Differences in selected blood parameters between brachycephalic and non-brachycephalic dogs

Abstract

Introduction: Cranial and upper-airway anatomy of short-nosed, flat-faced brachycephalic dogs predisposes them to brachycephalic obstructive airway syndrome (BOAS). Periodic apnoea increased inspiratory resistance, and an inability to thermoregulate effectively are characteristic of BOAS, but internationally accepted objective markers of BOAS severity are missing. The objective of this study was to compare the selected blood parameters between non-brachycephalic (NC) and brachycephalic (BC) dogs, exploring the possibility of developing a blood test for BOAS severity grading in the future.

Methods: We evaluated blood biochemistry, complete blood cell counts, red blood cell (RBC) indices, reticulocyte counts, a blood-born marker of intermittent hypoxia (glutathione, NO production), RBC hydration, deformability, and blood markers of metabolic changes and stress between BC (n = 18) and NC (meso- and dolichocephalic, n = 22) dogs.

Results: Reticulocyte counts and the abundance of middle-fluorescence immature reticulocytes were significantly (p < 0.05) higher in BC dogs compared to NC dogs. BC dogs had significantly more NO-derived NO${}_2^{-}$/NO${}_3^{-}$ in plasma than NC dogs. RBCs of BC dogs were shedding significantly more membrane, as follows from the intensity of eosin maleimide staining, and had a significantly higher mean corpuscular hemoglobin concentration than NC dogs. Intracellular reduced glutathione content in RBCs of BC dogs was significantly lower, while plasma lactate was significantly higher in BC dogs compared to NC dogs. Plasma cholesterol and triglycerides were significantly lower, and cortisol was significantly higher in BC dogs compared to NC dogs. Eosinophil counts were significantly lower and the neutrophil-to-lymphocyte ratio was higher in BC dogs compared to NC dogs.

Discussion: Taken together, our findings suggest that the brachycephalic phenotype in dogs is associated with alterations at the level of blood cells and, systemically, with oxidation and metabolic changes. The parameters identified within this study should be further investigated for their potential as objective indicators for BOAS.

Keywords: blood markers; brachycephalic obstructive airway syndrome; brachycephaly; dog; hypoxia; red blood cells; stress.

Figure 1

Oxygen-sensitive parameters in blood samples of brachycephalic (BC) and non-brachycephalic (NC) dogs shown as box-and-whiskers plots. Plasma levels of nitrite and nitrate RNO (A), reticulocytes count (B), Middle fluorescence intensity reticulocyte counts in % (C) Hemoglobin O₂ saturation in mixed venous blood (D), hemoglobin (E), and hematocrit (F). After the normality of distribution of the data was tested, Unpaired t-test or the Wilcoxon signed-rank test was performed to detect significance of differences between the BC and NC groups. P-values are shown for each parameter. Numbers above the bars are the number of study participants. Blue boxes are for the brachycephalic group and white ones for the non-brachycephalic group. Dotted lines indicate a healthy reference range.

Figure 2

Red blood cell distribution width (RDW) and carboxy-hemoglobin abundance are shown in panels (A, B) respectively as markers of RBC distress and hemolytic activity. Dotted lines indicate healthy reference range.

Figure 3

Membrane stability, RBC indices, cell deformability, and hydration markers. Membrane surface area using EMA staining (band 3 protein) normalized to human control (A), Mean cell hemoglobin [MCH, (B)], Mean cell hemoglobin concentration [MCHC, (C)], and Mean cell volume [MCV, (D)]. The LoRRca osmoscan mode was used to detect the following ektacytometry indices: Area [the range of osmolarities tolerated while able to deform, (E)], Maximal elongation index EImax [deformability, (F)], Maximal tolerated osmolarity O_hyper [hydration state of RBCs, (G)], and minimal tolerated osmolarity O_min [osmotic stability, (H)]. Unpaired Student's t-test was performed on the datasets except for the Area parameter. For the latter, Wilcoxon rank sum test was performed.

Figure 4

RBC oxidation markers. Intraerythrocytic non-protein reduced thiols represented mainly by reduced glutathione [GSH, (A)], and total (non-protein and protein) reduced thiols assessed as monobromobimane (MBBr) fluorescence intensity (B).

Figure 5

Plasma concentrations of the inflammatory marker C-reactive protein CRP, (A) and of a stress marker, cortisol (B).

Figure 6

Metabolic markers in plasma. Glucose (A) and lactate (B) concentrations in plasma of 21 NC and 18 BC dogs. Plasma concentrations of cholesterol (C) and triglycerides (D) for 21 NC and 18 BC dogs. Dotted lines indicate healthy reference range.

Figure 7

Time-dependent alterations in the intraerythrocytic GSH levels (A), and in the abundance of band 3 protein in RBC membrane measured as EMA fluorescence intensity (B) during the 24 h simulated transportation.