 2005).
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and moderate numbers of increased intra-capillary megakaryocytes. Additionally, 5/6 cats developed acute centrilobular hepatocellular necrosis, and mild lymphocyte apoptosis was observed in the spleen and/or intestinal Peyer’s patch in 5/6 of the cats (Table 3). No abnormalities were observed within the pancreas, kidney, heart, or skeletal muscle. 4. Discussion This study documents the systemic response to lowdose LPS infusion in cats. Endotoxin infusion resulted in the development of systemic inflammation demonstrated by fever; TNF, IL-6, CXCL-8 and IL-10 production; leucopenia, and neutropenia; hemodynamic derangement such as hypotension and relative bradycardia; metabolic dysfunction including hyperglycemia, hyperlactatemia; and early organ dysfunction including azotemia and histopathologic evidence of pulmonary and hepatic damage. Low-dose LPS infusion was chosen for this study to avoid several pitfalls found in high-dose bolus endotoxemia models, including: (1) acute overwhelming inflammation, (2) induction of an immediate severe hypodynamic circulatory response, (3) atypical massive cytokine production, (4) highly invasive instrumentation and (5) rapid mortality (Deitch, 1998). Additionally, the cats were not anesthetized to allow maintenance of the neuro-endocrine axis and unaltered assessment of cardiovascular function. Unlike models of overwhelming sepsis resulting in rapid mortality, this model is more likely to provide clinically useful data for the evaluation of novel biomarkers and therapeutics prior to evaluation in pet cats. Altered thermoregulation, hypothermia or fever, is a common finding in cats with naturally developing sepsis (Barrs et al., 2005; Brady et al., 2000; Costello et al., 2004; Sergeeff et al., 2004; Waddell et al., 2002). Cats in our study developed fever 3.5 h after the initiation of the LPS infusion, with a peak at 5 h; none developed hypothermia. Peak body temperature after intravenous bolus injection of LPS was reported to be at 4 h previously (McCann et al., 2005). The differences in timing of the peak febrile response between these two studies likely relates to the different methods of LPS administration (i.e. bolus injection versus infusion). The cats in this study had a biphasic hypotensive response to LPS infusion. Humans have an almost identical biphasic hypotensive response to LPS administration (Sufferedini et al., 1989). However, cats are unique in that there was no significant difference in heart rate after LPS administration; a finding that was especially interesting in light of the presence of hypotension. One would expect a hyperdynamic response to LPS characterized, in part, by tachycardia (Sufferedini et al., 1989). The findings of this study mimic those in cats with naturally developing sepsis (Brady et al., 2000). Low-dose LPS infusion resulted in significant increases in plasma TNF activity, IL-6, CXCL-8 and IL-10 concentrations. The peak plasma TNF activity at 1.5 h was at a similar time point as has been described previously in cats with endotoxemia (Otto and Rawlings, 1995). To the author’s knowledge, production of IL-6, CXCL-8 and IL-10 in
response to LPS in the blood have not been studied in cats previously. However, IL-6 has been documented to increase in the cerebrospinal fluid of cats after endotoxin challenge (Akarsu et al., 1998). In this study, the plasma IL6, CXCL-8 and IL-10 response exhibited a similar peak and duration to that in other species (DeClue et al., 2008; Kemna et al., 2005; LeMay et al., 1990; Reichenberg et al., 2002; Taniguchi et al., 2008; van Deventer et al., 1990). Interestingly, there was a limited plasma IL-1b response after LPS infusion in this study. There are several possible reasons for this. First, the assay utilized to measure IL-1b may not have been sensitive enough to detect the change in plasma IL-1b. Second, our sampling may have been too early in the course of inflammation to detect marked changes in plasma IL-1b. For the 2 cats that had detectable IL-1b concentrations, it was at 6 h after initiation of LPS. Finally, anti-inflammatory mediators like IL-10 suppress IL-1b production. The IL-1b response may have been blunted by the observed IL-10 expression or another unmeasured anti-inflammatory mediator. A similar phenomenon has been recognized in humans after LPS injection (Kemna et al., 2005). Cats with naturally developing sepsis may have a leucopenia, neutropenia and lymphopenia as was observed in this study; although leukocytosis is more commonly recognized (Brady et al., 2000; Costello et al., 2004; King, 1994; Waddell et al., 2002). Since laboratory evaluation of cats with naturally developing sepsis is rarely accomplished within the first few hours of the inflammatory process, it is not surprising that an acute leucopenic response, as demonstrated here, is often missed. Low-dose LPS infusion did not produce significant coagulation abnormalities. There was a significant prolongation of plasma PT, however this mild prolongation was not considered biologically important. These findings are