Abstract
The aim of this study was to evaluate changes in RBC aggregation and deformability over 24 hr and suggest specific shear stress values for detecting RBC deformability in a mouse endotoxemia model using lipopolysaccharide (LPS).
Six-week-old male BALB/c mice received LPS (20 mg/kg) intraperitoneally. Aggregation indices (AIs) and T1/2 were measured to assess RBC aggregation, and elongation indices (EIs) were used to assess RBC deformability at shear stress values of 0.3, 0.5, 1, 3, 7, 10, 15 and 20 Pascals (Pa) 0, 30 min, 1, 2, 4, 6, 9, 12, 18 and 24 hours after the LPS injection.
No significant differences were detected in the AIs during the study period, however, T1/2 shortened significantly 2, 6, 12, 18, and 24 hr after the LPS injection. The EIs increased significantly 24 hr after LPS injection at 0.5 and 1 Pa shear stress, whereas it decreased significantly at 10 Pa of shear stress 24 hr after the LPS injection.
Altered RBC deformability was detectable 24 hr after the LPS injection and T1/2 may be a sensitive marker for detecting changes in RBC aggregation. The EIs should be measured at 1 Pa to detect changes in RBC deformability in LPS-induced septic mice.
Introduction
Severe sepsis and septic shock are associated with an overall hospital mortality rate of 28.4% despite recent advances in our understanding of sepsis [14].
Sepsis is a complex pathophysiological process that involves changes in the microcirculation and the biochemical and physiological characteristics of the blood constituents [5, 21]. The development of medical imaging techniques together with data from clinical investigations have helped to determine that the microcirculation plays a key role in sepsis [19]. The microcirculation can be influenced not only by endothelial cell function and smooth muscle tone but also by blood components, including red blood cells (RBC), white blood cells (WBC), platelets, and plasma.
Several studies have demonstrated that derangements in microcirculatory perfusion and impaired RBC rheology (increased aggregation and decreased deformability) are seen in septic patients at the time of ICU admission [4, 21]. Persistent microcirculatory changes and reduced RBC deformability are associated with organ failure and increased mortality [4, 15]. However, most studies have investigated patients or animals at one moment in time, without specific information about the time delay from the onset of sepsis or about the time course of the RBC alterations. In addition, specific shear stress has not been recommended to detect RBC deformability, notwithstanding a dynamic study of RBC deformability in relation to shear stress applied to the membrane.
A better understanding of changes in RBC rheology during sepsis and their effects on oxygen transport may lead to new therapeutic strategies to reduce organ failure in patients with sepsis and septic shock [13]. Therefore, we evaluated changes in RBC aggregation and deformability over 24 hr and suggest specific shear stress values for detecting RBC deformability in a mouse endotoxemia model using lipopolysaccharide (LPS).
Materials and methods
Animals and blood sampling
Six-week-old male BALB/c mice (Hanlim Co. Ltd., Hwasung, South Korea) were used in this study. This study was approved by the Ethical Committee on Animal Research of the Korea University College of Medicine. All experimental procedures involving animals were carried out in accordance with the NIH guide for the Care and Use of Laboratory Animals issued by the Korea University School of Medicine. All animals were maintained in a constant-temperature (24±2°C) room with a 12-hr light/dark cycle. The mice had ad libitum access to food and water.
The LPS-induced sepsis model was created by injecting 20 mg/kg LPS (E. coli O127: B8; Sigma, St. Louis, MO, USA) intraperitoneally in 0.4 ml physiological saline. The animals were injected with LPS (n = 6, LPS group) or 0.5 ml saline (n = 4, saline group) and were observed for 30 min, 2, or 24 hr. The animals were anesthetized with an intraperitoneal injection of 50 mg/kg Zoletyl (Tiletamine and zolazepam, VirbacKorea, Korea). The inferior vena cava (IVC) was exposed through a median incision using a sterile surgical procedure, and blood was obtained directly from the IVC.
The mice were divided randomly into ten sub-experimental groups (n = 6/group) according to time (0, 30 min, 1, 2, 4, 6, 9, 12, 18, and 24 hr after LPS injection) to investigate the time course of the RBC alterations.
