Hypertonic saline resuscitation of hemorrhagic shock diminishes neutrophil rolling and adherence to endothelium and reduces in vivo vascular leakage

Abstract

Objective: To evaluate the in vivo effects of hypertonic saline (HTS) resuscitation on the interactions of endothelial cells (ECs) and polymorphonuclear neutrophils (PMNs) and vascular permeability after hemorrhagic shock.

Summary background data: The PMN has been implicated in the pathogenesis of EC damage and organ injury following hemorrhagic shock. Compared to Ringer's lactate (RL), HTS resuscitation diminishes PMN and EC adhesion molecule expression and organ sequestration of PMNs.

Methods: In a murine model of hemorrhagic shock (50 mmHg for 45 minutes followed by resuscitation) using intravital microscopy on cremaster muscle, the authors studied PMN-EC interactions and vascular leakage (epifluorescence after 50 mg/kg fluorescent albumin) in three resuscitation groups: HTS (shed blood + 4 cc/kg 7.5% HTS, n = 12), RL (shed blood + RL [2x shed blood volume], n = 12), and sham (no hemorrhage or resuscitation, n = 9). EC ICAM-1 expression was evaluated by immunohistochemistry. Data, presented as mean +/- SEM, were evaluated by analysis of variance with Bonferroni correction.

Results: There were no differences between groups in flow mechanics. Compared to RL, HTS animals (t = 90 minutes) displayed diminished PMN rolling and PMN adhesion to EC at time intervals beyond t = 0. There were no differences between the sham and HTS groups. Vascular leakage was 45% lower in HTS than in RL-resuscitated animals. Cremaster EC ICAM-1 expression was similar in the two groups.

Conclusions: Using HTS instead of RL to resuscitate hemorrhagic shock diminishes vascular permeability in vivo by altering PMN-EC interactions. HTS could serve as a novel means of immunomodulation in hemorrhagic shock victims, potentially reducing PMN-mediated tissue injury.

Figure 1. Representative examples of fluorescent intravital microscopic images contrasting the low leakage of FITC-labeled albumin from postcapillary venules in HTS (A) compared with RL (B) animals. I_P (perivenular light intensity) and I_V (venular light intensity) represent regions evaluated for fluorescence.

Figure 2. RL animals displayed more rolling neutrophils than either HTS or sham groups. Values are expressed as mean number of rolling neutrophils per 2-minute period ± SEM. **RL versus sham only, P < .05. ‡RL versus either HTS or sham, P < .05. There were no significant differences between HTS and sham at any time point.

Figure 3. As compared to HTS and sham, RL animals had higher numbers of neutrophils adhering to endothelial cells. Values are expressed as mean number of stationary PMNs on a given 100-μm vessel length ± SEM. **RL versus sham only, P < .05. ‡RL versus either HTS or sham, P < .05. There were no significant differences between HTS and sham at any time point.

Figure 4. HTS animals tended to have higher PMN rolling velocities than RL aniimals. Values are expressed as the mean velocity (μm/s) of 10 PMNs crossing a given vessel section ± SEM. **RL versus sham only, P < .05. There were no significant differences between HTS and sham at any time point.

Figure 5. Compared to the two resuscitation groups, sham animals had a significantly higher blood pressure 30 minutes after resuscitation and thereafter. Values are expressed as mean (mmHg) ± SEM. *Sham versus either HTS or RL, P < .05. †Sham versus either HTS or RL, P < .001. There were no significant differences between HTS and RL at any time point.

Figure 6. Representative examples of intravital microscopic images contrasting the lack of PMN rolling (arrowhead) and PMN adherence (arrow) in HTS (A) as compared to RL (B) vessels 90 minutes after resuscitation.

Figure 7. Vascular permeability index (PI) (where I_P/I_V = PI) is an estimation of vessel leakage and was higher in HTS as compared to sham animals, while RL animals had the highest PI of all. Values are expressed as % permeability index ± SEM. †Versus HTS, P < .05. *Versus RL, P < .01.