Aging Markers in Equine Red Blood Cells.

Sandra Kämpf,Elena Seiler,Anna Bogdanova,Jolanta Bujok,Regina Hofmann-Lehmann,Barbara Riond,Asya Makhro

doi:10.3389/fphys.2019.00893

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 Ca2+ 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 Ca2+ levels and its distribution within the cells. Based on the obtained findings, we suggest that intraerythrocytic Ca2+ 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.

Highlights

Equine red blood cells (RBCs) are surviving in the circulation for as long as 140–150 days (Carter et al, 1974) being exposed to shear stress, oxidation, and hyperthermia associated with high physical activity of these animals
L, M, and H fractions of equine and human RBCs (Figure 1A) were harvested and the cells forming them stained for RNA as a reticulocyte marker
Equine RBC forming L, M, and H fractions are presented with several potentially age-related features that confirm the increase of RBC density with aging and may be used for identification of RBC longevity

Summary

Introduction

Equine red blood cells (RBCs) are surviving in the circulation for as long as 140–150 days (Carter et al, 1974) being exposed to shear stress, oxidation, and hyperthermia associated with high physical activity of these animals. Multiple attempts to detect reticulocytes (RNA-positive cells or cells carrying transferrin receptor) in peripheral blood of horses failed even in the studies, where stress erythropoiesis was induced by phlebotomy, administration of phenylhydrazine, or erythropoietin administration (Lumsden et al, 1975a,b; Shull, 1981; Radin et al, 1986; Cooper et al, 2005). Detection of RBC life-span in horses in which stress erythropoiesis was induced by phlebotomy or erythropoietin administration, revealed a trend to faster clearance of cells produced upon stimulation of their de novo production (Lumsden et al, 1975a,b)

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Conclusion