Red blood cell flow properties include self-aggregation, deformability, and adherence to the vascular endothelium.26 All may adversely affect tissue perfusion. Microfluidic platforms have been used to study RBC aggregation and deformability and have shown significant differences between fresh RBC and stored RBC.27 The decline of these properties is related to the duration of RBC storage. There is minimal information on the effect of RBC storage on the adherence to the vascular endothelium, especially as it relates to the integrity of the glycocalyx layer. Previous studies using isolated endothelial cells to model the endothelial barrier function are limited by the dimensions of the glycocalyx established under standard conditions.28 Endothelial cells cultured within microchannels of microfluidic devices and subjected to physiologic fluid shear stress develop a glycocalyx layer with a thickness comparable to that found in micro capillaries in vivo. We have demonstrated the development of a hydrodynamically relevant glycocalyx layer with this platform in a previous study.29
It has been known for some time that major trauma is associated with endothelial glycocalyx injury as demonstrated by shedding of glycocalyx components into the systemic circulation.15 Naumann and colleagues30 have demonstrated that poor microcirculatory flow dynamics after T/HS are associated with endothelial cell damage and glycocalyx shedding. The impact of these changes in the microcirculation on fresh and stored RBC flow properties is unknown.
It has been shown that RBCs undergo progressive changes when stored for prolonged time. In our study, we demonstrate that the RBC glycocalyx undergoes degradation similar to the RBC cell membrane damage that has been well described. In vivo numerous structural changes of the RBC glycocalyx are known to occur as part of aging.32 These changes may contribute to removal of senescent cells from the circulation. The progressive loss of the RBC glycocalyx with storage may similarly lead to enhanced RBC aggregation and adhesion to the vascular endothelium. Under normal conditions, the anionic properties of the endothelial glycocalyx and the negatively charged RBC determine hydrodynamic resistance and effect the flow pattern of RBCs.23 Obertleiner demonstrated that endothelial and RBC glycocalyx may cause adverse effects when one or both are damaged. This interaction was also suggested by Yalcin et al.31 in a rat cremasteric muscle preparation. Stored but not fresh RBC led to microrheolgic disturbances which included a disruption of the RBC free layer and cell shear stress signals, both of which are related to a functional endothelial glycocalyx.
Our study further delineated the interrelationship of the respective eGC and RBC glycocalyx layers during flow conditions. Fresh versus stored RBC had significantly different glycocalyx layers and were perfused through microfluidic channels lined with control HUVEC with an intact glycocalyx or HUVEC exposed to H/R and Epi to mimic the microcirculation after T/HS. The H/R and Epi exposure resulted in a markedly diminished endothelial glycocalyx layer. We chose to study RBC adherence to the vascular endothelium rather than aggregation or deformability as this property is most reflective of a functional endothelial glycocalyx layer.33,34 Our study demonstrated that RBC endothelial adherence was more significant when both the RBC and endothelial glycocalyx layers were degraded.
The shear-dependent behavior of RBC adherence was demonstrated in our microfluidic system using a range of physiologically relevant shear rates. The effect of shear was most apparent in the HUVEC monolayer control group with either fresh or stored RBCs. However, RBC adherence to the endothelial monolayer subjected to H/R and Epi was significantly greater and relatively unaffected by increasing shear rates. This suggested a much firmer attachment to the HUVEC monolayer in these groups. In vivo, this would likely lead to prolonged compromise of the microvasculature. In summary, our model suggests that the microrheologic impact of stored RBCs is related to the status of the microcirculation and more importantly the endothelial glycocalyx. This may in part be due to the combined effects of increased NO scavenging following RBC storage and decreased endothelial production of NO due to shock induced endothelial glycocalyx degradation.35,36 Most clinical studies regarding detection of glycocalyx degradation from systemic blood is associated with adverse outcomes. However, little information exists regarding the actual glycocalyx thickness or integrity. Our previous study did show an association between endothelial glycocalyx thickness and concentrations of glycocalyx degradation products detected in the perfusate of our biomimetic model of hemorrhagic shock.29 The correlation of syndecan and other glycocalyx degradation products with microvascular glycocalyx thickness was also shown in a rodent model performed by Torres Filho and colleagues.37
There are several limitations to our study. Two dilute RBC solutions were used to quantitate RBC adherence to the endothelium. Viscosity has been shown to effect RBC flow properties which may not be significant when using dilute RBC solutions. Second, other blood elements, including leukocytes, platelets, and plasma, may impact RBC vascular adherence and were not included in the current study. Finally, the rectangular geometric design of the microchannels in our microfluidic plates do not recapitulate the complex branching pattern of vessels in the microcirculation, which may also effect RBC flow properties.
