Abstract
OBJECTIVE
To describe daily changes in serum concentrations of hyaluronic acid (HA), a biomarker of endothelial glycocalyx degradation, in dogs with septic peritonitis and to determine whether relationships exist among serum concentrations of HA and biomarkers of inflammation and patient fluid status.
ANIMALS
8 client-owned dogs.
PROCEDURES
Serum samples that had been collected for a previous study and stored at −80°C were used. Blood samples were collected at admission and daily thereafter during hospitalization and were analyzed for concentrations of HA and interleukins 6, 8, and 10. Patient data including acute patient physiologic and laboratory evaluation score, type and amount of fluids administered daily, and daily CBC and lactate concentration results were recorded. To determine the significant predictors of HA concentration, a general linear mixed model for repeated measures was developed.
RESULTS
All dogs survived to discharge. Concentrations of HA ranged from 18 to 1,050 ng/mL (interquartile [25th to 75th percentile] range, 49 to 119 ng/mL) throughout hospitalization. Interleukin-6 concentration was a significant predictor of HA concentration as was total administered daily fluid volume when accounting for interleukin-6 concentration. When fluid volume was analyzed independent of inflammatory status, fluid volume was not a significant predictor. Concentrations of HA did not significantly change over time but tended to increase on day 2 or 3 of hospitalization.
CONCLUSIONS AND CLINICAL RELEVANCE
Results supported the theory that inflammation is associated with endothelial glycocalyx degradation. Dogs recovering from septic peritonitis may become more susceptible to further endothelial glycocalyx damage as increasing fluid volumes are administered.
Introduction
Sepsis is characterized by severe systemic inflammation that puts patients at risk for the development of septic shock, multiorgan dysfunction, and death. The administration of IV fluids is central to the treatment of this physiologic state, namely correcting the macrohemodynamic derangements. However, recent focus has shifted to the cellular and microcirculatory dysfunction that occurs in sepsis,1 and several large-scale studies2–6 of people fail to show a clinical benefit to current liberal fluid resuscitation strategies. Aggressive fluid resuscitation may neglect to address or may exacerbate the pathological changes to the microcirculation.4,7
On the luminal surface of healthy vascular endothelium resides the EG, a multicomponent gel-like layer that consists of a cell-bound component and a surface layer of intricately linked glycosaminoglycans including HA, chondroitin sulfate, and heparan sulfate.8–13 The EG is not a static structure and remains in constant dynamic equilibrium of biosynthesis and degradation (loss) of its constituents.14 However, during pathological states, this equilibrium can be disrupted in favor of EG degradation, which may then exacerbate systemic inflammation and its associated consequences by stimulating cytokine production,15 disrupting coagulation,9–13 and increasing vascular permeability.10,16–18 Endothelial glycocalyx degradation is associated with an increased mortality rate and acts as an independent predictor of death in human trauma patients.19–22 Insult to the EG causes shedding of its constituents into circulation, and measurement of these glycosaminoglycans allows for indirect assessment of EG degradation.4,12,23–25 Syndecan-1, heparan sulfate, chondroitin sulfate, and HA are considered valid markers of EG integrity in people and are used in clinical research to identify EG degradation.14 The use of EG biomarkers in companion animals is in its infancy,26,27 with HA as the only EG biomarker that has been validated in dogs.28
Although establishing reliable methods of measuring EG degradation products is an essential first step, working to understand the cause of EG-constituent shedding is also necessary. Many factors including inflammatory mediators, atrial natriuretic peptide, fluid balance, fluid resuscitation strategies (ie, volume and rate of crystalloid, artificial colloid, and natural colloid solutions), and plasma administration have been implicated in EG degradation through human clinical trials and animal research models.8,20,22,29–32 Further exploration of EG biomarkers in veterinary patients and gaining an understanding of EG disruption in various stages of critical illness are increasingly relevant to the treatment of septic veterinary patients.
The primary objective of this preliminary study was to describe daily changes in serum HA concentration over the course of hospitalization in client-owned dogs with septic peritonitis. A secondary objective was to identify whether any relationships exist between serum HA and cytokine concentrations and daily fluid status. Concentration of HA was hypothesized to be highest at admission and to gradually decrease over time with treatment for septic peritonitis. Additionally, cytokine concentration and TADFV were predicted to positively correlate with increased concentrations of HA.
Materials and Methods
Case selection
The study was conducted at the Companion Animal Hospital of the Ontario Veterinary College Health Sciences Centre. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Guelph, and written consent was obtained from the dog owners prior to study enrollment.
Prospectively collected data and stored blood samples from client-owned dogs with confirmed septic peritonitis were used. Dogs were enrolled in a previously published study,33 and a subset of these dogs (n = 8) was used for the present study on the basis of the availability of stored serum samples.
Dogs had been hospitalized between January 2012 and June 2014. Enrollment criteria included a diagnosis of septic peritonitis that was based on the observation of overt intestinal leakage or necrosis during surgery, or the presence of at least one of the following criteria: positive result on bacterial culture of abdominal fluid, identification of intracellular bacteria with cytologic examination of abdominal fluid, a difference in glucose concentration of > 20 mg/dL between blood and abdominal fluid, or a difference in lactate concentration of > 18 mg/dL between blood and abdominal fluid. Glucose and lactate concentrations were determined with a point-of-care blood gas analyzer.33,a Dogs were enrolled at hospital admission (day 0) and had an exploratory celiotomy within a mean of 4.6 hours (range, 2 to 16 hours) of admission. A closed-suction abdominal drain was placed in all dogs for postoperative management. Blood samples were collected each morning until abdominal drain removal. Time of drain removal was determined by the attending clinician.
