Data from both military and civilian studies have associated significant survival benefit after massive transfusion with resuscitation of high ratio plasma to red blood cells (≤1:2 plasma:RBCs).1–5 This change in resuscitation strategy centers on the early and increased use of plasma and has led to an increase in early survival, though the mechanism of protection is unknown. The purpose of the current study was to investigate the role of plasma on the endothelial glycocalyx after hemorrhagic shock.
The endothelial glycocalyx is a complex network of soluble components that projects from the cell surface of the endothelium into the vessel lumen.6 It consists of proteoglycans and glycoproteins attached to the cell membrane. The proteoglycans provide the structural support for the glycocalyx and consist of a core protein, either syndecans or glypicans, to which the glycosaminoglycans attach. Syndecans are the major source of heparan sulfate proteoglycans for all cell types. Endothelial cell adhesion molecules, primarily the selectins and immunoglobulin superfamily (ICAMs), are the major glycoproteins of the glycocalyx and play a key role in pathologic neutrophil–endothelial cell interactions that occur with injury to the glycocalyx.7 The glycocalyx lines the entire endothelium, and its preservation has been implicated in multiple disease states. Other glycoproteins are important to coagulation, fibrinolysis, and hemostasis.
There is a dynamic equilibrium between the soluble components of the glycocalyx and the plasma component of blood. The area of the vessel lumen encompassed by the glycocalyx prohibits erythrocytes and leukocytes from interacting with the vessel wall and importantly reduces the flow of plasma, thus promoting plasma– endothelial cell interaction.8–10 We therefore hypothesized that the endothelial glycocalyx is injured after hemorrhagic shock and that resuscitation with plasma aids in restoring the glycocalyx. Injury to the endothelial glycocalyx has been demonstrated in laboratory models of ischemia/reperfusion but has not been investigated after hemorrhagic shock.11,12 This study now demonstrates for the first time that the endothelial glycocalyx is indeed injured after hemorrhagic shock and partially repaired by plasma in comparison with lactated Ringer's (LR) solution resuscitation.
Animal Model of Hemorrhagic Shock
All procedures performed were protocols approved by the University of Texas Houston Medical School Animal Welfare Committee. The experiments were conducted in compliance with the National Institutes of Health (NIH) guidelines on the use of laboratory animals. All animals were housed at constant room temperature with a 12:12-hour light–dark cycle with access to food and water ad libitum.
Male Sprague–Dawley rats weighing 200 to 300 g were fasted overnight with free access to water. Under isoflurane anesthesia, animals were placed on a heating blanket to maintain body temperature of 35°C to 37°C. Femoral arterial and venous catheters (Instech, Plymouth Meeting, PA) were placed, then flushed with 0.3 mL of 1% heparin. No further heparin was used. The catheters were then connected to the corresponding fluid reservoir and blood pressure monitor (BPA-400; Micro-Med, Louisville, KY).
A pressure-controlled model of shock was used as previously described.13 Rats were bled to a mean arterial blood pressure (MAP) of 30 mm Hg for 90 minutes, then resuscitated with either LR or fresh plasma to a MAP of 80 mm Hg and compared with shams or shock alone (with no resuscitation) (n = 5 per group). Shams underwent anesthesia and placement of catheters but were not subjected to hemorrhagic shock. Five animals were used for each group. A computer-controlled, low-flow peristaltic pump (model P720; Instech) was connected to the venous cannula through which blood was withdrawn and resuscitation fluids were administered.14 Shed blood and resuscitation fluids were held in separate reservoirs placed on a balance with fluid weights recorded every 5 seconds using a LabVIEW program (National Instruments, Austin, TX). Fluid volume was calculated from its weight on the basis of measured fluid density. After 2 hours the animals were killed by exsanguination and lungs harvested. For animals resuscitated with plasma, a separate set (n = 5) of Sprague–Dawley rats was used to obtain fresh plasma. Blood was obtained from an abdominal aortic puncture through a midline laparotomy. Blood was centrifuged at 500 g for 10 minutes at 4°C to obtain plasma. Plasma was then warmed to 37°C, then administered through the venous catheter.