There are a number of other potential uses of our microfluidic system to study RBC-endothelial interactions under flow conditions. These include assessment of the efficacy of current and future RBC storage solutions and RBC rejuvenation strategies.38,39 In addition, it may provide useful information regarding the flow properties of cryopreserved RBC when exposed to both normal and perturbed endothelial surfaces.40 The potential role of RBC microparticles in our study is unknown. Although microparticles including those from RBCs are normally continuously shed into the circulation, they are also produced during processing and storage of blood for transfusion. Because they may adhere to vascular endothelium, the impact of RBC microparticles in normal and perturbed microvasculature is under investigation in our laboratory.41,42
Trauma-related injury to the glycocalyx and endothelial vascular barrier occurs early after injury and is related to shock severity.17,43 The duration of the vascular barrier dysfunction is unknown. In vivo data suggest that recovery time of the glycocalyx endothelial barrier may require 5 days to 7 days.44 Spinella et al.45 have shown that use of fresh whole blood may be optimal for the early resuscitation of the patient with significant hemorrhage. This may in part be due to the improved perfusion offered by fresh RBCs as opposed to standard issue PRBC. Measurement of glycocalyx and endothelial parameters may help guide the use of “young” versus “old” banked blood during this vulnerable period.30
The microfluidic platform used in our in vitro study of the interaction of the vascular endothelium with RBCs from fresh and blood bank sources suggest that both entities are important in assessing the impact of the age of stored blood in the ability to restore microvascular perfusion in the transfusion therapy following severe trauma.
L.N.D. and D.L. made substantial contributions to the conception or design of the work; they also conceived and designed the experiments. L.N.D. and D.L. analyzed the data. D.L. and L.N.D. drafted the article. L.N.D. and D.L. critically revised the article for intellectual content. All authors provided final approval of the version to be published.
The authors declare no conflicts of interest.
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ROSEMARY A. KOZAR, M.D., Ph.D. (Baltimore, Maryland): Good afternoon. The authors have continued their investigations into the endothelial cell glycocalyx using their in-vitro microfluidic system.
In the current study the authors examined the interactions of endothelial cells and red blood cells under stress conditions with the hypotheses that older red cells would adversely affect this interaction.
The authors confirmed changes in glycocalyx thickness as well as syndecan-1 and hyaluronic acid shedding in activated endothelial cells and, interestingly, demonstrated changes in red blood cell glycocalyx with age of red cells.
They then examined red cell adherence in their activated endothelial cells and found there to be an increase in RBC adherence as age of the red cells increased.
I must admit I was not aware that there was a glycocalyx in red blood cells. I was wondering how the glycocalyx in red cells differed from that of the endothelial cell? What is the function of the red cell glycocalyx?
Second, the authors demonstrated changes in endothelial cell glycocalyx when the cells were activated and in red cell glycocalyx with duration of storage. But do you have evidence that the increased adherence of the red cells is causally related to the changes in the glycocalyx?
Third, as a control, did you look at the red cell adherence when cells were stimulated with the fresh red blood cells?
You showed us data for the 14 days and the 21 days but as a control did you look at how the fresh red blood cells were affected in the stimulated endothelial cells?
Lastly, could you hypothesize if the degree of red cell adherence that you demonstrate is sufficient to cause microvascular thromboses and subsequent organ damage that we see in some of these clinical studies?
Thank you. I appreciate the privilege of the presentation.
Lawrence Diebel M.D. (Detroit, Michigan): The Glycocalyx is a part of the red blood cell membrane. Storage of red blood cells is known to cause compositional changes of the red blood cell membrane which increase with time in storage.
Thus the results of this experiment add new information to the existing data regarding the effects of storage on red blood cells. Also the loss of the red blood cell glycocalyx does occur with senescence in vivo, and contributes to capture and removal in the splenic vasculature.
Regarding metabolic effects of storage, this is an entirely different storys. What I can tell you in studies we are doing comparing red blood cells in the obese with the metabolic syndrome that there are differences compared to red blood cells from non-obese patients. So this is something we will look at in the future.
The three different blood groups, fresh blood, banked blood with storage less than 14 days, or greater than 21 days were all perfused with normal endothelium and endothelium subjected to biomimetic conditions of shock...so all group variations were performed.
We use a hematocrit of 1.5% in these studies and have recently completed studies at a hematocrit of 21%. The lower hematocrit makes it easier to accurately count individual red blood cells. The studies with the higher hematocrit lead to significantly higher number of red blood cells adherent to the endothelium. And each microfluidic channel may be thought of as representing a single microcirculatory unit in vivo, I believe these results would be clinically relevant.