Analytical procedures
RBC aggregation and RBC kinetics were evaluated with a microchip-based cell aggregometer (Rheoscan-A; Rheo Meditech, Seoul, Korea). A laser backscatter light was used for the RBC aggregation measurements, in which the intensity of the reflection depended on whether the RBCs formed rouleaux at rest or if they were disaggregated and deformed at high shear rates. The computer program analyzed the aggregation parameters of the RBCs, which were based on a syllectogram, which is a curve of the relationship between laser backscatter intensity and time. A decrease in laser backscatter intensity represents progression of the aggregation process. The relevant part of the syllectogram reflects both static and kinetic parameters of aggregation. The following parameters specific to the aggregation process were estimated from the syllectogram: the aggregation index (AI in %), which shows the kinetics and amplitude of aggregation and aggregation half-time (T1/2 in sec), which is the time to half-maximal aggregation [17].
The AI and T1/2 were measured 0, 30 min, 1, 2, 4, 6, 9, 12, 18, and 24 hr after LPS injection.
RBC deformability was measured using a microfluidic ektacytometer (Rheoscan-D; Rheo Meditech, Seoul, Korea). A RBC suspension was prepared by mixing 6 μl of whole blood with 0.5 ml of 0.14 mM polyvinylpyrrolidone (molecular weight = 360,000), and RBC deformability was measured.
RBS images were recorded using a CCD camera linked to a frame grabber integrated with a computer during flow of the suspension in a disposable rectangular slit at shear stress of 0.09–20 Pascals (Pa). The diffraction pattern during shear stress was processed in elliptic form. Elongation indices (EIs; measure of RBC deformability) were determined from an isointensity curve in the diffraction pattern using an ellipse-fitting program: (A–B)/(A+B), where A and B are the major and minor ellipse axes [16]. EIs were tested at shear stress values of 0.3, 0.5, 1, 3, 7, 10, 15, and 20 Pa at 0, 30 min, 1, 2, 4, 6, 9, 12, 18, and 24 hr after LPS injection. EImax, a theoretical maximum EI value for cell deformation at infinite stress, was also calculated.
Serum pro-inflammatory cytokines (interleukin [IL]-6 and IL-12) levels were measured at 0, 30 min, 1, 2, 4, 6, 9, 12, and 24 hr after LPS injection. Serum cytokine levels were determined using commercial enzyme linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA). Blood was collected by IVC puncture and centrifuged at 4,000× g and 4°C for 10 min to collect the serum. The IL-6 and IL-12 detection ranges were 7.80–500 pg/mL, respectively.
Statistical analysis
The data analysis was carried out using SAS v 9.4 (SAS Institute, Cary, NC, USA). All results are expressed as mean±SEM. Wilcoxon rank-sum test and median test were used to compare AI, T1/2 and EIs between saline group and LPS group and repeated-measures analysis of variance was used for the LPS injected group. When a significant main effect was detected, contrasts were used in post-hoc testing. A p-value <0.05 was considered significant.
Results
AI values were significantly greater and the T1/2 was shorter in the LPS group than those in the saline group 24 hr after the LPS or saline injections (p = 0.01, respectively) (Fig. 1). The EI increased significantly in the LPS group compared to that in the saline group at 0.3 and 1 Pa of shear stress (p = 0.03 and p = 0.01), whereas the EI decreased significantly in the LPS group compared to that in the saline group at 15 and 20 Pa of shear stress 24 hr after the LPS or saline injections (p = 0.02, respectively) (Fig. 2). However, no difference in EImax was detected between the two groups.
No significant differences were detected in AIs within LPS-injected group over the study period, however, T1/2 was significantly shorter 2, 6, 12, 18, and 24 hr after LPS injection compared to the baseline value (before LPS injection) (p = 0.003, 0.01, 0.0009, 0.016, and <0.0001, respectively) (Fig. 3).
The EImax and EIs at 0.5, 1, 10, and 20 Pa of shear stress were significantly different over time (p = 0.0047, p = 0.04, p < 0.0001, p = 0.0003, and p = 0.0006, respectively). EIs increased significantly 24 hr after the LPS injection at 0.5 and 1 Pa shear stress (p = 0.0038 and p = 0.005, respectively), whereas the EIs decreased significantly 24 hr after the LPS injection at 10 Pa of shear stress (p = 0.02). The EIs were not different at any specific time when compared with baseline (before LPS injection) at 20 Pa of shear stress; however, EImax decreased significantly 24 hr after the LPS injection (p = 0.01) (Fig. 4).
IL-6 and IL-12 increased significantly over the study period (p < 0.0001 and p = 0.0001) (Fig. 5).