Data collection
Medical records were reviewed retrospectively for signalment, body weight at admission, and prior medical history. Illness severity score at the time of admission was determined with the full APPLE score, an illness severity stratification scoring system developed for dogs.34 Total administered daily fluid volume, defined as the volume (expressed as the number of milliliters per kilogram of body weight) of all fluids administered IV during the preceding day of hospitalization, was calculated at each blood sampling time point for each dog on the basis of information in the medical records. Fluid type (crystalloid, artificial colloid, or blood product administered IV) was recorded. The volume of drugs administered IV and the volume of fluids administered prior to presentation to the Ontario Veterinary College were not included in the calculation of TADFV. The calculated fluid volume for day 1 was the volume of IV fluids that was administered from the time of admission (day 0) until the following morning (day 1) and included all fluids administered during surgery. For all other days, the calculated volume included all fluids administered IV in the hospital over the previous 24 hours. Data collection was discontinued on the day of abdominal drain removal.
Biomarker determination
Blood samples (5 mL) were collected at the time of hospital admission and prior to surgery (day 0) and each morning thereafter until removal of the abdominal drain. On day 0, samples were collected during placement of an IV catheter and thereafter by saphenous or jugular venipuncture. Following collection, a portion of the blood sample was immediately transferred to a heparinized syringeb and another portion to a blood collection tube containing EDTA.c Within 10 minutes of sample collection, the heparinized sample was analyzed with a point-of-care blood gas analyzer.a The samples in the EDTA blood collection tubes were analyzed with an automated hematologic analyzer.d The remainder of the blood sample was transferred to a vacuum-sealed plastic tube without additivec and was allowed to clot for 15 minutes before being centrifuged at 1,500 × g for 7 minutes. The serum was then retrieved and stored at −80°C within 1 hour of collection. All samples were subjected to 1 previous freeze-thaw cycle.
Serum samples were batch analyzed. The samples were thawed at room temperature (21°C) and then vortexed and centrifuged for particulate removal prior to analyses. Serum HA concentration was determined with a commercially available ELISA kite that had been validated by the manufacturer for use with canine sera and used in determining the HA concentration in canine synovial fluid in a previous study.28 All samples were run in duplicate and according to the manufacturer's instructions. The assay's lower limit of detection reported by the manufacturer was 2.7 ng/mL. Serum concentrations of IL-6, IL-8, and IL-10 were measured in duplicate with a commercially available canine-specific multiplex cytokine immunoassay kitf that had been validated by the manufacturer. The assay was performed following overnight sample incubation at 4°C to maximize assay sensitivity according to the manufacturer's recommendations. The measured concentration of each analyte per sample was calculated from a standard curve generated from the standards and blank provided by the manufacturer. The assay's lower limits of detection provided by the manufacturer for IL-6, IL-8, and IL-10 were 3.7, 21.7, and 8.5 pg/mL, respectively.
Statistical analysis
Data were checked for normality with the Shapiro-Wilk test and examination of the residuals. If necessary, data were logarithmically transformed to meet the assumption of normality. To determine the significant predictors of serum HA concentration, a general linear mixed model for repeated measures was developed. Fixed parameters of interest included time in days as a class variable and IL-6, IL-8, and IL-10 concentrations as continuous variables tested separately as MFI and concentration (pg/mL), with each concentration that was below the lower limit of detection handled by assigning it a value of the limit of detection divided by the square root of 2. Despite the fact that results were similar for MFI and concentration, results for MFI were chosen for reporting cytokine interactions because the logarithmic transformation of MFI data yielded normally distributed data. Continuous parameters of total WBC count, segmented neutrophil count, band neutrophil count, lactate concentration, TADFV (mL/kg), and quadratic effects of each cytokine and cytokine and WBC interactions with TADFV were included in the model. The Akaike information criterion was used to determine which covariance structure to use to account for repeated measures over time for each dog. The autoregressive structure had the lowest Akaike information criterion and therefore was used for modeling the repeated measures. Nonsignificant (P > 0.15) variable effects were removed from the model in the development of the predictive equation for serum HA concentration. Each dog was treated as a random effect if the repeated error effect of each dog was not significant. All data were analyzed with commercially available software.g Values of P < 0.05 were considered significant.