Electron microscopy was performed on postcapillary venules obtained from the small bowel mesentery in a separate set of animals using the same groups as described above (at least 2 per group). The small bowel mesentery was chosen as an easily accessible organ to perfuse and isolate vessels to study the endothelium. At the conclusion of resuscitation, the superior mesenteric artery was cannulated, then infused with a fixative/staining solution of 2% lanthanum, 2% glutaraldehyde, 2% sucrose, and 0.1 M sodium cacodylate buffer.15 Lanthanum is a trivalent cation that binds to negatively charged glycoprotein moieties of the glycocalyx.16 A section of mesentery 10 cm from the ileocecal valve and adjacent to the bowel was excised and processed for electron microscopy following established protocols, except for the addition of a 2% osmium tetroxide, 2% lanthanum staining before dehydration and embedding. Venules were identified using a JEOL 1200 II electron microscope in sections poststained with uranyl acetate and lead citrate for contrast. Electron microscope magnifications varied accordingly to frame differently sized venule images squarely onto either a Gatan 1 k × 1 k or TVIP F215 2 k × 2 k CCD. Images recorded at different magnifications were then normalized to the same magnification by scaling images to the same pixel size using the proc2d program within the EMAN image analysis software package.17
Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA was extracted from lung tissue from each animal using TRI reagent (Sigma, St. Louis, MO). Equal amount of RNA was taken from each sample, then 1000-ng RNA from each group was reverse transcribed in a 20-μL reaction using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). The real-time polymerase chain reaction (PCR) was performed in 20 μL of a reaction mixture containing 50 ng cDNA (20 ng/μL), 10 μL of 2× TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ), 6 μL distilled water, and 1 μL of a primer and probe mixture, which located at 4 to 5 exon boundary of syndecan-1 (Mm00448920_g1, Applied Biosystems). Actin expression (actin probe, Mm00607939_s1, Applied Biosystems) was used as the internal reference. Real-time PCR assays were performed in triplicate using Applied Biosystems Step-One-plus real-time PCR system (Applied Biosystems) with the following program: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and at 60°C for 1 minute. A sequence detection program calculated the threshold cycle number (CT) at which the probe cleavage– generated fluorescence exceeded the background signal.18 Relative RNA expression levels were calculated using a comparative CT method. The set of gene primer and probe for syndecan-1was confirmed to have amplification efficiency equal to that of the internal reference gene. The relative expression level of syndecan-1 was normalized to that reference gene and to a calibrator sample that was run on the same plate.19 The normalized relative expression levels were automatically calculated using commercial software (StepOne Software V2.1, Applied Biosystems).
Cell Surface Syndecan-1 Expression
Lungs were harvested and sections fixed with 4% formaldehyde, then embedded in paraffin and sectioned. At least 2 sections from separate animals in each group were immunostained at the same setting with 1:200 rat antisyndecan-1 monoclonal antibody (BD, PharmingenTM), then incubated with 1:500 Alexa Fluor goat antirat IgG (H+L, Invitrogen). Images were obtained using an Olympus 1X71 microscope with SimplePCI6 software. The relative fluorescence intensity was quantified using Image J software (NIH) and reported as relative fluorescence units (RFU). A negative control was used as a technical control for staining without using primary antibody.
Lungs were harvested and sections fixed with 4% formaldehyde, then embedded in paraffin and sectioned. At least 2 sections from separate animals in each group were stained with hematoxylin and eosin, then scored using a 3-point scale based on alveolar thickness, capillary congestion, and cellularity20: alveolar wall's thickness, 0 = normally thin alveolar walls; 1 = mild thickening; 2 = clearly thickened walls; and 3 = thickening of the wall, with 50 to 100% extremely thick. For capillary congestion of the alveolar wall: 0 = exhibiting a normal red cell density; 1 = mildly congested; 2 = moderately congested; 3 = severely congested. For cellularity of the alveolar wall: 0 = normal variable but small number of cells; 1 = mild increase in cellularity; 2 = mild to moderately increased cell number in 50% to 75% the section; and 3 = an increase in cellularity of all alveolar walls. The overall lung injury score was calculated by averaging the 3 indices of injury.
