Effect of Paprika Xanthophyll Supplementation on Cognitive Improvement in a Multitasking Exercise: A Pilot Study for Middle-Aged and Older Adults

Ingestion of paprika xanthophyll supplement (PX), which has antioxidant effects, has been recently reported to maintain red blood cell deformability and improve oxygen delivery efficiency. Therefore, we hypothesized that the brain activation induced by multitasking exercise in middle-aged and older participants along with the improved erythrocyte oxygen-carrying efficiency induced by PX supplementation would show a synergistic effect, increasing oxygen supply to the brain and improving cognitive function more effectively. In study 1, cerebral blood flow measurements were conducted during the multitasking exercise and cognitive function tests to verify their effect on cognitive function. The results confirmed that cerebral blood flow increased during the exercise and cognitive function improved after the exercise. In study 2, we compared the effects of the multitasking exercise on cognitive function before and after PX supplementation in middle-aged and older participants to evaluate the effects of PX supplementation. The results suggested that PX supplementation enhanced the effects of active multitasking exercise on cognitive function. We speculate that the improvement of oxygen transport efficiency by PX resulted in more effective oxygen supply, allowing the multitasking exercise to occur more effectively, which was reflected as an improvement in the cognitive function.

Changes in blood cell deformability in Chorea-Acanthocytosis and effects of treatment with dasatinib or lithium

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of 5 ChAc patients compared to healthy controls during up to one-year individual off-label treatment with the tyrosine kinases inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results show that blood cell deformability - including both RBCs and leukocytes - in general is altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observe alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hints at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.

Different Involvement of Band 3 in Red Cell Deformability and Osmotic Fragility—A Comparative GP.Mur Erythrocyte Study

GP.Mur is a clinically important red blood cell (RBC) phenotype in Southeast Asia. The molecular entity of GP.Mur is glycophorin B-A-B hybrid protein that promotes band 3 expression and band 3–AQP1 interaction, and alters the organization of band 3 complexes with Rh/RhAG complexes. GP.Mur+ RBCs are more resistant to osmotic stress. To explore whether GP.Mur+ RBCs could be structurally more resilient, we compared deformability and osmotic fragility of fresh RBCs from 145 adults without major illness (47% GP.Mur). We also evaluated potential impacts of cellular and lipid factors on RBC deformability and osmotic resistivity. Contrary to our anticipation, these two physical properties were independent from each other based on multivariate regression analyses. GP.Mur+ RBCs were less deformable than non-GP.Mur RBCs. We also unexpectedly found 25% microcytosis in GP.Mur+ female subjects (10/40). Both microcytosis and membrane cholesterol reduced deformability, but the latter was only observed in non-GP.Mur and not GP.Mur+ normocytes. The osmotic fragility of erythrocytes was not affected by microcytosis; instead, larger mean corpuscular volume (MCV) increased the chances of hypotonic burst. From comparison with GP.Mur+ RBCs, higher band 3 expression strengthened the structure of RBC membrane and submembranous cytoskeletal networks and thereby reduced cell deformability; stronger band 3–AQP1 interaction additionally supported osmotic resistance. Thus, red cell deformability and osmotic resistivity involve distinct structural–functional roles of band 3.

Assessing the Physiologic Relevance of Red Blood Cell Deformability in Iron Deficiency Anemia

