The microcirculation is responsible for many essential physiologic functions.1 Endothelial cells line the microvasculature and synthesize a luminal surface layer called the endothelial glycocalyx (EGC).1 The EGC is a gel-like layer, consisting of a matrix of proteoglycans, glycosaminoglycans, and glycoproteins.1 Heparan sulfate and hyaluronan are the main glycosaminoglycans, which contribute to endothelial integrity and prevention of inappropriate thrombosis. Together, the EGC and microcirculatory structure make up the microcirculatory unit. Each component of the microcirculatory unit is key in maintaining its essential functions including regulating inflammation, coagulation, and maintenance of microcirculatory blood flow.2 In states of shock where blood flow to the microcirculation is decreased, hypoxia perpetuates an inflammatory response and subsequent endothelial dysfunction.3–4 Injury to and pathology of the microvasculature results in EGC shedding and degradation, resulting in increased permeability and pathological cellular interactions, ultimately playing a role in the development of systemic inflammatory response syndrome, coagulopathy, and multiple organ failure.2
Therefore, there may be significant clinical implications associated with EGC shedding and microcirculatory dysfunction. Further, normalization of macrocirculatory parameters does not necessarily equate to normalization of microcirculatory parameters and reversal of pathologic derangements.5 In other words, microcirculatory dysfunction may persist despite normalization of heart rate, blood pressure, and other macrocirculatory parameters.6–7 This is of particular importance because in humans continued microcirculatory derangement has been associated with the development of organ failure and worse outcomes.7–8 The ability to noninvasively evaluate the microcirculation and EGC at the bedside might allow for a more effective assessment of an ongoing shock state and determination of optimal resuscitation, ultimately improving outcomes.
Several techniques exist to evaluate the microcirculation and EGC.9–10 Sidestream dark field (SDF) sublingual videomicroscopy allows for visualization of microcapillaries under a thin layer of mucosa and can provide measurement of vessel density, estimation of flow, and estimation of EGC thickness.8,11,12 This technique provides an estimation of EGC thickness by measurement of the perfused boundary region (PBR).13 The PBR is, by definition, the lateral movement of RBCs into the most luminal layer of the EGC.13 When the EGC is damaged, it thins, increasing vessel lumen diameter allowing more RBC penetration into the EGC, and resulting in an increased PBR. In other words, the PBR is an inverse measure of the EGC thickness. SDF videomicroscopy has been used to evaluate the microcirculation in various disease states in both people and dogs.14–17
Measurement of EGC components in plasma or serum has also been utilized in the evaluation of EGC health. In hemorrhagic shock specifically, increases in circulating EGC components including heparan sulfate, syndecan-1, and hyaluronan have been demonstrated in rats, guinea pigs, dogs, and humans.18–21 Measurable components of the EGC include syndecan-1, hyaluronan, heparan sulfate, and VE-cadherin.22–23 Syndecan-1 is a transmembrane proteoglycan found on both epithelial and endothelial cells. It has important anti-inflammatory effects and has been found to be an independent marker of EGC degradation and mortality in humans.24–25 Heparan sulfate, a fragment of syndecan-1, promotes pathological inflammatory responses by playing a role in leukocyte activation and cytokine production, along with other immune-modulating functions.26 Studies18,27 evaluating EGC degradation have documented increases in heparan sulfate in rats, although no significant increases have been noted in humans. Hyaluronan is a critical component of the extracellular matrix and has been previously evaluated in conjunction with EGC damage in dogs and people.21–22 Hyaluronan stimulates chemokine and cytokine production and has both anti- and proinflammatory effects.28–29 Finally, VE-cadherin is a major endothelial adhesion molecule that plays a role in the control of vascular permeability and leukocyte extravasation and has been evaluated in humans.19,30,31 Although some of these biomarkers of EGC damage have previously been measured in dogs, the current study seeks to assess a breadth of biomarkers in a hemorrhagic shock model.21,32,33
The goal of this study was to assess microcirculatory insufficiency and EGC shedding following induction of hemorrhagic shock and subsequent fresh whole blood resuscitation in dogs, using SDF microscopy and concurrent comprehensive analysis of EGC components, including syndecan-1, hyaluronan, heparin sulfate, and VE-cadherin. For the purposes of this study, these will be referred to as “biomarkers.” We hypothesized that impacts on the microcirculation due to hemorrhagic shock in anesthetized dogs would lead to increased PBR and EGC plasma biomarker concentrations, along with microcirculatory derangements.