consistent with a previous study where high doses of LPS (3–10 mg/kg, IV) were administered to cats with no acute changes in coagulation parameters (Lucas and Kitzmiller, 1972). However, in this same study, cats did develop moderate thrombocytopenia after high dose LPS administration (Lucas and Kitzmiller, 1972). Because many cats in our study had clumping of their platelets, we were unable to accurately determine if low-dose LPS infusion results in thrombocytopenia as well. Metabolic dysfunction, namely hyperglycemia and hyperlactatemia, was observed after LPS infusion. These changes have been previously documented in cats with experimentally induced endotoxemia and are also seen in cats and humans with naturally developing sepsis (Costello et al., 2004; Hughes et al., 1981) The proposed pathogenesis of hyperglycemia during sepsis relates to a change in the balance between insulin and counter regulatory hormones, notably catecholamines (Nelson, 2002; Rand et al., 2002). The sepsis-induced hyperlactatemia observed in this study may be due to documented hypotension; or other mechanisms not specifically evaluated in this study such as microcirculatory derangement and mitochondrial dysfunction. Based on plasma biochemical analysis, there was little evidence of organ damage after LPS infusion. Urine specific gravity was significantly lower and serum creatinine was
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significantly higher in the LPS group, however this change is more likely due to decreased vasopressin sensitivity of the renal medulla and pre-renal azotemia as opposed to early renal failure (Grinevich et al., 2004). Additionally, the creatinine concentration remained within the reference interval for this laboratory. The lungs of all cats developed patchy alveolar congestion, multifocal acute alveolar epithelial necrosis, small foci of neutrophils within the alveoli, mild edema and moderate numbers of increased intra-capillary megakaryocytes. Respiratory failure is the most rapid type of organ failure in humans with sepsis (Bernard et al., 1997). In a retrospective study of severe sepsis in cats, 13/22 cats had necrosis or inflammation in the lung despite having a non-pulmonary source of infection (Brady et al., 2000). Additionally, Lucas described similar pulmonary pathology after administration of 3 and 10 mg/kg of E. coli LPS into the vena cava of anesthetized cats (Lucas and Kitzmiller, 1972). Given the findings of this study, those of Brady and Lucas, and the high incidence of sepsis-induced lung injury in humans, further study evaluating the importance of lung injury during sepsis and other forms of critical illness in cats is needed (Brady et al., 2000; Lucas and Kitzmiller, 1972). Centrilobular hepatic necrosis was evident in 5/6 cats after LPS infusion and ALT was mildly increased but not significantly so. In other species, LPS induced hepatocellular injury is accompanied by neutrophilic inflammation which was not a characteristic finding in this study (Kaur et al., 2006). Because of the lack of neutrophilic inflammation, the acute hepatocellular necrosis likely represents acute ischemic injury related rather than direct injury from LPS. There are several limitations to this study that should be mentioned. A cross-over design was employed in the current study to limit the number of cats utilized; with this design, the placebo was administered prior to LPS to avoid residual inflammatory effects. Therefore, we were unable to randomize the cross-over design. Baseline values for each variable measured were not statistically different between the two groups, but a time effect cannot be completely excluded. Additionally, there was no placebo control for the histologic evaluation of tissues. It is possible that the histologic changes noted in this study resulted from a process unrelated to LPS administration and thus these data should be interpreted with caution. In conclusion, administration of a low-dose infusion of LPS to cats results in systemic inflammation, hemodynamic derangement, metabolic dysfunction and mild organ damage that mimics many of the clinical alterations seen during naturally developing sepsis in cats. Although the inflammatory mediator response is similar to that of other species, cats have a unique cardiovascular response to LPS. In the future, this model may be used for evaluation of early diagnostics and novel therapeutics for sepsis prior to administration to pet cats. Conflict of interest The authors certify that they do not have any conflicts of interest related to this study.
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Acknowledgements The authors would like to acknowledge Dr. Leona Rubin for her generous donation of mouse fibroblast cells; Dr. Keith Branson for kindly providing infusion pumps; Matt Haight for his technical expertise; and Drs. Heather Markway, Kelley Thiemann and Rebecca Plamondon, and Rachael Cohen for their laboratory assistance. A portion of the funding for this study was from the University of Missouri Pi Chapter of Phi Zeta.
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