The endothelial glycocalyx is a microthin gel lining that coats the luminal surface of all endothelial cells. It is comprised of a complex network of soluble components, primarily proteoglycans (syndecan and glypican), glycosaminoglycans, glycoproteins, and plasma proteins (albumin and antithrombin), that extend from the cell surface of the endothelium into the vessel lumen.1 A healthy endothelial glycocalyx plays a homeostatic role by maintaining a balanced permeability and regulating blood vessels’ pro- and anti-inflammatory as well as pro- and anticoagulant functions.2,3 Multiple studies4–6 have reported that trauma-induced inflammation, ischemia, and hypovolemia can break down up to 59% of endothelial glycocalyx. This leads to capillary leakage, edema, platelet aggregation, hypercoagulation, and loss of vascular responsiveness. Ultimately, this promotes a proinflammatory and immunosuppressive state, leading to increased morbidity and mortality for trauma patients.2
Changes in microcirculation and endothelial glycocalyx health have been demonstrated in numerous animal models, such as mice, rats, pigs, and dogs, in various conditions, from hemorrhage to sepsis.2 Two practical methods of measuring the endothelial glycocalyx are accepted: (1) sidestream dark field (SDF) video microscopy and (2) measuring circulating biomarkers of glycocalyx injury.3 Electron microscopy can be utilized in research settings, although it is not practical clinically. Sidestream dark field video microscopy is a noninvasive test that visualizes RBCs within the microvasculature and then incorporates a proprietary software (GlycoCheck) to calculate multiple values that determine the health of the microvasculature: perfused boundary region (PBR), total vessel density, total vessel density (count), capillary blood volume absolute (CBVABS) (µm/s/mm2), capillary blood volume relative (CBVREL), and flow (µm/s/mm2). The most commonly used value is the PBR, which is calculated by measuring, in micrometers, the lateral movement of RBCs within the microvasculature. A healthy endothelial glycocalyx limits the RBC lateral movement to approximately 1 RBC width, facilitating optimal flow. However, when the endothelial glycocalyx is damaged, the RBCs are allowed more lateral movement, enabling them to encroach closer to the endothelium. As a result, the PBR (RBC lateral movement) increases. Put simply, PBR has an inverse relationship with endothelial glycocalyx health; as PBR increases, the health of the endothelial glycocalyx decreases and vice versa.7
Multiple human and animal studies8,9 have evaluated the microvasculature posthemorrhage using SDF. However, these studies had a limited window of SDF recordings postresuscitation. Furthermore, these studies only used fresh whole blood or packed RBCs (pRBC) as the resuscitation media.
Our study aimed to evaluate glycocalyx injury via SDF in a crossover canine model of hemorrhagic shock with various resuscitation strategies. This is the first study to evaluate the microvasculature in a controlled hemorrhagic shock model while resuscitating with multiple resuscitation strategies, including various combinations of pRBCs, fresh frozen plasma (FFP), chilled whole blood (CWB), lyophilized blood products (platelets and plasma), and lactated Ringer’s solution (LRS)/hydroxyethyl starch (HES) solution over 3 hours.
We hypothesized that there will be no statistically significant difference in PBR measurements when traditional blood products (CWB or pRBC/FFP) are administered versus when lyophilized blood products (freeze-dried plasma [FDP]/hemoglobin-based oxygen carrier [HBOC]/lyophilized platelets [LPs] or FDP/HBOC) are administered for resuscitation. Similarly, we hypothesize that there will be no significant difference in PBR measurements when traditional blood products are administered to dogs versus when LRS/HES solution is administered for resuscitation.
Methods
This protocol was approved by the University of Utah IACUC (protocol number 21-01012) and received second-level approval by the Department of Defense. Six male, purpose-bred dogs, ages 10 to 14 months old and between 25 to 33 kg, were used for a fixed-pressure crossover study design utilizing a novel hemorrhagic shock model from September 2021 through June 2022.
An 18- to 20-gauge IV catheter was placed in a cephalic or saphenous vein to facilitate induction. Animals were premedicated with midazolam (0.3 mg/kg) and fentanyl (5 μg/kg) IV while receiving preoxygenation. Anesthesia was then induced with IV propofol (titrated to a maximum of 8 mg/kg). Following orotracheal intubation, anesthesia was maintained via IV constant rate infusion of propofol (1 to 20 mg/kg/h), fentanyl (0.05 to 0.3 μg/kg/min), and midazolam (0.1 to 0.5 mg/kg/h), titrated to allow for spontaneous ventilation. Lactated Ringer solution was also administered IV at 5 mL/kg/h.
