Sepsis impairs microvascular autoregulation and delays capillary response within hypoxic capillaries

Abstract

Introduction: The microcirculation supplies oxygen (O2) and nutrients to all cells with the red blood cell (RBC) acting as both a deliverer and sensor of O2. In sepsis, a proinflammatory disease with microvascular complications, small blood vessel alterations are associated with multi-organ dysfunction and poor septic patient outcome. We hypothesized that microvascular autoregulation-existing at three levels: over the entire capillary network, within a capillary and within the erythrocyte-was impaired during onset of sepsis. This study had three objectives: 1) measure capillary response time within hypoxic capillaries, 2) test the null hypothesis that RBC O2-dependent adenosine triphosphate (ATP) efflux was not altered by sepsis and 3) develop a framework of a pathophysiological model.

Methods: This was an animal study, comparing sepsis with control, set in a university laboratory. Acute hypotensive sepsis was studied using cecal ligation and perforation (CLP) with a 6-hour end-point. Rat hindlimb skeletal muscle microcirculation was imaged, and capillary RBC supply rate (SR = RBC/s), RBC hemoglobin O2 saturation (SO2) and O2 supply rate (qO2 = pLO2/s) were quantified. Arterial NOx (nitrite + nitrate) and RBC O2-dependent ATP efflux were measured using a nitric oxide (NO) analyzer and gas exchanger, respectively.

Results: Sepsis increased capillary stopped-flow (p = 0.001) and increased plasma lactate (p < 0.001). Increased plasma NOx (p < 0.001) was related to increased capillary RBC supply rate (p = 0.027). Analysis of 30-second SR-SO2-qO2 profiles revealed a shift towards decreased (p < 0.05) O2 supply rates in some capillaries. Moreover, we detected a three- to fourfold increase (p < 0.05) in capillary response time within hypoxic capillaries (capillary flow states where RBC SO2 < 20 %). Additionally, sepsis decreased the erythrocyte's ability to respond to hypoxic environments, as normalized RBC O2-dependent ATP efflux decreased by 62.5 % (p < 0.001).

Conclusions: Sepsis impaired microvascular autoregulation at both the individual capillary and erythrocyte level, seemingly uncoupling the RBC acting as an "O2 sensor" from microvascular autoregulation. Impaired microvascular autoregulation was manifested by increased capillary stopped-flow, increased capillary response time within hypoxic capillaries, decreased capillary O2 supply rate and decreased RBC O2-dependent ATP efflux. This loss of local microvascular control was partially off-set by increased capillary RBC supply rate, which correlated with increased plasma NOx.

Fig. 1

Relationship between capillary red blood cell (RBC) supply rate (SR) and plasma NO₂ ⁻ + NO₃ ⁻ (NOx). RBC supply rate (RBC/s) at the arteriolar end of capillaries in hind limb skeletal muscle was measured using a functional microvascular imaging system, as described in the Methods. Plasma NOx was measured in arterial blood samples, as described in the Methods. Linear regression detected a significant relationship between capillary RBC supply rate and plasma NOx level in septic animals (black boxes). White circles = sham

Fig. 2

Capillary red blood cell (RBC) supply rate, O₂ saturation and capillary O₂ supply rate profiles. Four representative 30-second profiles of capillary RBC supply rate (SR, blue circles), RBC hemoglobin O₂ saturation (SO₂, red squares) and O₂ supply rate (qO₂, green triangles) are shown in panels a–d. Based on 95 % confidence intervals (see Additional file 5: Table S1 in sham animals), SR was assessed as either slow, average or fast, while qO₂ was assessed as being either low, average, or high. Capillaries were also categorized according to their functional capillary density, as either continuous (RBC SR >0 for 30 seconds, panels a,c), intermittent (if RBC supply rate came to arrest for at least one second, panel b, where oval marks interval of stopped-flow) or stopped (if RBCs remained at arrest, panel d, provided RBCs remained at arrest for 30 seconds). e,f qO₂ distributions for sham and cecal ligation and perforation (CLP) animals, respectively. Legend for 30-second profile statistics (mean (coefficient of variation)). A significant difference between qO₂ distributions was determined by Chi-squared analysis, p < 0.05

Fig. 3

Capillary response time within hypoxic capillary (red blood cell oxygen saturation <20 %). a–e Five 30-second capillary red blood cell (RBC) supply rate (SR, blue circles), RBC hemoglobin O₂ saturation (SO₂, red squares) and O₂ supply rate (qO₂, green triangles) (SR–SO₂–qO₂) profiles. Capillary response time with low RBC saturation (SO₂ < 20 %) was assessed as the time required for a capillary to return to a state where RBC SO₂ > 20 %. The dashed horizontal red line shown in SR–SO₂–qO₂ profiles, panels a–e, at SO₂ = 20 % is the threshold used to quantify the response. Ovals indicate time intervals in the SR–SO₂–qO₂ profiles where capillary RBC SO₂ had fallen below 20 %. f The capillary response times at both the arteriolar (art-end) and venular (ven-end) ends of capillary networks in sham and cecal ligation and perforation (CLP) animals. Legend with profile statistics (mean (coefficient of variation)), capillary response time is mean (SD). NA not applicable