Results
The study included 33 samples from 8 dogs. Mean age of dogs was 5.7 years (range, 0.5 to 11 years), and mean body weight was 24 kg (range, 7.4 to 31.6 kg). Three dogs were male (castrated, n = 2; sexually intact, 1), and 5 dogs were female (spayed, 4; sexually intact, 1). Dogs included mixed breeds (n = 3) and a Boxer, Doberman Pinscher, German Shepherd Dog, Miniature Schnauzer, and Nova Scotia Duck Tolling Retriever (1 each). Previous or concurrent medical conditions included increased activities of serum liver enzymes (n = 2) and the presence of a heart murmur (2). At admission, a full APPLE score was recorded for 7 dogs, with a median full APPLE score of 26 (range, 18 to 40). All patients underwent exploratory celiotomy and intestinal surgery, had placement of a closed-suction abdominal drain, and recovered in the intensive care unit. Mean time from admission to removal of the abdominal drain was 3.25 days (range, 3 to 4 days; removed on day 3 for 2 dogs and on day 4 for 6 dogs). All patients survived to hospital discharge. Blood collected at the time of admission (day 0) from 4 dogs was not available for analysis; therefore, the number of dogs per analyte per day varied (Table 1).
Mean (SD)* or median (range)† of various analytes determined for up to 8 dogs (No. in parentheses) with septic peritonitis confirmed after presentation to the hospital (day 0).
| Analyte | Day | ||||
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | |
| HA (ng/mL)† | 71.4 (18.4–87.2) (4) | 69.2 (38.5–192.5) (8) | 105.9 (46.2–239.4) (8) | 66.8 (24.3–1,050) (8) | 53.4 (31.3–170) (5) |
| IL-6 (pg/mL)† | 54.1 (21.5–474.2) (4) | 66.9 (11–519.4) (8) | 18.8 (8.8–197.7) (7) | 19.6 (6.5–522.4) (8) | 9.9 (0–43.8) (5) |
| IL-8 (pg/mL)† | 7,880 (1,030–10,106) (4) | 6,300 (9–13,625) (8) | 6,232 (237.4–18,130) (7) | 10,510 (6–20,680) (8) | 2,808 (1 – 15,515) (5) |
| IL-10 (pg/mL)† | 44.5 (32.3–178.7) (4) | 12.6 (4–54.6) (8) | 22.6 (12.9–40) (5) | 11.2 (0.6–98.1) (8) | 6 (2.7–28.2) (5) |
| WBC (× 103 cells/μL; RI, 4.9 × 103 to 15.4 × 103 cells/μL)* | 13.9 (10.1) (7) | 20.2 (6.9) (8) | 22.7 (7.4) (7) | 19.8 (5.5) (8) | 25.7 (5.4) (6) |
| Neutrophils (× 103 cells/μL; RI, 2.9 × 103 to 10.6 × 103 cells/μL)* | 12 (8.9) (7) | 17 (6.8) (8) | 19.1 (6.8) (7) | 15.5 (5.5) (8) | 21 (5.1) (6) |
| Band neutrophils (× 103 cells/μL; RI, 0 × 103 to 0.3 × 103 cells/μL)* | 2 4 (1.8) (7) | 4 6 (3.1) (8) | 1.1 (1.7) (7) | 0.56 (0.4) (8) | 0.87 (0.6) (6) |
| Lactate (mmol/L; RI, < 2.5 mmol/L)* | 2.4 (0.7) (7) | 1.5 (0.9) (8) | 1.1 (0.5) (8) | 11 (0.1) (8) | 0.9 (0.3) (5) |
| Fluid volume (mL/kg)* | — | 113 (75) (8) | 120 (46) (8) | 78 (25) (8) | 58 (30) (8) |
Serum HA concentration was first determined at hospital admission (day 0) prior to undergoing exploratory celiotomy and placement of a closed-suction abdominal drain, and determination of analytes ceased after the abdominal drain was removed (removed on day 3, n = 2; removed on day 4, 6). For 4 dogs, blood collected at day 0 was not available for analysis. The number of dogs that were available each day for each analyte determination varied depending on the availability of blood samples and day of abdominal drain removal.
— = Unknown volume administered prior to presentation and study enrollment.
RI = Reference interval.
Daily serum HA concentrations and biomarkers of inflammation were summarized (Table 1). A significant (P = 0.34) association was not identified between HA concentration and day. However, although not significant, a trend of increased HA concentration was noted on day 2, compared with day 1, for 5 dogs on the basis of visual review of a graph of HA concentration versus day (Figure 1). Concentration of HA then decreased on day 3 for 3 of those dogs and increased on day 3 for 2 of those dogs. Of the 33 samples, the number of samples that had concentrations of IL-6, IL-8, and IL-10 below the lower limit of detection for the assays were 2, 4, and 8, respectively. Only IL-6 concentration was a significant (P < 0.001) predictor of increased serum HA concentration; no significant association was identified between HA concentration and IL-8 (P = 0.75) or IL-10 (P = 0.74) concentrations. Total WBC count (P = 0.31), segmented neutro-phil count (P = 0.30), and band neutrophil count (P = 0.50) were not significant predictors of HA concentration.
Serum HA concentrations in 8 dogs confirmed with septic peritonitis on day 0 and up to day 4 of hospitalization. Serum HA concentration was first determined at hospital admission (day 0) prior to the dogs undergoing exploratory celiotomy and placement of a closed-suction abdominal drain, and determination ceased after the abdominal drain was removed (removed on day 3, n = 2; removed on day 4, 6). For 4 dogs, blood collected at day 0 was not available for analysis. Each colored line indicates the serum HA concentrations for each dog; the black line indicates the median serum HA concentrations for all dogs.