A 7-Fr, 20-cm triple port jugular catheter was placed in the right or left jugular via a routine Seldinger technique. A 5-Fr femoral arterial catheter was placed percutaneously with ultrasound guidance to monitor invasive arterial blood pressure. A Foley urinary catheter was placed to monitor urine output. Animals were monitored with continuous ECG, end-tidal carbon dioxide, and pulse oximetry. Heat support was provided to maintain a core temperature > 36.6 °C.
Animals were subjected to hemorrhage over 60 minutes through the central venous catheter to achieve a mean arterial blood pressure of 35 mm Hg or removal of 40% of their blood volume (estimated as 0.4 X 90 mL/kg), whichever occurred first. They were then maintained in shock for 45 minutes before receiving 1 of 5 resuscitation strategies chosen at random: 1 L LRS + 250 mL HES (30 mL/kg and 7.5 mL/kg), 1 U of CWB (14 mL/kg), 1 U of FFP (7 mL/kg) + 1 U of pRBC (7 mL/kg), 1 U FDP + 1 U HBOC, or 1 U FDP + 1 U HBOC + 1 U LPs. All resuscitation strategies were administered over 30 minutes. Animals were recovered from anesthesia after 30 additional minutes to ensure normotension (Figure 1). Each dog was allowed to recover after the hemorrhage and resuscitation for 4 to 6 weeks before they underwent the procedure again. This was repeated until each dog was resuscitated with each strategy. Sidestream dark field video microscopy measurements were obtained at the beginning of the procedure (T0), after hemorrhage (T60), after shock (T105), after resuscitation (T135), and at the end of the procedure (T180).
An SDF video microscope was connected to GlycoCheck acquisition and analysis software to acquire video of the sublingual circulation automatically. Each measurement consisted of 10 2-second videos of 40 frames/video each, from which the software calculated PBR, total vessel density (count), CBVABS (µm/s/mm2), CBVREL, and flow (µm/s/mm2). Perfused boundary region is the only value analyzed in this manuscript. Raw data for total density, CBVABS, CBVREL, and flow are reported in Supplementary Material S1 for transparency purposes only. All values were calculated via the proprietary SDF software.
Statistical analysis
Treatment group differences were assessed statistically by using a generalized linear mixed model. Values were analyzed using a model that contained a fixed effect for treatment, a random effect for dogs, and a repeated measures effect for the time points. Various repeated measures covariance matrices (compound symmetry, unstructured, autoregressive, etc) were used to determine which provided the best model fit. Pairwise treatment differences were sliced at each time point and compared using a step-down version of the Tukey-Kramer multiple comparisons procedure. Pairwise time point differences versus baseline were sliced at each treatment and compared using a step-down version of the Dunnett multiple comparisons procedure. Statistical significance for all analyses was determined using 2-sided tests and a family-wise false positive error rate of 0.05. Statistical analyses were performed using SAS, version 9.4, procedure PROC GLIMMIX (SAS/STAT, version 15.2; SAS Institute Inc).
Model residuals were assessed for normality and homogeneity of variance using a combination of descriptive plots (histogram, quantile-quantile plot, linear predictor) and the appropriate statistical tests (eg, Shapiro-Wilk, Anderson-Darling, D’Agostino-Pearson). A lognormal distribution was used instead of a normal distribution where departures from normality were detected.
A normal distribution was used for PBR; a compound symmetry covariance matrix was used for all analyses. Values are expressed as mean and 95% CI. Due to missing data, group sample sizes were n = 5 unless otherwise noted.
Results
Logistical constraints during the coronavirus disease 2019 pandemic limited the availability of the SDF system during a portion of the study. As a result, the following number of measurements were available at each time point: the beginning of the procedure (n = 23), after hemorrhage (n = 24), T105 (n = 24), T135 (n = 24), and T180 (n = 22). Similarly, the following SDF measurements were available for each respective resuscitation arm: LRS/HES (n = 25), CWB (n = 23), FFP/pRBC (n = 20), FDP/HBOC (n = 24), and FDP/HBOC/LPs (n = 25).