All data are presented as mean ± SEM, with significance at P < 0.05. Results were analyzed by 1-way analysis of ariance (ANOVA) with Tukey post hoc tests, n = 5 per group.
Hemorrhagic Shock Model
All experimental groups had approximately 50% of total blood volume withdrawn during the shock period (50.2%–53.8%) to achieve a MAP = 30 mm Hg. Shams had no blood withdrawn. The plasma group required significantly less volume (29.5 ± 2.8 mL/kg/h) to maintain the MAP during resuscitation in comparison with the LR group (73.9 ± 10.0 mL/kg/h) (Fig. 1, A and B).
Glycocalyx Is Partially Restored by Plasma Resuscitation
Electron microscopic images (Fig. 2) of the glycocalyx are shown in Figure 2A and the quantitative data in Figure 2B. Images revealed that shams had an intact glycocalyx that was virtually ablated by hemorrhagic shock. LR resuscitation did not result in repair, whereas plasma resuscitation demonstrated early signs of restoration of the glycocalyx by 2 hours of resuscitation: sham versus shock, P < 0.001; sham versus LR, P = < 0.001; sham versus plasma, P = 0.01; shock versus plasma, P < 0.001; LR versus plasma, P < 0.001.
Syndecan-1 Expression in Lung Is Enhanced by Plasma
In comparison with shams (3.03 ± 0.22), the relative expression level of lung syndecan-1 mRNA was significantly reduced by shock alone (1.39 ± 0.22, P = 0.02) and even more so by LR (0.82 ± 0.03, P < 0.001) but enhanced by plasma resuscitation (2.76 ± 0.03), back to sham levels. Both the shock (P = 0.03) and LR (P < 0.001) groups had significantly less mRNA than did the plasma group, suggesting that plasma restores the backbone of the endothelial glycocalyx (Fig. 3). Similar findings were demonstrated after tissue immunostaining and were consistent with plasma restoration of syndecan-1 (Fig. 4). Both sham (2707 ± 226 RFU) and plasma (2172 ± 146 RFU) had significantly more syndecan-1 staining than did either shock (624 ± 72 RFU, P < 0.001, sham versus shock, and P < 0.001, plasma versus shock) or LR (1262 ± 173 RFU, P = 0.002, sham versus LR, and P = 0.014, plasma versus LR).
Lung Injury Is Lessened by Plasma Resuscitation
Shock animals had significantly increased lung injury, as assessed by alveolar wall thickness, capillary congestion, and cellularity. In comparison with shams, shock and LR resuscitation worsened lung injury, and injury was attenuated by plasma (Table 1and Fig. 5).
In this study we demonstrated for the first time that hemorrhagic shock injures the endothelial glycocalyx, which is partially restored by plasma but not LR resuscitation. Similarly, pulmonary syndecan-1 mRNA and alveolar cell surface syndecan-1 expression were higher in animals resuscitated with plasma in comparison with LR and correlated with reduced lung injury.
Syndecan is the major cell membrane protein of the glycocalyx. Its extracellular domain is substituted with heparan sulfate chains and promotes interaction with plasma proteins.21 Syndecans are expressed on virtually every cell in the body, and there are 4 members (syndecan 1 to 4) that compose the syndecan family.22 Syndecan-1 has been detected in patients immediately after aortic surgery, and we have published preliminary data demonstrating that it is shed in patients after hemorrhagic shock.15,23
Injury to Syndecan-1 and the Glycocalyx
Injury to syndecan-1 and the glycocalyx has important implications. The glycocalyx serves as an effective barrier to leukocyte–endothelial cell and platelet adhesion by providing steric hindrance between receptor and ligand, thereby blocking adhesion to the underlying endothelium.24 Processes that degrade the glycocalyx—such as cytokines, tumor necrosis factor-α, ischemia/reperfusion, and now hemorrhagic shock—uncover adhesion molecules, which allow direct interaction between the endothelial cell surface and circulating neutrophils and plasma proteins.25,26 Importantly, Chappell et al. recently demonstrated protection of the glycocalyx by hydrocortisone and antithrombin, which reduced postischemic leukocyte adhesion,27 suggesting that interventions to preserve or repair the glycocalyx may have important therapeutic implications.