Fig. 4

Erythrocyte O₂-dependent ATP efflux decreased in sepsis. a Plasma ATP levels under normoxic (red blood cell (RBC) exposed to 5 minutes 21 % O₂) and hypoxic conditions (RBC exposed to 5 minutes 0 % O₂) in sham and cecal ligation and perforation (CLP) animals. b The % change in RBC O₂-dependent ATP efflux. ATP efflux was normalized as the hypoxia/normoxia ratio (H/N) and is summarized in Table 2

Fig. 5

Model of microvascular autoregulation. a Pathways involved in microvascular autoregulation. At the model center (green square) is red blood cell (RBC) O₂-dependent ATP efflux, where RBCs act as signal transducers responding to local O₂ gradients, shear stress and metabolic conditions. Blue dots (A–A2) indicate normal microvascular function whereby partial pressure of oxygen (PO₂) gradients or RBC deformation [–38] induce RBCs to release ATP, triggering a conducted vascular response leading to increased capillary RBC supply rate [10, 11, 16], matching local O₂ delivery with demand. Red dots indicate negative feedback on RBC O₂-dependent ATP efflux by nitric oxide (NO) [24], lactate [43] and decreased RBC deformability [42] (dashed red boxes). Multiple effects of NO on microvascular autoregulation, O₂ transport and O₂ consumption (orange dots (S1–S6)) include: S1, inhibiting RBC O₂-dependent ATP efflux [24]; S2, reducing conducted vascular response [9, 25]; S3, decreasing RBC deformability [22]; S4, inhibiting mitochondrial function [26] and O₂ consumption [2]; S5, inducing vasodilation, but altering vasoreactivity by inducing arteriolar hyporesponsiveness [–30]. Sepsis increases lactate (S6) via tissue hypoxia or pyruvate dehydrogenase (PDH) phosphorylation [31], which decreases RBC O₂-dependent ATP efflux. Sepsis impaired microvascular autoregulation is manifested by increased capillary response time within hypoxic capillaries, attenuated RBC O₂-dependent ATP efflux, increased capillary stopped-flow [2, 4, 5] and low capillary venular end O₂ supply rates. b A flow chart of the model, where blue arrows trace normal microvascular autoregulation, red arrows show negative feedback on RBC O₂-dependent ATP efflux and orange arrows indicate NO-mediated effects. Dashed lines show relationships to microvascular function and autoregulation during sepsis. i/nNOS Inducible/neuronal nitric oxide synthase, qO ₂ Capillary O₂ supply, VO ₂ O₂ consumption

Fig. 6

Metabolic, erythrocyte and microvascular changes at the 6-hour end-point. The figure summarizes the metabolic, RBC and microvascular functional changes observed at the 6-hour end-point in this study. ATPe(H/N) RBC O₂-dependent ATP efflux ratio (hypoxia/normoxia; see methods for description), CDstop Capillary density of stopped-flow capillaries, CLP Cecal ligation and perforation, NOx Nitrite + nitrate, t(SO ₂  < 20) Capillary response within hypoxic capillaries, where t is the time required for a capillary to return to a state where RBC oxygen saturation (SO₂) >20 %, qO ₂ (v) Venular end RBC O₂ supply rate. *p < 0.05

Fig. 7

Model of three-levels of microvascular autoregulation. a–c Schematics of three levels of microvascular autoregulation: 1) the overall skeletal muscle capillary network, 2) the capillary and 3) the erythrocyte, respectively. a Microvascular autoregulation at the capillary network level is viewed as the integrated conducted vascular response over the entire network feeding back to the resistance vessels [45], where nitric oxide (NO) relaxes smooth muscle vasodilating feeding arterioles, causing downstream increases in capillary red blood cell (RBC) supply rate (SR) [10, 11]. The dashed green line represents the conducted vascular response. b At the capillary level is the interaction between vascular ATP (released by hypoxic RBCs) and purinergic type 2 (P2Y) receptors on endothelial cells, which trigger the conducted vascular response. c At the level of the hypoxic erythrocyte is the interaction (metabolic switch) between deoxyhemoglobin and cdb3 at the inner RBC membrane, where deoxyhemoglobin displaces glycolytic enzymes, triggering glycolysis and ATP release [–15, 57, 58]. (Note, RBC O₂-dependent ATP release is inhibited by glycolytic inhibitors, CO [13] and NO [24]). The dashed circle in c shows two additional RBC mechanisms. While NO₂ ^– has been reported to function in hypoxic vasodilation whereby deoxyhemoglobin converts NO₂ ^– to NO [46, 56], its role in sepsis is unclear. Similarly, it is unclear how hemoglobin-derived S-nitrosothiol [59] would function as a vasodilator in the capillary network, as capillaries are not surrounded by smooth muscle. Art-end Arteriolar end of capillaries, cdb3 Cytoplasmic domain of band 3, eNOS endothelial nitric oxide synthase, GE Glycolytic enzymes, Hb Hemoglobin, LDH Lactate dehydrogenase, NO ₂ ^– Nitrite, PFK Phosphofructokinase, qO ₂ Capillary oxygen supply rate, R Relaxed Hb state, Smc Smooth muscle cell, SO ₂ Oxygen saturation, T Tense Hb state, Ven-end Venular end of capillaries