Background: Iron deficiency anemia (IDA), which affects individuals of all ages worldwide, is an often overlooked and undertreated component of chronic disease, despite data correlating its association with adverse outcomes in patients with cardiovascular disease (von Haehling, Nat Rev Cardiol, 2015). While red blood cells (RBCs) in IDA are known to be smaller and contain less hemoglobin than healthy RBCs, how RBC deformability is altered in IDA remains poorly understood; some ektacytometry studies have observed impaired deformability in iron deficient RBCs (idRBCs), while others described either unchanged or increased deformability (Brandão, Clin Hemorheol Microcirc, 2009). Here we ask: can single cell biophysical techniques definitively determine whether idRBCs are less deformable than healthy RBCs and how heterogenous that phenomena may be? Recent investigations into IDA's role in cardiovascular disease have generally focused on the myocardium and coronary vasculature, yet much regarding other physiologic implications remains unknown, including whether idRBCs cause microvascular obstruction or vasculopathy. To address such questions, we leveraged a suite of microvascular models we developed. Methods: We first coupled our microfluidic capillary model with μEXACT, our customized automated particle tracking program for hematologic cell-based assays, to collect high-throughput velocity tracking of single RBCs from a healthy control and 2 IDA patients (anemic for age, ferritin <10 ng/mL) to create a single cell deformability index (sDI) for each RBC (Fig 1). Next, whole blood samples collected in EDTA tubes from the control and IDA patients were perfused into both straight 100μm wide channels (mimicking large venules) and branching 30μm wide microfluidic devices (mimicking smaller venules) at a constant shear rate for 30 minutes to observe if any occlusions or obvious alterations in flow were observed (Fig 2). Finally, using the straight 100μm channel microfluidic devices, human umbilical vein endothelial cells (HUVECs) were cultured throughout each microchannel and RBCs from a healthy control and 3 IDA patients were perfused in parallel microchannels for 4 hours. The endothelialized models were then fixed, permeabilized, and immunostained with antibodies against VCAM-1 and E-selectin, known markers of endothelial inflammation. Mean fluorescence intensity was measured to quantify endothelial inflammation (Fig 3). Results: sDI distribution histograms were obtained for healthy and IDA patient RBCs. The mean sDIs for IDA patient RBCs were decreased in comparison to the healthy RBCs. Additionally, both IDA patient's RBCs lacked a subpopulation of highly deformable RBCs, likely reticulocytes, seen in the healthy RBCs (Fig 1C). There was no evidence of microchannel occlusion for the healthy control or IDA patient whole blood samples in either the straight 100μm microchannels or branching 30μm microfluidic devices (Fig 2D). Finally, in our endothelialized microfluidic model, endothelium exposed to IDA patient RBCs exhibited increased VCAM-1 and E-selectin expression over endothelium exposed to healthy RBCs (Fig 3B). Conclusions: By utilizing an array of microfluidic models we can develop a more comprehensive understanding of the role idRBCs play systemically on microvasculature. Our combined microfluidic and image analysis system demonstrated decreased deformability in idRBCs and can offer detection of subpopulation differences that cannot be fully characterized with bulk techniques such as ektacytometry. So far, our data demonstrates that while no microvascular occlusion occurs, idRBCs contribute to endothelial inflammation. Additionally, the observation that physical interactions between endothelial cells and idRBCs are sufficient to cause endothelial inflammation warrants further investigation, as generally idRBCs had not been viewed as pro-inflammatory. Ongoing studies will couple unique sDI distribution curves with the degree of endothelial inflammation, as well as elucidate how these changes are associated with the degree of IDA or clinical events such as the initiation of iron supplementation. Utilizing atomic force microscopy to better understand how the idRBC membrane impacts deformability and developing biophysical computer simulations to determine if increased idRBC-endothelium interactions are observed in silico are also planned. Figure 1 Figure 1. Disclosures Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company.

Degradation of Red Blood Cell Deformability during Cold Storage in Blood Bags

RBC transfusions are a life-saving procedure, aiding both chronic and acute patients in restoring tissue oxygenation. The ability to store collected RBC units for prolonged periods has been one of the most transformative advances in medicine, significantly improving the reliability and the speed of access to blood. However, RBCs undergo a number of metabolic, structural, and biochemical changes during storage, collectively known as the storage lesion, that is detrimental to the quality of the RBC. A major challenge is the ability to evaluate the extent of the storage lesion, and thus the quality of the stored RBC unit directly prior to transfusion. The storage lesion can directly or indirectly reduce the ability of the RBC to deform through the small openings in the microvasculature. Rigid RBCs pose a risk of sequestration in capillaries, impeding blood flow and reducing tissue oxygenation, and are more likely to be cleared out by endothelial macrophages. Studies have shown that there is a loss in RBC deformability during storage and that the rate of RBC deformability loss is donor-dependent. Thus, RBC deformability can be a valuable and reliable biophysical marker of RBC unit quality. Currently, there is a need for a reliable measurement technique that is repeatable and sensitive enough to observe individual differences in RBC deformability in healthy donors, to enable quality control testing of RBC units. We have developed the microfluidic ratchet device, which sorts RBCs based on their deformability, allowing the measurement of both rigid and deformable sup-populations of RBCs within the sample, and generating a unique deformability curve. Here, we use this assay to predict the quality of stored RBC units. We assessed the deformability of 14 healthy donor RBC units through 8 weeks of cold storage at 4°C, which is 2 weeks beyond the Canadian Blood Services approved 6-week standard in Canada. We measured RBC deformability, standard hematological parameters (MCV, MCHC, MCH, and RDW), and hemolysis levels at the time of RBC unit manufacture (week 0), followed by weeks 2, 4, 6, and 8. The microfluidic ratchet device operates by forcing RBCs to deform and travel through rows of tapered constrictions. Constriction size changes from 7.5 to 1.5 µm and is reflective of the microvasculature and vessel opening sizes encountered by RBCs in circulation. RBCs are sorted into 12 distinct outlets based on their deformability. Distribution of RBCs in outlets 1-12 can be quantified and used to calculate the cumulative distribution curve. The cumulative distribution curve provides a distinct deformability signature of each individual RBC sample, which can be defined as rigidity score (RS). RS provides an easy metric to compare the changes in RBC deformability throughout storage (ΔRS) in a single donor as well as across multiple donors. We show that there are both donor- and sex-specific differences in the RBC deformability signatures of stored RBC units. We observed significant inter-donor variability in RBC deformability measured on the day of the RBC unit manufacture, where male donors showed a more stable RBC deformability range (n=8, RS=3.00±0.18) compared to female donors (n=6, RS= 3.29±0.48). The average RS scores were stable between weeks 0-2 (ΔRS 0.07) and showed a reduction in deformability between weeks 1-6 (ΔRS 0.35), with the greatest loss seen between weeks 6-8 (ΔRS 0.42) of cold storage. Interestingly, the response to cold storage is variable, with ΔRS 0.22 to 0.90, suggesting that some donors are more susceptible to storage related changes in RBC deformability than others. Notably, the change in RS over time was donor-specific and did not correlate with RBC deformability at week 0. The majority of RBCs from male donors (ΔRS 0.485, p<0.05), but none of the female donors (ΔRS 0.172) showed changes in deformability during cold storage, suggesting that RBCs from female donors degrade at a slower rate compared to RBCs from male donors. The ability to profile RBC deformability at the individual blood bag level may help identify more stable RBCs for use in chronic and sensitive patients, or RBC units that can be safely stored beyond the 6-week storage window. Disclosures No relevant conflicts of interest to declare.

