Resuscitation of traumatic hemorrhagic shock patients with hypertonic saline-without dextran-inhibits neutrophil and endothelial cell activation

Abstract

Posttraumatic inflammation and excessive neutrophil activation cause multiple organ dysfunction syndrome (MODS), a major cause of death among hemorrhagic shock patients. Traditional resuscitation strategies may exacerbate inflammation; thus, novel fluid treatments are needed to reduce such posttraumatic complications. Hypertonic resuscitation fluids inhibit inflammation and reduce MODS in animal models. Here we studied the anti-inflammatory efficacy of hypertonic fluids in a controlled clinical trial. Trauma patients in hypovolemic shock were resuscitated in a prehospital setting with 250 mL of either 7.5% hypertonic saline (HS; n = 9), 7.5% hypertonic saline + 6% dextran 70 (HSD; n = 8), or 0.9% normal saline (NS; n = 17). Blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Multicolor flow cytometry was used to quantify neutrophil expression of cell-surface activation/adhesion (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers as well as oxidative burst activity. Circulating concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVACM-1), P- and E-selectins, myeloperoxidase (MPO), and matrix metalloproteinase 9 (MMP-9) were assessed by immunoassay. Multiple organ dysfunction syndrome, leukocytosis, and mortality were lower in the HS and HSD groups than in the NS group. However, these differences were not statistically significant. Hypertonic saline prevented priming and activation and neutrophil oxidative burst and CD11b and CD66b expression. Hypertonic saline also reduced circulating markers of neutrophil degranulation (MPO and MMP-9) and endothelial cell activation (sICAM-1, sVCAM-1, soluble E-selectin, and soluble P-selectin). Hypertonic saline + 6% dextran 70 was less capable than HS of suppressing the upregulation of most of these activation markers. This study demonstrates that initial resuscitation with HS, but neither NS nor HSD, can attenuate posttraumatic neutrophil and endothelial cell activation in hemorrhagic shock patients. These data suggest that hypertonic resuscitation without dextran may inhibit posttraumatic inflammation. However, despite this effect, neither HS nor HSD reduced MODS in trauma patients with hemorrhagic shock.

Figures

Fig. 1. Leukocyte and neutrophil counts were significantly elevated in all trauma patients

Peripheral blood samples were collected in EDTA vacutainers from healthy controls (n=20) and trauma patients resuscitated with NS (n=17), HSD (n=8), or HS (n=9) at the time of emergency department admission (≤3 h post-resuscitation) and 12 and 24 h post-resuscitation. Total leukocyte counts (A) and neutrophil (B; PMN) differential counts were determined with a Beckman Coulter Hematology Analyzer. Statistics: aP < 0.05 vs. age-matched healthy controls; bP < 0.05 vs. time-matched NS-treated patients; cP < 0.05 vs. time-matched HS-treated patients, by ANOVA.

Fig. 2. Neutrophil cell-surface activation, adhesion, and degranulation markers

Cell-surface expression of CD11b (A), CD62L (B), CD35 (C), CD63 (D), CD64 (E), and CD66b (F) were assessed by multiparameter flow cytometry using freshly drawn heparinized whole blood. Results were expressed as mean fluorescence intensity (MFI ± SEM) in arbitrary units (a.u.). The corresponding percentage of receptor-positive cells is shown in Table 2. Blood was sampled serially from patients resuscitated with normal saline (NS; n=17), hypertonic saline-dextran (HSD; n=8), or hypertonic saline (HS; n=9) upon hospital admission (ED ≤3 h) and 12 and 24 h after resuscitation. Blood samples from age and gender matched healthy volunteers served as control (n=20). Statistical analyses: aP < 0.05 vs. age-matched healthy controls; bP < 0.05 vs. time-matched NS-treated patients; cP < 0.05 vs. time-matched HS-treated patients, by ANOVA.

Fig. 3. Neutrophil oxidative burst activity

Neutrophil oxidative burst activity was measured by assessing intracellular rhodamine (Rho)123 mean fluorescence intensity (MFI ± SEM) in arbitrary units (a.u.) of neutrophils in whole-blood samples from healthy controls (n=20) and trauma patients resuscitated with NS (n=17), HSD (n=8), or HS (n=9) at the time of emergency department admission (≤3 h post-resuscitation) and 12 and 24 h after resuscitation. Samples were incubated at 37°C with N-formyl-methionine- leucine-phenylalanine (fMLP, 5 μM) as a weak stimulus or with phorbol 12-myristate 13-acetate (PMA, 8.1 μM) as a strong stimulus. Rho-123 MFI values were expressed as a percentage of unstimulated control samples. Statistical analyses: §P < 0.05 vs. unstimulated control values; aP < 0.05 vs. age-matched healthy controls; bP < 0.05 vs. time-matched NS-treated patients; cP < 0.05 vs. time-matched HS-treated patients, by ANOVA.

Fig. 4. Circulating myeloperoxidase and matrix metallopeptidase 9 levels

Plasma concentrations of MPO and MMP-9 were determined in healthy controls (n=20) and trauma patients resuscitated with NS (n=17), HSD (n=8), or HS (n=9) at the time of emergency department admission (≤3 h post-resuscitation) and 12 and 24 h after resuscitation, using electrochemiluminescence-based immunoassay detection methods. Statistical analyses: data are shown as mean ± standard error mean (SEM); aP < 0.05 vs. age-matched healthy controls; bP < 0.05 vs. time-matched NS-treated patients; cP < 0.05 vs. time-matched HS-treated patients, by ANOVA.

Fig. 5. Soluble endothelial adhesion molecules

Serum concentrations of soluble (s)ICAM-1 (A), sVCAM-1 (B), sE-selectin (C), and sP-selectin (D) were determined in healthy controls (n=20) and trauma patients resuscitated with NS (n=17), HSD (n=8), or HS (n=9) at the time of emergency department admission (≤3 h post-resuscitation) and 12 and 24 h after resuscitation. Statistics: data are shown as mean ± standard error mean (SEM); aP < 0.05 vs. age-matched healthy controls (n = 20); bP < 0.05 vs. time-matched NS-treated patients; cP < 0.05 vs. time-matched HS-treated patients, by ANOVA.