Citation: American Journal of Veterinary Research 82, 7; 10.2460/ajvr.82.7.566
Mean (SD) lactate concentration at the time of admission was 2.4 mmol/L (0.7 mmol/L; Table 1). Lactate concentration was not significantly associated with serum HA concentration. The duration of time for which TADFV on day 1 was calculated ranged from 6 to 18 hours. The TADFV was not a significant (P = 0.23) predictor of HA concentration. However, when TADFV was incorporated into the predictive model that also accounted for IL-6 concentration, TADFV had a significant (P = 0.04) effect on HA concentration. An association was not noted between IL-6 concentration and TADFV.
Types of IV fluids administered throughout hospitalization were crystalloid solutions, natural and synthetic colloid solutions, and blood products. All patients received a balanced crystalloid solutionh as the primary replacement fluid. Three patients received an additional crystal-loid solution (lactated Ringer solution, n = 1; saline [0.9% NaCl] solution, 2; half-strength balanced crystalloid solutionh [50% of the volume of crystalloid solution manually removed and replaced with sterile water], 2). Four patients received a synthetic colloid solution (6% hydroxyethyl starch,i n = 1; pentastarch,j 4). Three patients received a blood product (fresh frozen plasma, n = 2; packed RBCs, 1; whole blood, 1). Sample size was not sufficient to determine whether fluid type affected serum HA concentration.
Discussion
The present study included an evaluation of the changes in serum HA concentrations from hospital admission through recovery in dogs with septic peritonitis. The results did not support the hypothesis that peak serum HA concentrations would be noted at admission; however, significant interactions between HA concentration, TADFV, and cytokine concentration were identified. The results supported the use of serum HA as a biomarker of EG degradation in dogs. As a major component of the extracellular matrix, HA is present in all tissues and can be produced by many cell types.35 Hyaluronic acid is widely distributed throughout the body and is involved in many physiologic functions. It is also actively produced during tissue injury and activates inflammatory cells (innate immune response.)35 In people, increased serum HA concentrations are noted in various disease states including pneumonia,36 hepatitis,37 diabetes mellitus,38 and end-stage kidney failure.39 Some of these conditions increase production or decrease clearance of HA; therefore, HA is not specific to the EG. Interpretation of HA concentration may be difficult in critically ill people with comorbid conditions.40 In dogs with histologic evidence of cirrhosis, serum HA concentration is increased, compared with concentration in dogs with noncirrhotic liver disease41; also, it is increased in dogs with progressive worsening of liver fibrosis42 and in dogs with congenital portosystemic shunts, compared with concentration in healthy dogs.43 Liver disease leads to increased production and decreased clearance of HA.43 In a hemorrhagic shock model with healthy dogs, changes in HA concentration as a marker of EG shedding were noted.26 The full extent of conditions aside from EG disruption that result in increased serum HA concentrations in dogs remains unknown. However, the present study provided an opportunity to evaluate a cohort of dogs with septic peritonitis and without extensive comorbidities that often complicate evaluation of the EG in septic patients in an intensive care unit. Additionally, each dog served as its own control.
The present study revealed daily changes in serum HA concentrations in septic dogs from hospital admission to recovery. A reference interval for serum HA concentration is not yet established in dogs; however, each dog served as its own control for evaluating changes in serum HA concentration over time. Although direct comparisons cannot be made because of assay variations, serum HA concentrations obtained in the present study were concordant with those previously reported for dogs.26,43,44 In the present study, serum HA concentration initially increased, compared with baseline, and concentrations peaked at day 2 or 3 after hospital admission. Similarly, people have an initial increase in serum HA concentration following hospital admission, and the timing of attaining peak concentration is variable.40,45 One study40 reveals that serum HA concentration peaks 3 hours after admission to the emergency department and positively correlates with cumulative IV-administered fluid volumes. Another study45 of people with septic shock shows that plasma HA concentrations are markedly increased on the first day after hospital admission and decreased by day 4, compared with day 1. Interestingly, results of a third study46 indicate that survivors demonstrate a daily decrease in serum HA concentration, compared with baseline, whereas nonsurvivors demonstrate a tendency toward an increase in serum HA concentration over hospitalized days. Although the present study did not identify significant changes in serum HA concentrations over time, such identification may have been confounded by the heterogeneity of IV fluid therapies for the small number of enrolled dogs. In a hemorrhagic shock model with healthy dogs, time of peak HA concentrations varies on the basis of the chosen resuscitation fluid.26