The mean PBR at baseline for all resuscitation arms was 2.19 µm (range, 2.03 µm to 2.43 µm). When each resuscitation arm was compared to its own baseline, the only arm with a significant difference in PBR was the LRS/HES arm at T135 and T180 (Table 1). The change in PBR between these points was +0.54 µm (P = .03; 95% CI, 0.02 to 1.06) and +0.63 µm (P = .01; 95% CI, 0.12 to 1.15), respectively (Figure 2). Furthermore, when the PBR of each resuscitation arm was compared to other resuscitation arms, the only significant differences were at T180, with LRS/HES solution being higher than CWB (+0.67 µm; P = .03; 95% CI, 0.01 to 1.31) and FFP/pRBC (+0.69 µm; P = .03; 95% CI, 0.038 to 1.35) (Table 2; Figure 1). No other significant differences in PBR were identified regardless of whether the resuscitation arm’s PBRs were compared to each other or if they were compared to themselves at different time points.
Perfused boundary region values for each time point for each resuscitation arm evaluated from September 2021 through June 2022.
Measurement | Time point | Treatment | N | Mean (range) |
---|---|---|---|---|
PBR | T0 (baseline) | LRS/HES | 5 | 2.13 (1.92–2.24) |
CWB | 4 | 2.21 (2.03–2.42) | ||
FFP + pRBC | 4 | 2.02 (1.76–2.37) | ||
FDP + HBOC | 5 | 2.43 (2.06–2.77) | ||
FDP + HBOC + LP | 5 | 2.18 (1.98–2.37) | ||
PBR | T60 (hemorrhage) | LRS/HES | 5 | 2.10 (1.81–2.41) |
CWB | 5 | 2.13 (1.68–2.51) | ||
FFP + pRBC | 4 | 2.04 (1.52–2.41) | ||
FDP + HBOC | 5 | 2.12 (1.47–2.81) | ||
FDP + HBOC + LP | 5 | 1.98 (1.50–2.37) | ||
PBR | T105 (shock) | LRS/HES | 5 | 2.34 (1.58–2.85) |
CWB | 5 | 2.05 (1.36–2.73) | ||
FFP + pRBC | 4 | 2.08 (1.66–2.43) | ||
FDP + HBOC | 5 | 2.08 (1.33–2.46) | ||
FDP + HBOC + LP | 5 | 2.12 (1.49–2.56) | ||
PBR | T135 (resuscitation) | LRS/HES | 5 | 2.67 (2.25–3.00) |
CWB | 5 | 2.17 (1.61–2.84) | ||
FFP + pRBC | 4 | 2.46 (2.18–2.71) | ||
FDP + HBOC | 5 | 2.34 (1.90–2.78) | ||
FDP + HBOC + LP | 5 | 2.18 (2.07–2.31) | ||
PBR | T180 (stablization) | LRS/HES | 5 | 2.76 (2.45–2.94) |
CWB | 4 | 2.10 (1.64–2.86) | ||
FFP + pRBC | 4 | 2.06 (1.90–2.29) | ||
FDP + HBOC | 4 | 2.48 (2.22–2.81) | ||
FDP + HBOC + LP | 5 | 2.45 (2.18–2.64) |
CWB = Chilled whole blood. FDP = Freeze-dried plasma. FFP = Fresh frozen plasma. HBOC = Hemoglobin-based oxygen carrier. HES = Hydroxyethyl starch. LP = Lyophilized platelet. pRBC = Packed RBCs. T0 = Baseline. T60 = Posthemorrhage. T105 = Postshock. T135 = Postresuscitation. T180 = Poststabilization.
Perfused boundary region (PBR) values are expressed in micrometers with upper and lower range. Each time point is expressed in minutes after the start of the procedure.
Summary of statistically significant perfused boundary region (PBR) measurements when comparing resuscitation strategies (statistical significance set at P < .05) from September 2021 through June 2022.
Measurement | Time point | Treatment group comparison | P value |
---|---|---|---|
PBR | T180 | LRS/HES:CWB | .033 |
T180 | LRS/HES:FFP/pRBC | .033 |
LRS = Lactated Ringer’s solution.
Non–statistically significant differences are not included for ease of interpretation. Treatment groups being compared are separated by a colon.
Discussion
In this study, we accepted our null hypothesis that there would be no significant difference in PBR between the resuscitation arms involving lyophilized blood products (FDP/HBOC or FDP/HBOC/LPs) and traditional blood products (CWB or FFP/pRBCs) across all time points measured. This supports the idea that lyophilized blood products may preserve the endothelial glycocalyx in a manner similar to traditional blood products.8 On the other hand, we rejected our null hypothesis that there would be no significant difference in PBR between the resuscitation arms utilizing traditional blood products and the resuscitation arm with LRS/HES solution, indicating that LRS/HES solution does not preserve the endothelial glycocalyx as effectively as traditional blood products. This finding is consistent with numerous other studies10–16 that demonstrated the inferiority of clear fluids compared to plasma or whole blood in preserving the endothelial glycocalyx during hemorrhagic shock resuscitation secondary to their hemodilution effects of plasma proteins, release of atrial natriuretic peptides, and promotion of inflammation.