Deformability of Stored Red Blood Cells

Red blood cells (RBCs) deformability refers to the cells’ ability to adapt their shape to the dynamically changing flow conditions so as to minimize their resistance to flow. The high red cell deformability enables it to pass through small blood vessels and significantly determines erythrocyte survival. Under normal physiological states, the RBCs are attuned to allow for adequate blood flow. However, rigid erythrocytes can disrupt the perfusion of peripheral tissues and directly block microvessels. Therefore, RBC deformability has been recognized as a sensitive indicator of RBC functionality. The loss of deformability, which a change in the cell shape can cause, modification of cell membrane or a shift in cytosol composition, can occur due to various pathological conditions or as a part of normal RBC aging (in vitro or in vivo). However, despite extensive research, we still do not fully understand the processes leading to increased cell rigidity under cold storage conditions in a blood bank (in vitro aging), In the present review, we discuss publications that examined the effect of RBCs’ cold storage on their deformability and the biological mechanisms governing this change. We first discuss the change in the deformability of cells during their cold storage. After that, we consider storage-related alterations in RBCs features, which can lead to impaired cell deformation. Finally, we attempt to trace a causal relationship between the observed phenomena and offer recommendations for improving the functionality of stored cells.

Elongational Stresses and Cells

Fluid forces and their effects on cells have been researched for quite some time, especially in the realm of biology and medicine. Shear forces have been the primary emphasis, often attributed as being the main source of cell deformation/damage in devices like prosthetic heart valves and artificial organs. Less well understood and studied are extensional stresses which are often found in such devices, in bioreactors, and in normal blood circulation. Several microfluidic channels utilizing hyperbolic, abrupt, or tapered constrictions and cross-flow geometries, have been used to isolate the effects of extensional flow. Under such flow cell deformations, erythrocytes, leukocytes, and a variety of other cell types have been examined. Results suggest that extensional stresses cause larger deformation than shear stresses of the same magnitude. This has further implications in assessing cell injury from mechanical forces in artificial organs and bioreactors. The cells’ greater sensitivity to extensional stress has found utility in mechanophenotyping devices, which have been successfully used to identify pathologies that affect cell deformability. Further application outside of biology includes disrupting cells for increased food product stability and harvesting macromolecules for biofuel. The effects of extensional stresses on cells remains an area meriting further study.

Adhesion and Stiffness of Detached Breast Cancer Cells In Vitro: Co-Treatment with Metformin and 2-Deoxy-d-glucose Induces Changes Related to Increased Metastatic Potential

Metastatic cancer cells can overcome detachment-induced cell death and can proliferate in anchorage-independent conditions. A recent study revealed that a co-treatment with two drugs that interfere with cell metabolism, metformin and 2-deoxy-D-glucose, promotes detachment of viable MDA-MB-231 breast cancer cells. In the present study, we analyzed if these detached viable MDA-MB-231 cells also exhibit other features related to cancer metastatic potential, i.e., if they are softer and more prone to adhere to epithelial cells. The cell mechanics of attached cells and floating cells were analyzed by optical tweezers and cell deformability cytometry, respectively. The adhesion was assessed on a confluent monolayer of HUVEC cells, with MDA-MB-231 cells either in static conditions or in a microfluidic flow. Additionally, to test if adhesion was affected by the state of the epithelial glycocalyx, HUVEC cells were treated with neuraminidase and tunicamycin. It was found that the treated MDA-MB-231 cells were more prone to adhere to HUVEC cells and that they were softer than the control, both in the floating state and after re-seeding to a substrate. The changes in the HUVEC glycocalyx, however, did not increase the adhesion potential of MDA-MB-231.