Methods
Animals
Eight purpose-bred, female spayed beagles were used in this study. Each dog was determined to be healthy based on physical examination, CBC, and serum biochemistry panel.
All procedures were approved by the Institutional Animal Care and Use Committee at Colorado State University (IACUC No. 2237).
Dogs were premedicated with hydromorphone (0.1 mg/kg, IM) and preoxygenated. General anesthesia was induced with propofol (5 to 10 mg/kg, IV) to effect. Dogs were orotracheally intubated, and anesthesia was maintained using isoflurane vaporized in 100% fraction of inspired oxygen.
After anesthesia induction, a 16-gauge over-the-needle catheter was placed in the jugular vein for sampling, and a 20-gauge over-the-needle catheter was placed in the dorsal metatarsal artery for direct blood pressure monitoring. Continuous monitoring included ECG, end-tidal carbon dioxide, pulse oximetry, and arterial blood pressure (Suntech Waveline Touch; DRE Inc). A 7.5-French Swan-Ganz catheter (Biosensors International Inc) was placed in the pulmonary artery via the opposite jugular vein with a flow-directed technique, and the location of the catheter was confirmed with pressure waveform analysis. Cardiac output (CO) was measured using thermodilution according to manufacturer guidelines. A temperature probe was placed in an ice bath of syringes containing 5 mL of 0.9% NaCl. A 5-mL bolus of 0.9% NaCl was injected into the proximal port of the Swan-Ganz catheter, and the CO was calculated by system software (Hemosphere Advanced Monitoring Platform; Edwards LifeSciences). The average of duplicate CO measurements was used for statistical analysis. A forced air warming device and heated water blanket were used to maintain normothermia.
Experimental model
The current study was part of a larger project that also included evaluation of a CO monitor and shock index in a hemorrhagic shock model.34 Measurements were obtained at 5 time points in our study. Time point 0 (TP0) was before the induction of general anesthesia. Data for time point 1 (TP1) were collected after induction of general anesthesia and instrumentation, once mean arterial pressure (MAP) was stable for at least 10 minutes. Blood was then collected from the jugular catheter using an aseptic technique into bags containing citrate phosphate dextrose adenine (CPDA-1) for later return to the dogs. Blood removal occurred until a MAP of 40 mm Hg was reached or 60% of blood volume was removed, whichever point was reached first. After 10 minutes, time point 2 (TP2) data were collected. Then, one-half the volume of shed blood was infused, 10 minutes were allowed for equilibration, and measurements were obtained for time point 3 (TP3). Next, infusion of previously removed blood was completed. Ten minutes afterward, final measurements were collected for time point 4 (TP4). Blood was infused in 2 steps (50% then 100%) to assess multiple volume states. After completion of measurements, the dogs were recovered from anesthesia. All dogs survived without complication. The experimental design with time points, data collection, blood removal, and autotransfusion is depicted (Figure 1).
Experimental design with time points, data collection, blood removal, and autotransfusion including blood collection from baseline (T0) through anesthesia (T1), hemorrhagic shock (T2), and resuscitation (T3 to T4) for measurement of endothelial glycocalyx components in plasma and sidestream dark field (SDF) measurements of the microcirculation at TP1 to TP4.
Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0134
For assessment of EGC biomarker concentrations, 4 mL of blood was collected in 2 EDTA blood collection tubes at TP0 to TP4. Tubes were inverted 8 to 10 times, maintained at 4 °C, and then centrifuged within 2 hours of acquisition at 1,000 X g for 15 minutes at room temperature. Plasma was removed immediately after centrifugation and stored at 2 to 8 °C until aliquoted. Plasma was aliquoted within 12 hours of collection and stored at –80 °C until batch analysis within 12 months of collection. Sample aliquots were thawed just before analysis and maintained on ice until they were added to the assay plates by a single operator (TLW). Samples were run in duplicate. Individual assays were completed on the same day with a fresh sample aliquot. A second operator coordinated and verified sample identification and assisted with assay completion (JDT). Concentrations of canine heparan sulfate (AMS.E08H1352; AMSbio), canine VE-cadherin (Canine Cadherin 5; AMS.E08C0500; AMSbio), and hyaluronan (DHYALO; R&D Systems) were determined by ELISA per manufacturer instructions, other than the delayed plasma separation.21 Masks and gloves were worn when completing the hyaluronan ELISA to prevent saliva contamination as recommended by the manufacturer. Syndecan-1 (DY2780; R&D Systems) was measured with a human ELISA kit previously utilized on canine samples according to manufacturer instructions.35
Microcirculatory variables were evaluated at TP1 to TP4 with a SDF videomicroscope (Glycocheck; MicroVascular Health Solutions Inc), placed on the sublingual mucosa. The Glycocheck videomicroscope uses a 540-nm green light-emitting stroboscopic diode to detect hemoglobin of passing RBCs. Images are captured using an 5X objective with 0.2 numerical aperture, providing a 325-fold magnification in 720 X 576 pixels. The software records 1-second clips, each consisting of 23 frames. Recording begins automatically once the software detects images of sufficient quality, meaning that the intensity and focus are appropriate for calculations and that the camera is held sufficiently still. Measurement points are defined at 10-µm intervals and the software automatically detects vessels, providing calculations for vessels 5 to 25 µm wide, only. Acquisition is complete when 3,000 measurement points have been acquired. All SDF measurements were performed by the same individual experienced in performing sublingual measurements with the device (JG). Care was taken to limit pressure artifacts by ensuring erythrocytes could be seen traveling through blood vessels during measurements. Images were recorded only in the absence of air bubbles, excessive amounts of saliva, excessive numbers of vessels in the frame, and excessive numbers of large venules. All measurements in each dog at each time point were performed within 5 minutes. The analysis of all images was performed using proprietary software (GlycoCheck version 5.2.2.21301; Microvascular Health Solutions Inc).
Microcirculatory SDF images collected at each time point were processed by the Glycocheck software to produce values for RBC flow, total vessel density (TVD), capillary blood volume relative (CBVrel), capillary blood volume absolute (CBVabs), and PBR.12,18,36,37 RBC flow was defined as the velocity of a RBC within an individual vessel. It was determined by dividing the displacement of the RBC by the time between video frames and expressed as micrometers per second.37 TVD was determined by multiplying the number of vascular segments containing RBCs by the capillary segment length.16,37 Vessels containing more than 50% RBCs with a velocity greater than 0 were detected by the software, and the density of 4- to 25-µm vessels was analyzed.37 The CBVabs was determined from the number of capillary segments multiplied by the capillary segment length and the segment-specific capillary cross-sectional area.37 CBVrel was a functional estimate using the absolute capillary blood volume and comparing the average volume of RBCs in capillaries (5 to 7 µm) to larger blood vessels (10 to 25 µm) in the same frame.37 The PBR was calculated by the software as the dynamic lateral movement of RBCs into the permeable part of the EGC. The superficial, luminal layer of the glycocalyx is penetrable to RBCs and hence is called the PBR.13 An intact glycocalyx is thicker and less penetrable to RBCs, resulting in a smaller PBR, while a larger PBR is noted with a damaged EGC. The PBR is thus an inverse measure of the EGC thickness. The proprietary software calculates the PBR from the intensity profile at every measurement point.13 A more gradual increase in intensity profile indicates a thicker PBR and thus a thinner EGC.13
Statistical analysis
Normality of continuous variables was tested using the D'Agonisto-Pearson test. Normally distributed variables are presented with mean and SD, and nonnormally distributed variables are presented as median and range. Differences between time points for each variable were tested using an ANOVA for repeated measures with Bonferroni correction after log transformation if not normally distributed, as recommended for a small sample size.38 If epsilon (Greenhouse-Geisser estimate of sphericity) was more than 0.75, then the Huynh-Feldt correction was used. If epsilon was less than 0.75, then the more conservative correction according to Greenhouse-Geisser was used.39 Some samples of syndecan-1 resulted in concentrations below detection; these results were reported as 0 for statistical analysis. Two variables (syndecan-1 and VE-cadherin) included results that were “0,” and the variables were nonnormally distributed, so they could not be log transformed, and a Friedman test was performed as appropriate. Below detection results were represented as “0.” All statistical analyses were performed with commercially available software (MedCalc Version 20.026; Medcalc Software Ltd). Significance was set at P < .05.
