Aging Markers in Equine Red Blood Cells

Abstract

Detection of hematopoietic activity in horses is a challenge due to the lack of cells carrying reticulocyte markers such as RNA remnants or CD71 in the circulation. In this study, we fractionated equine red cells according to their density and analyzed the cells forming low (L), medium (M), and high (H) density fractions for markers of aging such as membrane loss, oxidation, and alterations in the intracellular free Ca 2+ levels. Cells forming L and M fraction were highly heterogeneous in projected areas and shapes, and had higher propensity to swell in response to hypo-osmotic challenge than the cells from the H fraction. The densest cells were deprived of band 3 protein compared to the cells within L or M fraction. Furthermore, the equine red cells from the H fraction were hyper-oxidized compared to the cells within M and L fractions as follows from an increase in autofluorescence characteristic for oxidized damaged hemoglobin and from thiol oxidation as detected using monobromobimane. The lightest cells showed lower free thiol content compared to the red blood cells from the M fraction, but did not contain oxidized hemoglobin. Finally, the majority of red blood cells forming L, M, and H fraction prominently differed from each other in intracellular free Ca 2+ levels and its distribution within the cells. Based on the obtained findings, we suggest that intraerythrocytic Ca 2+ levels and its subcellular distribution, eosin-5-maleimide binding test for band 3 abundance, and autofluorescence of cells along with the changes in red blood cell indices, distribution width and creatine levels may become potential markers of regenerative erythropoiesis in horses. Validation of the power of these potential markers of red cell aging is pending.

Simple Assessment of Red Blood Cell Deformability Using Blood Pressure in Capillary Channels for Effective Detection of Subpopulations in Red Blood Cells

“…As a demonstration, the present method was applied to measure the RBC deformability in fractions of RBCs of different densities obtained from the same sample. To separate RBCs based on their density, a simple method was developed that uses centrifugation-driven separation on the Percoll density gradient, which is currently used for RBC fractionation and disease diagnostics. ,, Based on the protocol for fractionation on the Percoll density gradient, RBCs donated from a healthy donor were separated into subpopulations of cells with low, high, and medium densities, as shown in Figure A.…”

“…Finally, to quantify the contribution of RBC density to RBC deformability, normal blood was fractioned with respect to the RBC density difference, as shown in Figure Ai. Fractionation of RBCs was performed in a continuous density gradient of an isotonic solution of Percoll (GE Healthcare, Sweden) . After setting the tube vertically in the gravitational direction, two fractions of RBCs (F 1 , normal density; and F 2 , high density) were collected in the middle and lower regions, respectively.…”

“…Fractionation of RBCs was performed in a continuous density gradient of an isotonic solution of Percoll (GE Healthcare, Sweden). 37 After setting the tube vertically in the gravitational direction, two fractions of RBCs (F 1 , normal density; and F 2 , high density) were collected in the middle and lower regions, respectively. Two blood samples were prepared by mixing RBCs with autologous plasma.…”

The protective effect of the spleen in sickle cell patients. A comparative study between patients with asplenia/hyposplenism and hypersplenism

“…Morphological characteristics of RBC and reticulocytes, phosphatidylserine (PS) exposure on outer membrane leaflet representing early apoptotic RBCs was measured by annexin V+ RBCs, intracellular Ca 2+ and oxidative stress measured by reduced glutathione (GSH) levels in RBC were investigated by FC as described elsewhere ( Kuypers et al, 1996 ; Kämpf et al, 2019 ). Briefly, 2 µl of whole blood suspended in a 1 ml plasma-like medium was loaded with 1.5 µl of either: - Intracellular Ca 2+ dye, Fluo-4 AM [1 mM stock, (Thermo Fisher Scientific].…”

Killing them softly with EPO: a new role for erythropoietin in the homeostasis of red blood cells

“…As for RBC destruction, mammalian RBCs are devoid of most intracellular structures [5, 6], so they escape the canonical way to remove cells from tissues, which is apoptosis [7, 8]. Instead, they are eliminated by macrophages of the splenic and hepatic sinusoids when they reach a critical species-specific age (around 60 days in rats, 70 in cats, 120 in humans, 150 in horses, and 160 in bovines [9, 10]). The marked regularity in RBC lifespan among mammal species led to the prevalent idea that RBC lifespan is fixed [11, 12].…”