Understanding the time course of EG damage may be useful in determining treatment interventions, such as IV fluid therapy, for these patients. In the present study, a significant association was noted between TADFV and serum HA concentration when accounting for a patient's inflammatory status, namely the concentration of IL-6. As inflammation progressed (IL-6 concentration increased), the TADFV required to cause a significant increase in HA concentration may have been lower. This suggests that patients with more severe inflammation have more extensive EG degradation and that these patients are more susceptible to the exacerbating effects of liberal fluid administration. However, the reverse situation, in which more extensive EG shedding contributes to inflammation, may also be true. In previous studies,20,40,47 the independent effects of IV fluid administration on the EG have also been established. Results of a study40 that includes a comparison between people with simple infections and people with sepsis indicate an increase in serum HA concentration independent from illness severity with increasing cumulative administered fluid volumes after adjustment for biomarkers of systemic inflammation. In healthy dogs, plasma HA concentrations increase after experimental induction of hemorrhagic shock and fluid resuscitation.26 This study26 also reveals possible induction of an inflammatory response, as determined mainly by high plasma concentrations of IL-6 and IL-10, following IV administration of a bolus of a crystalloid fluid. In the present study, however, no association was noted between inflammatory cytokine concentrations and TADFV, making induction of inflammation after fluid administration less likely. Yet, because of the small sample size and variability in cytokine concentrations, a type 2 error cannot be excluded. The relationship between inflammation, fluid administration, and EG degradation is likely complex and multifactorial; however, the results of the present study suggested that fluid administration had a negative effect on the EG in dogs with septic peritonitis and that degradation of the EG had continued over the first few days of hospitalization, such that particular attention to IV fluid therapy may be required during this point of peak HA concentration (ie, high TADFV can lead to worsening of EG degradation). Results may further support the current strategy of rapid de-escalation of fluid therapy to avoid overzealous fluid administration and subsequent worsening of EG degradation during the first few days of hospitalization.2,8,48,49
The present study also included an investigation of various biomarkers of inflammation and their association with serum HA concentrations. A significant association was noted between serum concentrations of IL-6 and HA. Interestingly, although IL-6 increased in association with HA, serum concentrations of IL-8 and IL-10 did not. In vitro findings do not necessarily correlate to an in vivo immune response, and the inconsistent timing of blood sampling may have contributed to variability among the results of the present study and those of previous studies. In an in vitro model, canine leukocytes experimentally exposed to lipopolysaccharide have an early, dramatic increase in IL-6 production almost immediately followed by an increase in IL-10 production.50,51 That study50 shows that lipopolysaccharide-induced leukocyte production of IL-6 and IL-10 is significantly lower within 24 hours of hospitalization in critically ill dogs, compared with healthy dogs, suggesting immune dys-function caused by critical illness. Dogs with sepsis reportedly have higher plasma IL-6 concentrations and lower plasma IL-10 concentrations at the time of hospital admission, compared with healthy dogs.52 That study52 also shows that serum IL-8 concentrations at the time of admission did not differ between healthy dogs, dogs with sepsis, and dogs with non-infectious inflammatory response syndrome. Yet, another study53 reveals that IL-8 production increases in septic dogs, compared with healthy dogs.
Peak cytokine concentrations may be time dependent or related to illness severity. In the present study, peak cytokine concentrations may not have been identified at the time of presentation or at predetermined times of blood sampling. To provide context for the interpretation of reported findings, the results of 1 study54 indicate the reference intervals for serum concentrations of IL-6, IL-8, and IL-10 in healthy dogs, as follows: IL-6, 0 to 133 pg/mL; IL-8, 0 to 3,775 pg/mL; and IL-10, 0 to 2,064 pg/mL. That study54 also reveals the median serum concentrations of IL-6, IL-8, and IL-10 to be 22 pg/mL (range, 0 to 311 pg/mL), 1,719 pg/mL (0 to 11,117 pg/mL), and 0 pg/mL (0 to 3,200 pg/mL), respectively, in septic dogs at 1 unspecified time point. The results of the present study indicated that serum IL-6 and IL-10 concentrations were within reference intervals for septic dogs, but IL-8 concentrations were higher at all time points. Peak cytokine concentrations can vary, and because the time of collection was not provided in the previous study,54 direct comparison of results to those of the present study was not possible.
Inflammation in septic patients is known to contribute to EG degradation through various mechanisms, and EG degradation is well documented in human clinical trials and animal research models.55,56 The association between serum IL-6 and HA concentrations identified in the present study was consistent with previously reported24,57 findings in which EG shedding increases with systemic inflammation. However, because of the nature of this preliminary study, determining the primary contributor to increased serum HA concentrations was not possible. Measurement of other biomarkers may have helped clarify and support the findings of the present study.
Hyaluronic acid is not a specific biomarker of EG health. Therefore, the use of additional EG biomarkers may have strengthened the finding that increased serum HA concentrations were associated with EG degradation; however, research of the EG is in its infancy in veterinary medicine. At the time of the present study, HA was the only EG biomarker validated for use in dogs; other EG biomarkers including syndecan-1, heparan sulfate, and chondroitin sulfate are validated for use in people.14 Disruption of the EG can range in severity from selective cleavage of heparan sulfate and chondroitin sulfate to major disturbances represented by removal of entire syndecan core proteins.56 Syndecan-1 is commonly evaluated in studies of people and is related to endothelial damage and EG degradation. Because syndecan-1 is a transmembranous core protein, it is considered a biomarker of more extensive EG damage.55 Its concentration also correlates with serum concentrations of inflammatory cytokines, including IL-10, in people.16 Therefore, measurement of syndecan-1 would have been an interesting addition to the present study to facilitate an understanding of the associations between EG biomarkers and inflammatory markers. Unfortunately, previous attempts58 to use the commercially available syndecan-1 ELISAs validated for use with people showed that the assay did not perform adequately for use with dogs.