Results
Data were obtained from 8 healthy, female beagle dogs aged 2.5 to 5.5 years old, with a mean weight of 8.9 kg (SD ± 1.56 kg). The mean volume of blood withdrawn to induce hypovolemic shock was 40 ± 12 mL/kg. Mean MAP at TP1, TP2, TP3, and TP4 was 74.25 ± 7.17 mm Hg, 49.50 ± 13.74 mm Hg, 63.50 ± 13.29 mm Hg, and 71.38 ± 8.77 mm Hg, respectively. CO measured by thermodilution at TP1, TP2, TP3, and TP4 was 2.57 ± 1.01 L/min, 0.8 ± 0.36 L/min, 1.81 ± 0.57 L/min, and 2.93 ± 1.22 L/min, respectively. CO was significantly different from TP1 to TP2 (P < .001) and not different from TP1 to TP4 (P = .71). The mean time from initiation of general anesthesia to TP1 was 73.0 ± 16.0 minutes. The mean time from instrumented baseline under anesthesia (TP1) to hemorrhage (TP2) was 58.6 ± 9.0 minutes. All 8 dogs were included in the descriptive data analysis. Descriptive analysis of EGC plasma biomarkers (Figure 2) and microcirculatory variables (Figure 3) revealed variable changes over time. Descriptive SDF microcirculatory data at each time point are summarized (Table 1). Descriptive biomarker data at each time point are summarized (Table 2). Due to the device running out of battery during the TP4 measurement, dog 8 had missing data for all microcirculatory variables at TP4 and was therefore excluded from the repeated measures analysis for SDF variables (n = 7). Measurements obtained for flow, TVD, CBVrel, CBVabs, and PBR were not significantly different over the 4 time points (P = 1.00, P = .86, P = .16, P = .88, and P = .78, respectively). The blood sample from TP3 for dog 2 was misplaced. Therefore, dog 2 was excluded from the repeated measures analysis for EGC biomarker concentrations (n = 7). Heparan sulfate was statistically different over time (P = .016), with pairwise comparisons showing a statistical difference between time points TP1 and TP2 (P = .031). Mean hyaluronan values were not statistically different over the 5 time points (P = .091). Syndecan-1 and VE-cadherin were also not statistically different over the 5 time points (P = .230 and P = .272, respectively).
Plasma biomarker measurements at each time point for all dogs (n = 8) from baseline through anesthesia, hemorrhagic shock, and resuscitation. A—Hyaluronan concentration over time. B—Heparan sulfate concentration over time. C—VE-Cadherin concentration over time. Bars indicate median, IQR, minimum values, and maximum values.
Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0134
Microcirculatory measurements at each time point for all dogs (n = 8) from anesthesia, hemorrhagic shock, and resuscitation. A—RBC flow (Flow) over time. B—Capillary blood volume absolute (CBVabs) over time. C—Capillary blood volume relative (CBVrel) over time. D—Total vessel density (TVD) over time. E—Perfused boundary region (PBR) over time. Bars indicate median, IQR, minimum values, and maximum values.
Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.06.0134
Microvascular variable data including RBC flow (Flow), total vessel density (TVD), capillary blood volume relative (CBVrel), capillary blood volume absolute (CBVabs), and perfused boundary region (PBR) at each time point.
