Prehospital hypertonic saline resuscitation attenuates the activation and promotes apoptosis of neutrophils in patients with severe traumatic brain injury

Abstract

Background: Activation of polymorphonuclear neutrophils (PMNs) is thought to contribute to traumatic brain injury (TBI). Since hypertonic fluids can inhibit PMN activation, we studied whether hypertonic fluid resuscitation can reduce excessive PMN activation in TBI patients.

Methods: Trauma patients with severe TBI were resuscitated with 250 mL of either 7.5% hypertonic saline (HS; n = 22), HS + 6% dextran-70 (HSD; n = 22), or 0.9% normal saline (NS; n = 39), and blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Polymorphonuclear neutrophil activation (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers and oxidative-burst activity, as well as spontaneous PMN apoptosis were measured by flow cytometry.

Results: Relative to healthy controls, TBI patients showed increased PMN activation and decreased apoptosis of PMNs. In the HS group, but not in the HSD group, markers of PMN adhesion (CD11b, CD64) and degranulation (CD35, CD66b) were significantly lower than those in the NS group. These effects were particularly pronounced 12 h after resuscitation. Treatment with HS and HSD inhibited PMN oxidative burst responses compared with NS-treated patients. Hypertonic saline alone partially restored delayed PMN apoptosis. Despite these differences, the groups did not differ in clinical outcome parameters such as mortality and Extended Glasgow Outcome Scale.

Conclusions: This study demonstrates that prehospital resuscitation with HS can partially restore normal PMN activity and the apoptotic behavior of PMNs, whereas resuscitation with HSD was largely ineffective. Although the results are intriguing, additional research will be required to translate these effects of HS into treatment strategies that improve clinical outcome in TBI patients.

Fig. 1. TBI induces sustained neutrophilia

Peripheral blood samples were collected in EDTA vacutainers from healthy controls (n=20) and trauma patients resuscitated with NS (n=39), HSD (n=22), or HS (n=22) at the time of emergency department (ED) admission (≤3 h post-resuscitation) and 12 and 24 h post-resuscitation. Both the percentage (square symbols) of neutrophils among all white blood cells and the concentration (circles) of neutrophils (PMNs) in the peripheral blood were elevated relative to levels in healthy controls (triangles) throughout the sampling period, as determined by a Beckman Coulter Hematology Analyzer. Statistics: ^ap < 0.05 vs. age-matched healthy controls by ANOVA.

Fig. 2. HS, HSD, and NS differentially modulate neutrophil activation and adhesion molecule expression after TBI

Cell-surface expression of CD11b (A), CD62L (B), and CD64 (C), were assessed by multiparameter flow cytometry using freshly drawn heparinized whole blood samples. Results were expressed as both the mean fluorescence intensity (MFI, bars) in arbitrary units (a.u.) and percentage antigen-positive (%, lines) neutrophils (PMNs). Blood was sampled serially from patients resuscitated with normal saline (NS; n=39), hypertonic saline-dextran (HSD; n=22), or hypertonic saline (HS; n=22) 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 (triangles; 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 degranulation marker expression is differentially affected by HS and HSD

Cell-surface expression of CD35 (A), CD63 (B), and CD66b (C) were assessed by whole-blood multiparameter flow cytometry. Results are expressed as both the mean fluorescence intensity (MFI, bars) in arbitrary units (a.u.) and percentage antigen-positive (%, lines) neutrophils (PMNs). Blood was sampled serially from patients resuscitated with normal saline (NS; n=39), hypertonic saline-dextran (HSD; n=22), or hypertonic saline (HS; n=22) upon hospital admission (ED ≤3 h) and 12 and 24 h after resuscitation. Blood samples from matched healthy volunteers served as controls (triangles; 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. 4. Hypertonic fluid treatment reduces oxidative burst activity of neutrophils after TBI

Neutrophil (PMN) 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=39), HSD (n=22), or HS (n=22) at the time of emergency department (ED) 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 moderate 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: ^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. Hypertonic saline partially restores neutrophil apoptosis after TBI

Spontaneous apoptosis was assessed as the total percentage (mean ± SEM) of Annexin-V-positive (AnV⁺) neutrophils (PMNs) in unstimulated whole-blood samples from healthy controls (n=20) and trauma patients resuscitated with NS (n=39), HSD (n=22), or HS (n=22) at the time of emergency department (ED) admission (≤3 h post-resuscitation) and 12 and 24 h after resuscitation. Samples were incubated at 37°C for 20 h prior to staining and flow cytometric analysis. 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.