Another helpful addition to the present study would have been measurement of atrial natriuretic peptide, which increases secondary to increased blood volume and precipitates shedding of EG constituents.30 The results of the present study suggested that as inflammation increased, a dog's susceptibility to EG damage from IV fluid administration increased. However, an alternative explanation to this finding may be transient volume overloading. In people with good cardiopulmonary health, volume loading with 20 mL of a synthetic colloid solution/kg over 15 minutes results in a significant increase in plasma atrial natriuretic peptide and serum HA concentrations, compared with baseline.20 These findings indicate that EG shedding can be induced by fluid administration in people with low-level inflammation as well. With respect to volume loading, the present study included the TADFV for determining the effect of fluid volume on serum HA concentrations. However, knowledge of the fluid administration rate and the timing of fluid administration in relation to blood-sampling time points may have been enlightening. Knowledge of these details coupled with atrial natriuretic peptide concentrations may have provided additional insight of the stimuli for increased HA concentrations.
The present study had several limitations, including a small sample size, the study's observational design, the variable treatments administered prior to hospital admission, and the use of only 1 biomarker of EG degradation. Therefore, conclusions may not be definitive and may not be extrapolated to a larger population. Also, prolonged storage time may have had an effect on cytokine and HA concentrations. All serum samples were stored at −80°C from the time of collection and were subjected to 1 freeze-thaw cycle before analyses. Cytokine stability in frozen serum samples from dogs is not known. However, degradation of ≥ 50% of the initial concentrations for many inflammatory cytokines of people is expected within 2 to 3 years of storage; specifically, IL-8 appears to be subject to more rapid deterioration after 1 year.59 Yet, 1 freeze-thaw cycle of the samples in the present study was unlikely to have caused relevant variation in cytokine concentrations. Research59 indicates that IL-6 and IL-10 concentrations are stable throughout many freeze-thaw cycles, and IL-8 starts to decrease after > 1 freeze-thaw cycle. The HA ELISA kite used in the present study was approved for use with human samples that undergo up to 3 freeze-thaw cycles, and the canine-specific multiplex cytokine immunoassayf was approved for use with canine samples that undergo up to 2 freeze-thaw cycles.
In summary, the present study was the first to report, to the authors' knowledge, serum HA concentrations throughout the course of septic peritonitis in dogs. An association was noted between serum concentrations of the proinflammatory cytokine IL-6 and HA, and a possible increase in susceptibility to EG disruption was noted secondary to IV fluid administration. Evidence challenging the rationale of early aggressive fluid resuscitation in septic patients is increasing. However, whether restrictive fluid strategies serve to prevent direct damage to the EG or whether an already disrupted EG requires a different approach remains unclear. The present study may have provided a foundation for understanding the complex changes in the EG during the course of sepsis in dogs. Future prospective investigation of the EG is warranted and may increase knowledge of the pathophysiology of sepsis and insight into developing more tailored fluid strategies for septic dogs.
Acknowledgments
This manuscript represents a portion of a thesis submitted by Dr. Shaw to the Department of Clinical Studies, Ontario Veterinary College, as a partial fulfillment of the requirements for a Master of Science degree.
Funded by the Ontario Veterinary College Pet Trust Fund. The funding agency had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that there were no conflicts of interest.
Presented online in abstract form at the European Veterinary Emergency and Critical Care Congress, June 2020.
The authors thank Michelle Beaudoin-Kimble for technical assistance.
Abbreviations
| APPLE | Acute patient physiologic and laboratory evaluation |
| EG | Endothelial glycocalyx |
| HA | Hyaluronic acid |
| IL | Interleukin |
| MFI | Mean fluorescence intensity |
| TADFV | Total administered daily fluid volume |
Footnotes
ABL800 FLEX, Radiometer Canada, London, ON, Canada.
AirLife reduced heparin arterial blood sampler, Vyaire Medical Inc, Mettawa, Ill.
Becton Dickinson and Co, Franklin Lakes, NJ.
Advia 2120, Siemens, Burlington, ON, Canada.
TECOmedical AG, Sissach, Switzerland.
MilliporeSigma, Burlington, Mass.
SAS/STAT, version 9.4, SAS Institute Inc, Cary, NC.
Plasma-Lyte A, Baxter Corp, Mississauga, ON, Canada.
Voluven, Fresenius Kabi Canada, Toronto, ON, Canada.
Pentaspan 10%, Bristol-Myers Squibb Canada, Montreal, QC, Canada.
References
1. Ince C. The microcirculation is the motor of sepsis. Crit Care 2005;9:S13–S19.
2. Mouncey PR, Osborn TM, Power GS, et al. Trial of early, goal-directed resuscitation for septic shock. N Engl J Med 2015;372:1301–1311.
3. Peake SL, Delaney A, Bailey M, et al. Goal-directed resuscitation for patients with early septic shock. N Engl J Med 2014;371:1496–1506.
4. Wu X, Hu Z, Yuan H, et al. Fluid resuscitation and markers of glycocalyx degradation in severe sepsis. Open Med (Wars) 2017;12:409–416.
5. Yealy DM, Kellum JA, Huang DT, et al. A randomized trial of protocol-based care for early septic shock. N Engl J Med 2014;370:1683–1693.
6. Maitland K, Kiguli S, Opoka RO, et al. Mortality after fluid bolus in African children with severe infection. N Engl J Med 2011;364:2483–2495.
7. Rivers EP, Kruse JA, Jacobsen G, et al. The influence of early hemodynamic optimization on biomarker patterns of severe sepsis and septic shock. Crit Care Med 2007;35:2016–2024.