| TP1 (n = 8) | TP2 (n = 8) | TP3 (n = 8) | TP4 (n = 7) | |
|---|---|---|---|---|
| Flow (µm/s) | 266.30 ± 94.53 (103.30, 419.10) | 174.01 ± 42.63 (89.60, 214.10) | 204.75 (129.70, 584.50)† | 309.80 (154.60, 543.30)†† |
| TVD (mm/m2) | 230.64 ± 74.50 (103.50, 334.30) | 235.50 (51.70, 1,914.30)† | 224.36 ± 110.97 (99.20, 453.90) | 222.30 (140.30, 348.70)†† |
| CBVrel (103 μm3) | 1.22 ± 0.15 (0.94, 1.38) | 1.32 ± 0.11 (1.21, 1.53) | 1.23 ± 0.07 (1.10, 1.32) | 1.36 (1.06, 1.50)†† |
| CBVabs (103 μm3) | 16.01 ± 6.37 (4.40, 21.90) | 18.50 (1.50, 94.30)† | 15.15 ± 5.95 (7.40, 24.00) | 12.20 (8.70, 30.80)†† |
| PBR (μm) | 2.27 ± 0.27 (1.77, 2.59) | 2.32 ± 0.22 (1.86, 2.60) | 2.40 ± 0.22 (2.11, 2.74) | 2.29 (1.75, 2.69)†† |
Normally distributed data are reported as mean ± SD and range. Nonnormally distributed data and data not able to be tested for normality are presented as median (range) as denoted.
TP1 = Anesthetized, postinstrumentation; TP2 = Hemorrhagic shock. TP3 = Retransfusion (50% shed blood). TP4 = Retransfusion (100% shed blood).
Biomarker plasma concentrations at each time point.
| TP0 (n = 8) | TP1 (n = 8) | TP2 (n = 8) | TP3 (n = 7) | TP4 (n = 8) | |
|---|---|---|---|---|---|
| Heparan sulfate (ng/mL) | 53.6 ± 15.9 (24.8, 77.7) | 51.2 ± 14.4 (26.2, 72.8) | 37.7 ± 11.1 (26.0, 60.4) | 35.6 (26.0, 41.3)†† | 37.2 ± 9.9 (25.2, 56.5) |
| Hyaluronan (ng/mL) | 36.7 ± 35.6 (5.8, 105.1) | 16.8 (7.1, 61.7)† | 14.7 (6.0, 64.6)† | 20.2 (5.8, 61.7)†† | 28.6 ± 19.9 (6.8, 68.2) |
| Syndecan-1 (pg/mL) | 0 (below detection, 1,545.7)† | 0 (below detection 758.0)† | 0 (below detection, 638.9)† | 0 (below detection, 768.0)†† | 0 (below detection, 574.6)† |
| VE-cadherin (ng/mL) | 0.85 ± 0.94 (0, 2.52) | 0.18 (0, 2.30)† | 0.58 ± 0.63 (0, 1.83) | 0.12 (0, 2.11)†† | 0.12 (0, 2.25)† |
See Table 1 for remainder of key.
Discussion
In this hemorrhagic shock model, expected increases in measured circulating EGC biomarkers were not appreciated, and there was no significant change in microcirculatory variables or PBR over time as assessed by SDF microscopy.
Evaluation of microcirculatory variables with SDF imaging has previously been used to evaluate the microvasculature in dogs in health and disease.17,40–43 In this study, measured PBR, used as a surrogate for EGC thickness, was comparable to published results in dogs, cats, and people.12,36,37,41,44 One study36 found a median PBR in 5- to 25-µm vessels of 2.39 µm in healthy, sedated cats. A recent abstract44 on anesthetized dogs found a mean PBR of 2.04 µm in anesthetized normal dogs. An additional study41 in healthy dogs showed the PBR of the jejunum was approximately 2.17 µm, which is comparable to measurements that have been found in infants (approx 2.5 µm) and human adults (approx 2.0 to 2.2 µm).
In contrast to the microcirculatory and PBR findings in the current study, several previous studies6,17,45 investigating hemorrhagic shock in humans, animal models, and dogs have demonstrated microcirculatory derangement and EGC degradation with SDF imaging. However, different SDF imaging technologies vary in the variables they measure and their evaluation of the microcirculation, depending on the software employed. For example, the Glycocheck software (MicroVascular Health Solutions) is an all-in-one digital video microscope camera, dedicated computer system, and patented glycocalyx measurement software system. Even within this software system, there are changes with consecutive updates. In contrast, other SDF imaging modalities do not necessarily integrate microcirculatory analysis and/or allow measurement of PBR, and separate software is often required to analyze captured images. Although the Glycocheck device has been utilized to evaluate the microcirculation and EGC in healthy cats and dogs, no previous veterinary studies have utilized the Glycocheck device in a canine hemorrhagic shock model.36,44 Nevertheless, a recent study46 was able to detect a change in PBR using the Glycocheck in dogs undergoing cardiopulmonary bypass.