The syndecan-1/glycocalyx endothelial layer also plays a key role in maintaining vascular stability. Injury after shock can lead to enhanced vascular permeability and tissue edema, which may predispose to organ failure and death.28 Hayasida et al. reported increased lung edema after lipopolysacharide administration in syndecan-1 knockout mice in comparison with wild-type mice,29 and van den Berg et al. showed that enzymatic degradation of the glycocalyx in myocardial capillaries led to myocardial edema.30 Preserving the microvascular barrier as a therapeutic target for sepsis is being explored.31 A recent in vitro study by our group demonstrated that plasma preserved pulmonary endothelial cell permeability in comparison with LR in a cell culture model of hypoxia/reoxygenation.32 Whether plasma's potential vascular stabilizing properties translate into reduced morbidity or mortality is unclear but will be the subject of future investigation. Using the lung as an end organ to study does have an important limitation; the lung comprises both endothelial and epithelial cells, and therefore we cannot determine the contribution of endothelial syndecan-1 in pulmonary protection by plasma. Data from mesenteric venules, however, suggest that endothelial cells play an important role.
Plasma Partially Restores Syndecan-1 and Glycocalyx: Potential Mechansims
Lastly, we have demonstrated that plasma resuscitation is effective in partially restoring syndecan-1 and the glycocalyx, though the precise mechanism by which this occurs is unknown. It is feasible that systemic syndecan-1 or other soluble plasma components physically interact with the endothelial membrane to repair components of the syndecan backbone. There are soluble components of the glycocalyx composing proteins and proteoglycans, which may be derived either from the endothelium or from blood constituents directly.6 It is also conceivable that plasma is not directly contributing to the structural integrity of the glycocalyx but rather is restoring cell surface syndecan-1 by mobilizing an intracellular pool of preformed syndecan-1. Finally, plasma may also stimulate endothelial cell syndecan-1 transcription but with signs of restitution demonstrated by 2 hours of resuscitation, this is a less plausible explanation. Additional investigation is clearly needed. However, one limitation of the current study is that only MAP was used as an end point of resuscitation in this pilot study. Future studies should include more traditional end points such as hemoglobin and base deficit.
In conclusion, we have demonstrated in a rodent model that hemorrhagic shock degrades the endothelial glycocalyx. LR resuscitation resulted in few signs of repair, whereas plasma resuscitation demonstrated early signs of restoration. In support of these findings, pulmonary syndecan-1 mRNA and alveolar cell surface syndecan-1 expression were higher in animals resuscitated with plasma in comparison with LR and correlated with reduced lung injury. Taken together, our findings support the protective effects of plasma seen clinically and may be due in part to its ability to restore the endothelial glycocalyx and preserve syndecan-1 after hemorrhagic shock.
Name: Rosemary A. Kozar, MD, PhD.
Contribution: Study design, data analysis, manuscript preparation.
Name: Zhanglong Peng, MD, PhD.
Contribution: Study conduct, data analysis.
Name: Rongzhen Zhang, MD, PhD.
Contribution: Study conduct.
Name: John B. Holcomb, MD.
Contribution: Study design, manuscript preparation.
Name: Shibani Pati, MD, PhD.
Contribution: Study design, manuscript preparation.
Name: Pyong Park, PhD.
Contribution: Study design and manuscript preparation.
Name: Tien C. Ko, MD.
Contribution: Study design and manuscript preparation.
Name: Angel Paredes, PhD.
Contribution: Conduct of the study and manuscript preparation.
The authors thank Bernard Becker from Ludwig-Maximilians-University Munich, Munich, Germany, and Patricia Navarro and Glenn Decker from the University of Texas, Houston, Texas, for their assistance with glycocalyx staining and Min Ye for her technical assistance with scoring lung injury.
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