8. Acheampong A, Vincent JL. A positive fluid balance is an independent prognostic factor in patients with sepsis. Crit Care 2015;19:251.
9. Alphonsus CS, Rodseth RN. The endothelial glycocalyx: a review of the vascular barrier. Anaesthesia 2014;69:777–784.
10. Curry FE, Adamson RH. Endothelial glycocalyx: permeability barrier and mechanosensor. Ann Biomed Eng 2012;40:828–839.
11. Pries AR, Secomb TW, Gaehtgens P. The endothelial surface layer. Pflugers Arch 2000;440:653–666.
12. Rahbar E, Cardenas JC, Baimukanova G, et al. Endothelial glycocalyx shedding and vascular permeability in severely injured trauma patients. J Transl Med 2015;13:117.
13. Weinbaum S, Tarbell JM, Damiano ER. The structure and function of the endothelial glycocalyx layer. Annu Rev Biomed Eng 2007;9:121–167.
14. Gaudette S, Hughes D, Boller M. The endothelial glycocalyx: structure and function in health and critical illness. J Vet Emerg Crit Care (San Antonio) 2020;30:117–134.
15. Lenart M, Rutkowska-Zapala M, Baj-Krzyworzeka M, et al. Hyaluronan carried by tumor-derived microvesicles induces IL-10 production in classical (CD14++CD16-) monocytes via PI3K/Akt/mTOR-dependent signalling pathway. Immunobiology 2017;222:1–10.
16. Haywood-Watson RJ, Holcomb JB, Gonzalez EA, et al. Modulation of syndecan-1 shedding after hemorrhagic shock and resuscitation. PLoS One 2011;6:e23530.
17. Nieuwdorp M, van Haeften TW, Gouverneur MCLG, et al. Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 2006;55:480–486.
18. Schött U, Solomon C, Fries D, et al. The endothelial glycocalyx and its disruption, protection and regeneration: a narrative review. Scand J Trauma Resusc Emerg Med 2016;24:48.
19. Burke-Gaffney A, Evans TW. Lest we forget the endothelial glycocalyx in sepsis. Crit Care 2012;16:121.
20. Chappell D, Bruegger D, Potzel J, et al. Hypervolemia increases release of atrial natriuretic peptide and shedding of the endothelial glycocalyx. Crit Care 2014;18:538.
21. Johansson PI, Stensballe J, Rasmussen LS, et al. A high admission syndecan-1 level, a marker of endothelial glycocalyx degradation, is associated with inflammation, protein C depletion, fibrinolysis, and increased mortality in trauma patients. Ann Surg 2011;254:194–200.
22. Nelson A, Berkestedt I, Schmidtchen A, et al. Increased levels of glycosaminoglycans during septic shock: relation to mortality and the antibacterial actions of plasma. Shock 2008;30:623–627.
23. Becker BF, Jacob M, Leipert S, et al. Degradation of the endothelial glycocalyx in clinical settings: searching for the shed-dases. Br J Clin Pharmacol 2015;80:389–402.
24. Ostrowski SR, Gaïni S, Pedersen C, et al. Sympathoadrenal activation and endothelial damage in patients with varying degrees of acute infectious disease: an observational study. J Crit Care 2015;30:90–96.
25. Padberg JS, Wiesinger A, di Marco GS, et al. Damage of the endothelial glycocalyx in chronic kidney disease. Atherosclerosis 2014;234:335–343.
26. Smart L, Boyd CJ, Claus MA, et al. Large-volume crystalloid fluid is associated with increased hyaluronan shedding and inflammation in a canine hemorrhagic shock model. Inflammation 2018;41:1515–1523.
27. Yini S, Heng Z, Xin A, et al. Effect of unfractionated heparin on endothelial glycocalyx in a septic shock model. Acta Anaesthesiol Scand 2015;59:160–169.
28. Plickert HD, Bondzio A, Einspanier R, et al. Hyaluronic acid concentrations in synovial fluid of dogs with different stages of osteoarthritis. Res Vet Sci 2013;94:728–734.
29. Barelli S, Alberio L. The role of plasma transfusion in massive bleeding: protecting the endothelial glycocalyx? Front Med (Lausanne) 2018;5:91.
30. Bruegger D, Jacob M, Rehm M, et al. Atrial natriuretic peptide induces shedding of endothelial glycocalyx in coronary vascular bed of guinea pig hearts. Am J Physiol Heart Circ Physiol 2005;289:H1993–H1999.
31. Chappell D, Hofmann-Kiefer K, Jacob M, et al. TNF-α induced shedding of the endothelial glycocalyx is prevented by hydrocortisone and antithrombin. Basic Res Cardiol 2009;104:78–89.
32. Torres LN, Sondeen JL, Ji L, et al. Evaluation of resuscitation fluids on endothelial glycocalyx, venular blood flow, and coagulation function after hemorrhagic shock in rats. J Trauma Acute Care Surg 2013;75:759–766.
33. Guieu L-VS, Bersenas AM, Brisson BA, et al. Evaluation of peripheral blood and abdominal fluid variables as predictors of intestinal surgical site failure in dogs with septic peritonitis following celiotomy and the placement of closed-suction abdominal drains. J Am Vet Med Assoc 2016;249:515–525.