Evaluation of microcirculatory parameters during experimental canine blood loss using SDF imaging has been performed in 2 previous studies.17,42 The first study42 utilized 12 healthy dogs and evaluated the microcirculation after a median of 14 mL/kg (range, 13 to 50) of blood was removed. No equilibration period was specifically stated, with microcirculatory parameters measured after completion of blood donation, approximately 15 minutes after baseline images were captured. No significant changes in microcirculatory parameters were identified.42 In the second study17 of 12 dogs, splenectomy and 30-minute equilibration were followed by hemorrhagic shock induction and an additional 60-minute equilibration before acquisition of images. Significant reductions were seen in all microcirculatory variables measured, including TVD, proportion of perfused vessels, perfused vessel density, and microcirculatory flow index using a first-generation SDF device.40 In contrast, though the current study involved significant blood loss, the duration of hemorrhagic shock was only 10 minutes and microcirculatory parameters were not significantly different. This might suggest that the period of hemorrhagic shock was too short to elicit measurable changes in PBR and microcirculatory variables. The current study showed a nonsignificant increase in PBR from a median of 2.27 µm at TP1 to 2.40 µm at TP3. As previously discussed, an increase in PBR corresponds to decreased EGC thickness and suggests EGC degradation. The lack of statistical significance may be due to variance among dogs or a small sample size. A type II error cannot be excluded.
Biomarker measurement is another tool used to assess the integrity of the EGC. In the current study, EDTA plasma levels of syndecan-1, heparan sulfate, VE-cadherin, and hyaluronan were evaluated. These EGC biomarkers are known to shed in trauma and blood loss in a variety of species including humans, dogs, and rats, resulting in elevated blood concentrations.21,24,25,47,48 However, they have not been evaluated comprehensively in canine hemorrhagic shock. In the current study, hyaluronan, syndecan-1, and VE-cadherin did not change significantly, while heparan sulfate significantly decreased between TP1 and TP2. Ranges for hyaluronan were similar to those previously found in dogs.21 However, syndecan-1 values were much lower than reported in previous canine studies32,35 and were often below the level of assay detection. When measuring biomarkers peripherally, it is important to consider that many have alternative sources of shedding and that their physiology may involve varied movement between the interstitium and circulation.26 The partially understood roles of these biomarkers may help to explain the unexpected lack of change in the measured biomarkers overall and the decrease in heparan sulfate values from stable general anesthesia to hypotensive shock (TP1 to TP2). For example, a large amount of hyaluronan exists in the interstitium, and it has been proposed that fluid therapy might cause movement from the interstitial to intravascular space, potentially resulting in increased measurements.26 The lack of bolus crystalloid fluid therapy in the current study might have resulted in unchanged values. EGC component consumption in the acute phase of bleeding is also considered as a potential hypothesis. Failure to detect elevations in all 4 biomarkers could also suggest that the hemorrhagic shock model in this study did not result in significant EGC damage, that measurements were performed too early to detect plasma biomarker elevations, that the specific ELISAs utilized or processing methods did not result in accurate measurement of the biomarkers in question, or a combination of the above. Specifically, syndecan-1 values were below the level of assay detection in a large number of samples. Additionally, we used EDTA plasma and plasma separation occurred within 2 hours rather than the assay-recommended 30 minutes, which could have affected our results for some biomarkers.