Discussion
Our results confirm that RBC aggregation and deformability changed in the LPS induced sepsis animal model and revealed that the change could be detected using the T1/2 and EIs at 1 Pa 24 hr after the LPS injection.
RBC aggregation, as reflected by AI values, was greater in the LPS-injected mice than that in the saline-injected mice, however, no differences were found in the AIs of the LPS-injected mice over the study period. Recent data show that AIs are greater in septic than in non-septic patients, however, they did not reveal the time difference over the study period (1–3 days) in the patients with sepsis [4]. These findings seem to be comparable to our results. T1/2 decreased significantly in the LPS-injected mice compared to that in the saline-injected mice and a significant difference was detected over the study period. A shorter aggregation half time (T1/2) indicates that RBC aggregates formed a rouleaux a shorter time after their disaggregation, and that their spontaneous ability to aggregate is higher.
The direct action of endotoxin (LPS) on the vascular endothelium and the acute increase in capillary wall permeability result in enhanced filtration of intracapillary fluid and plasma components form the vascular bed into the intercellular spaces [2]. At the same time, the capillary wall is three times more permeable to albumins, compared to globulins. Consequently, the concentrations of high-molecular proteins (fibrinogens and globulins) rise in microvessels which accounts for the enhanced intravascular aggregation of RBCs through formation of intererythrocyte bridges [11, 18]. RBC aggregation should be discriminated from aggregability which denotes the ability of RBCs to form aggregates under specific conditions [8]. Our results suggest that T1/2 is a more sensitive indicator than the AI value for assessing RBC aggregability.
RBC deformability decreases during sepsis, which makes it difficult for the RBCs to pass through small capillaries [1, 18]. However, our results show that EIs increased under low shear stress (≤1 Pa) and decreased at >10 Pa in the septic animal model. Interestingly, a similar pattern was shown in patients with sepsis. EIs increase significantly in patients with sepsis compared to those in non-septic patients at 0.3 and 0.48 Pa of shear stress and decreased significantly at >3 Pa in patients with sepsis compared to those in non-septic patients [4]. Reggiori et al. reported that EIs increase significantly at 0.48 Pa in patients with sepsis compared to those in volunteers but were significantly lower in patients with sepsis than those in volunteers for virtually all shear stress values (except 0.48 and 0.76 Pa) [13]. Another clinical study demonstrated no differences between sepsis and control groups at low shear stresses of 0.3 and 0.6 Pa, but significant decreases in RBC deformability at shear stresses of 1.2–60 Pa [12]. RBCs appear to have a tendency to be more deformable at low shear stress and stiffer at high shear stress during sepsis.
Under LPS-induced sepsis reactive oxygen species and secondary mediators (tumor necrosis factor-α, IL-1, IL-6, IL-12, etc.) released by activated WBCs and the LPS component attack RBC membranes causing dramatic impairment of their membrane resilience-elastic properties [7, 12].
Baskurt et al. reported that EIs are significantly lower in patients with sepsis than those in volunteers only at shear stress <5 Pa, but EIs decreased significantly at 0.5 Pa 6 hr after cecal ligation-puncture (CLP), and only at 0.5 and 1.58 Pa 18 hr after CLP in an experimental animal model [1].These discrepancies in specific shear stress may be due to different species, deformability measurement methods, and experimental methods that caused the sepsis [3, 12]. Altered RBC rheology occurs at different times according to the sepsis model, endotoxin type (LPS, or live bacteria), doses, and administration methods. An intraperitoneal injection of endotoxin (3 mg/100 g body weight) induces a change in RBC deformability within 6 hr after the LPS injection [6] and altered RBC deformability was detected 10 min after intravascular administration of 5 mg/kg LPS [20]. An intravascular injection or excessive doses of endotoxin or live bacteria result in rapid evolution of sepsis [10].
Our results show that alterations in RBC deformabilty occurred at 1 Pa 24 hr after an intraperitoneal LPS (2 mg/kg) injection and changes in RBC aggregation were detected 12 hr after the LPS injection.
Conclusively, our results reveal that altered RBC deformability was detectable 24 hr after the LPS injection in mice. These changes followed a dual pattern according to shear stress; thus, shear stress should be considered to detect RBC deformability. T1/2 seems to be more sensitive than AIs for detecting RBC aggregation, as the change was detected earlier in the septic animal model.
Footnotes
Acknowledgments
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (No. NRF-2015R1A2A2A01006734).