34. Hayes G, Mathews K, Doig G, et al. The acute patient physiologic and laboratory evaluation (APPLE) score: a severity of illness stratification system for hospitalized dogs. J Vet Intern Med 2010;24:1034–1047.
35. Liang J, Jiang D, Noble PW. Hyaluronan as a therapeutic target in human diseases. Adv Drug Deliv Rev 2016;97:186–203.
36. Inokoshi Y, Tanino Y, Wang X, et al. Clinical significance of serum hyaluronan in chronic fibrotic interstitial pneumonia. Respirology 2013;18:1236–1243.
37. Guéchot J, Poupon RE, Giral P, et al. Relationship between procollagen III aminoterminal propeptide and hyaluronan serum levels and histological fibrosis in primary biliary cirrhosis and chronic viral hepatitis C. J Hepatol 1994;20:388–393.
38. Mine S, Okada Y, Kawahara C, et al. Serum hyaluronan concentration as a marker of angiopathy in patients with diabetes mellitus. Endocr J 2006;53:761–766.
39. Turney JH, Davison AM, Forbes MA, et al. Hyaluronic acid in end-stage renal failure treated by haemodialysis: clinical correlates and implications. Nephrol Dial Transplant 1991;6:566–570.
40. Smart L, Macdonald SPJ, Burrows S, et al. Endothelial glycocalyx biomarkers increase in patients with infection during emergency department treatment. J Crit Care 2017;42:304–309.
41. Kanemoto H, Ohno K, Sakai M, et al. Blood hyaluronic acid as a marker for canine cirrhosis. J Vet Med Sci 2009;71:1251–1254.
42. Glińska-Suchocka K, Orłowska A, Spużak J, et al. Suitability of using serum hialuronic acid concentrations in the diagnosis of canine liver fibrosis. Pol J Vet Sci 2015;18:873–878.
43. Seki M, Asano K, Sakai M, et al. Serum hyaluronic acid in dogs with congenital portosystemic shunts. J Small Anim Pract 2010;51:260–263.
44. Nganvongpanit K, Itthiarbha A, Ong-Chai S, et al. Evaluation of serum chondroitin sulfate and hyaluronan: biomarkers for osteoarthritis in canine hip dysplasia. J Vet Sci 2008;9:317–325.
45. Sallisalmi M, Tenhunen J, Kultti A, et al. Plasma hyaluronan and hemorheology in patients with septic shock: a clinical and experimental study. Clin Hemorheol Microcirc 2014;56:133–144.
46. Anand D, Ray S, Srivastava LM, et al. Evolution of serum hyaluronan and syndecan levels in prognosis of sepsis patients. Clin Biochem 2016;49:768–776.
47. Hippensteel JA, Uchimido R, Tyler PD, et al. Intravenous fluid resuscitation is associated with septic endothelial glycocalyx degradation. Crit Care 2019;23:259.
48. Kelm DJ, Perrin JT, Cartin-Ceba R, et al. Fluid overload in patients with severe sepsis and septic shock treated with early goal-directed therapy is associated with increased acute need for fluid-related medical interventions and hospital death. Shock 2015;43:68–73.
49. Sirvent J-M, Ferri C, Baró A, et al. Fluid balance in sepsis and septic shock as a determining factor of mortality. Am J Emerg Med 2015;33:186–189.
50. Hoffman D, Amorim J, DeClue A. Immune function in critically ill dogs. J Vet Intern Med 2018;32:208–216.
51. Deitschel SJ, Kerl ME, Chang CH, et al. Age associated changes to pathogen associated molecular pattern induced inflammatory mediator production in dogs. J Vet Emerg Crit Care (San Antonio) 2010;20:494–502.
52. DeClue AE, Sharp CR, Harmon M. Plasma inflammatory mediator concentrations at ICU admission in dogs with naturally developing sepsis. J Vet Intern Med 2012;26:624–630.
53. Goggs R, Letendre JA. Evaluation of the host cytokine response in dogs with sepsis and noninfectious systemic inflammatory response syndrome. J Vet Emerg Crit Care (San Antonio) 2019;29:593–603.
54. Johnson V, Burgess B, Morley P, et al. Comparison of cytokine responses between dogs with sepsis and dogs with immune-mediated hemolytic anemia. Vet Immunol Immunopathol 2016;180:15–20.
55. Chelazzi C, Villa G, Mancinelli P, et al. Glycocalyx and sepsis-induced alterations in vascular permeability. Crit Care 2015;19:26.
56. Kolářová H, Ambrůzová B, Švihálková Šindlerová L, et al. Modulation of endothelial glycocalyx structure under inflammatory conditions. Mediators Inflamm 2014;2014:694312.
57. Steppan J, Hofer S, Funke B, et al. Sepsis and major abdominal surgery lead to flaking of the endothelial glycocalix. J Surg Res 2011;165:136–141.
58. Briganti A, Di Franco C, Meucci V. Endovascular shedding markers in critically ill patients, in Proceedings. 17th Annu Euro Vet Emerg Crit Care Cong 2019;S36–S37.
59. de Jager W, Bourcier K, Rijkers GT, et al. Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays. BMC Immunol 2009;10:52.