Measurement of circulating EGC biomarkers in canine hemorrhagic shock has been performed in 1 previous study,21 which utilized a canine hemorrhagic shock model to measure changes in plasma biomarkers with various resuscitation techniques. Biomarkers evaluated included hyaluronan, IL-6, IL-8, and IL-10 and tumor necrosis factor-α, monocyte chemoattractant protein-1, keratinocyte chemokine-like, and atrial natriuretic peptide. Twenty-seven greyhounds were anesthetized, and blood was removed until a MAP of 50 mm Hg was achieved. Hypotension was sustained for 60 minutes before resuscitation with either autologous fresh whole blood, hydroxyethyl starch, succinylated gelatin, or isotonic crystalloids.21 Hyaluronan was measured at baseline and after 60 minutes of hypotension. Additional measurements were obtained immediately after fluid administration, as well as 40, 100, and 160 minutes later. Hyaluronan levels at baseline under anesthesia ranged from 10 to 48 ng/mL, as measured in heparin plasma separated 1 hour after collection. These values are comparable to those found at baseline under anesthesia in the current study (eg, 7.10 to 61.70 ng/mL). Further, the hyaluronan range in the current study after 10 minutes of hemorrhagic shock was 6.0 to 64.6 ng/mL, compared to 16 to 129 ng/mL after 60 minutes of hypotension in the study of interest. In the 2018 study,21 an increase in hyaluronan values from baseline was noted after 60 minutes of hypotension, but the significance of this change was not evaluated, making it difficult to draw direct comparisons.
Other than the aforementioned study,21 no other canine hemorrhagic shock models evaluate multiple biomarkers of EGC degradation. However, there are veterinary studies evaluating serum levels of syndecan-1, heparan sulfate, and hyaluronan as indicators of EGC health in various other disease states including sepsis and parvovirus.32,35,49 Serum hyaluronan levels and EDTA plasma heparan sulfate levels previously found in septic dogs are similar to those found in the current study.35,49 Further, EDTA plasma heparan sulfate levels in the current study were slightly higher than those in serum from puppies with parvovirus.32 In contrast, the syndecan-1 values in our study are much lower than the serum values from puppies with parvovirus and the EDTA plasma values from dogs in septic shock.32,35 Discordant results could be due to the disease process studied, the model employed, assay methodology (eg, plasma versus serum, delayed plasma separation, variable sample processing, specific ELISA utilized), or patient age. Based on comparable human studies,19,47 the duration and severity of hemorrhagic shock induced in the current study may not have been severe or long enough to elicit significant changes in the EGC. Changes in microcirculatory variables, PBR, and circulating levels of EGC biomarkers may take time to become apparent with the employed modalities, and any change that occurs in the acute setting may not occur in measurable levels. In the current study, microcirculatory images and blood samples for EGC biomarker analysis were collected after a short equilibration period once hemorrhagic shock had been induced. Previous studies17,21 have evaluated the microcirculation and markers of EGC health in the hours after trauma (rather than minutes) and after hemostasis.
Additionally, studies in rats,48 dogs,21 and humans26 have demonstrated that resuscitation in and of itself, along with the type and volume of fluid used, can contribute to EGC degradation. Considering that animals and humans in need of resuscitation likely already have EGC alterations due to inflammation, it may be difficult to assess which aspects of trauma, shock, and resuscitation are most contributing to EGC degradation at any given time.26 The majority of evidence regarding EGC degradation and resuscitation has focused on fluid therapy with “clear” fluids, postulating that degradation is due to hemodilution and natriuretic peptides.26 In clinical practice, patients would likely receive a crystalloid fluid bolus before resuscitation with blood products, and the lack of such crystalloid therapy in the current study may limit its clinical applicability and represent a limitation. Nevertheless, both hypovolemia and resuscitation have been shown to affect the EGC, complicating the investigation of EGC damage. Further, in hemorrhagic shock, splenic contraction occurs, increasing circulating blood volume, CO, and arterial oxygen content. This mechanism provides a physiologic means to cope with blood loss in the short term. As the current study protocol did not include splenectomy, our hemorrhagic shock model may have allowed for more compensation during the short equilibration period and thus more EGC preservation than previous models.
Several limitations may have impacted the results of this study. The study participants were all healthy, young, female spayed beagles. The effects of breed, age, and sex on the parameters measured have not been fully evaluated. Additionally, the study sample size was small, consisting of 8 dogs. Therefore, a type II error cannot be excluded. A power analysis determined that using PBR measurements, 34 paired samples are necessary to show a difference between 2.3 ± 0.2 and 2.4 ± 0.2 using a paired sample t test with a power of 20% and an alpha of 0.05. Further, there are limitations concerning the ELISA assays used. Time to plasma separation was longer than the time recommended in the ELISA instructions, which may have affected measured results. Although the human syndecan-1 ELISA kit utilized in this study has been used previously in the setting of canine septic shock, it has not been validated in dogs.35 Also, ELISAs utilize light spectrophotometry, which can be impacted by plasma coloration secondary to hemolysis, lipemia, or icterus; variations in plasma color were noted in some of the study samples.