For this fixed-pressure hemorrhagic shock model, the baseline’s mean PBR value of 2.19 µm aligns with PBR baselines of other canine studies.8,9,17 This consistency is essential since the SDF acquisition system has undergone several mandatory software upgrades since its original release. However, the various versions do not appear to significantly alter the algorithm in which the system calculates PBR, at least compared to the aforementioned studies.
When evaluating each resuscitation arm to each other via the Tukey step-down method for multiple comparisons, there was no statistically significant change in the PBR until T180, at which point the LRS/HES resuscitation arm PBR was significantly higher than the CWB and pRBC/FFP arms. Prior to T105, procedurally, all resuscitation arms were identical. Therefore, it took 75 minutes after administration of the LRS/HES solution before there was enough degradation of the endothelial glycocalyx to alter PBR to a level of statistical significance. These findings indicate the adverse effects of clear fluids on the endothelial glycocalyx are not immediate. Similarly, the difference in PBR of the resuscitation arms utilizing lyophilized blood products compared to the PBR of the traditional blood products increased throughout the study, though not to a level of statistical significance. A study18 in rats showed that it took 120 minutes (45 minutes longer than the last reading of our study) before early signs of endothelial glycocalyx restoration were observable on electron microscopy. Future studies are required to evaluate if extending SDF measurements beyond 75 minutes after shock would show the endothelial glycocalyx continuing to degrade. It is also worthwhile to mention that another study19 evaluating the endothelial glycocalyx in rats, albeit via endothelial biomarkers rather than SDF video microscopy, showed statistically significant damage that occurred during hemorrhagic shock. Although our results did not show impairment of the endothelial glycocalyx until postresuscitation, it is probable that the initial damage occurred during shock. However, at that point, the impairment was not severe enough to alter the endothelial glycocalyx’s functionality as detected by SDF.
Although multiple veterinary studies have evaluated a healthy endothelial glycocalyx via SDF video microscopy, only 1 study evaluated it through similar conditions. This study utilized this technology in a hemorrhagic shock model and found no statistically significant change in PBR when comparing prehemorrhage to postresuscitation with fresh whole blood.8 Similarly, 1 study,20 which was over a longer duration, showed no significant changes in PBR with CWB resuscitation. The lack of change in PBR is likely attributed to whole blood’s ability to protect the endothelial surface matrix in hemorrhagic shock. In contrast, another study21 detected an increase in PBR in canines subjected to massive transfusions while undergoing cardiopulmonary bypass. However, their transfusions consisted of pRBCs and autotransfusions for resuscitation.
Multiple limitations exist in this study. First, the limited population size encompassing only young, healthy male dogs increases the odds of a type II error. Second, the controlled hemorrhage shock model utilized did not accurately mimic the inflammatory response seen in naturally occurring trauma that includes tissue damage, which is known to exacerbate endothelial glycocalyx damage.22 Furthermore, inhalant and IV anesthesia alters microcirculation.23 In our study, these effects were minimized by not utilizing an inhalant anesthetic. However, propofol, which was utilized, also alters the hepatosplanchnic microcirculation and the inflammatory cascade.24 This study also evaluated the glycocalyx via SDF using PBR as a principal surrogate for its health. The other SDF calculations, such as flow, CBV, and density, were not interpreted due to the current limited understanding of these values in veterinary medicine. Finally, comparing the PBR values to researched endothelial glycocalyx biomarkers, such as Syndecan-1, could further validate the reliability of this methodology.25
Future research areas include comparing SDF measurements with various endothelial glycocalyx biomarkers that are shed during hemorrhagic shock. This should be evaluated in both controlled and uncontrolled hemorrhagic conditions.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
None reported.
Disclosures
The views expressed in the manuscript are those of the authors and do not reflect the official policy or position of the US Medical Department, the Department of the Army, the Department of Defense, or the US Government.
No AI-assisted technologies were used in the generation of this manuscript.
Funding
Support for this work was provided by the US Department of Defense (contract No. W81XWH-21-C-0002). This research was supported in part by an appointment to the Research Participation Program at the US Army Institute of Surgical Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Army Institute of Surgical Research.
ORCID
K. E. Hall https://orcid.org/0000-0001-5399-932X
J. Guillaumin https://orcid.org/0000-0001-8622-4387
G. Hoareau https://orcid.org/0000-0002-8635-3960
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