Another limitation to consider is that the hemorrhagic shock model used in this study is not entirely indicative of true conditions in an emergency setting. Hemorrhage was closely controlled in our canine population, with dogs being under general anesthesia and provided 100% fraction of inspired oxygen throughout the shock period. Studies50 have shown that anesthesia affects the microcirculation in both normovolemia and hypovolemia. Additionally, given the length of time from TP0 to TP1 (mean, 73 minutes), anesthesia alone could have had an independent effect on the microcirculation. The dogs then received a freshly collected, full-volume autotransfusion, which would not be the case for most hemorrhagic shock patients in clinical practice, where resuscitation might involve crystalloids, colloids, and/or various blood products. Clinically, many canine hemorrhagic shock cases also involve the inflammation associated with trauma or neoplasia. This is particularly relevant as it has been previously established that tissue injury and associated inflammation contribute to EGC shedding and microcirculatory derangement.26 In fact, many previous shock models have included some element imparting inflammation, such as tissue damage (splenectomy), naturally occurring trauma, or septic shock.17,37,47 Therefore, the lack of tissue injury and subsequent inflammation in the current study may have prevented measurable EGC degradation. However, in the setting of trauma, where both hemorrhagic shock and tissue trauma exist, it may be difficult to determine how each of these 2 factors is contributing to EGC damage.
Although the model in the current study did not simulate naturally occurring trauma, it allowed investigation of the effects of hemorrhage alone on the EGC. The model utilized may provide insight regarding the role of blood loss alone in EGC degradation, although one must consider that compensatory splenic contraction may have blunted the effect of hemorrhage. Finally, the Glycocheck device has not been validated for use in dogs in estimating EGC thickness. The lack of significant results could suggest that the device was not effective in the evaluation of the EGC in dogs or was not sensitive enough to detect changes in a hemorrhagic shock state. Although the ability to compare our measurements with a gold standard would have provided increased validity for our results, there is no gold standard for the evaluation of the EGC in living animals.
The results of the current study contribute unique and important information to the existing literature regarding microcirculatory and EGC assessment in dogs with hemorrhagic shock, with translational applications for human trauma patients. Considering current and previous study results, future studies evaluating the microcirculation in hemorrhagic shock should further investigate the time in which measurable EGC damage occurs, the effects of blood loss versus posttraumatic inflammation, and the correlation between specific patient factors and changes in microcirculatory variables, PBR, and EGC biomarkers. Our syndecan-1 results suggest that a validated canine syndecan-1 assay is needed for accurate assessment of this biomarker in dogs, and further studies utilizing the Glycocheck software are needed to investigate its use in hemorrhagic shock. Further elucidation of the pathophysiology of EGC damage and microcirculatory derangement is essential in determining the best management strategies and improving patient outcomes.
The effect of trauma on the EGC and microcirculation is an active and ongoing area of research in canine and human patients. Currently, although the evidence base is growing, we have only scratched the surface of understanding the relevant pathophysiology and its relationship to outcome. Considering that EGC components may have other sources of shedding or alternative pathways of upregulation, evaluation of one or several biomarkers in isolation may not reflect dynamics at the level of the microcirculation.26 Therefore, the current study aimed to fill a gap by more comprehensively investigating the effects of hemorrhage on microcirculation. Through the measurement of multiple biomarkers (syndecan-1, hyaluronan, heparan sulfate, and VE-cadherin) in conjunction with SDF imaging of the microcirculation and estimation of EGC thickness, we sought to better characterize the changes that occur with hemorrhage.
In light of the previously discussed limitations and within the constraints of our study, our results suggest that short-term hemorrhagic shock in an otherwise healthy dog followed by complete resuscitation with a whole blood autotransfusion does not cause damage to the EGC that is measurable by the employed SDF and plasma biomarker assay protocols. However, further work is necessary to validate the use of biomarker assays and Glycocheck in this species and further studies are needed to investigate the effects of longer duration hemorrhagic shock.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding for this study was provided by the Koster Endowment and a College Research Council Grant.
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