It is generally accepted that severe head injury causes a profound inflammatory response within the brain leading to breakdown of the BBB with peripheral leukocyte invasion [5, 48]. There is also abundant evidence for reciprocal alterations between CNS and peripheral inflammatory cells/mediators, which initiate systemic inflammatory cascades [4, 49, 50]. Growing recognition of the limitations of isotonic fluids for cerebral resuscitation has led to the search for alternative osmotic agents aimed at restoring cerebral perfusion and reducing intracranial pressure, while simultaneously conferring neutroprotection. In this randomized controlled trial, we investigated the potential immunomodulatory effects of prehospital HSD resuscitation on the production and interplay of leukocyte and endothelial adhesion molecules, cytokines, and coagulation cofactors in severe TBI patients. Although there is extensive preclinical and clinical experience evaluating the use of hypertonic fluids for their superior volume-expanding properties following extra-cranial insults, little is known concerning the potential impact of hyperosmolar therapy on acute immunoinflammatory response after isolated head injury [17, 51]. HS/D has been shown to dampen posttraumatic hyperinflammatory responses and aberrant leukocyte-EC interactions, leading to reduced tissue cytotoxicity and end-organ damage in animal models of resuscitated hemorrhagic shock ; and recently the beneficial immunoregulatory properties of HS/D have also begun to translate into successful clinical trials of trauma patients [12, 14]. However, to our knowledge, this is the first study to investigate the effects of HSD on the activation of cellular and molecular inflammatory/coagulation cascades initiated by TBI.
Leukocyte and endothelial cell activation/adhesion molecules
Posttraumatic leukocyte adherence and activation are critical mediators of the pathogenesis of host tissue injury. In this study, we assessed the role of specific leukocyte and endothelial-derived activation/adhesion molecules in resuscitated TBI patients. Our results provide evidence that the expression of cell-associated and soluble adhesion molecules in severe TBI is differentially modulated depending on the type of fluid administered. After conventional crystalloid resuscitation, we found that relative to healthy controls, brain-injured patients exhibit extensive leukocyte mobilization, with significantly enhanced PMN and monocyte surface expression of L-selectin (CD62L) and β2-integrin (CD11b) adhesion molecules, along with exocytosis of the specific granular protein (CD66b). Along with increased expression of membrane-bound adhesion molecules, our analysis revealed that brain trauma caused elevated circulating levels of sE-selectin (sCD62E), sICAM-1 (sCD54) and sVCAM-1 (sCD106), but lower sL-selectin levels. A major finding of the present trial was that HSD markedly attenuated TBI-induced changes in these vital cell-associated and soluble molecules compared to NS.
The prominent neutrophilia and monocytosis observed in TBI patients is a well-known feature of acute inflammation that is consistently reported in patients sustaining polytrauma and closed head-injury . This reflects the sympathetic hormonal surge following severe tissue damage, which massively mobilizes leukocytes into the circulation from the marginal pool in the microvasculature. As observed in the current study , we previously reported that early resuscitation with HSD greatly reduces shock-associated leukocyte redistribution via inhibition of catecholamine release. The ability of HSD to attenuate the magnitude of post-injury leukocytosis is clinically relevant since elevated leukocyte counts on hospital admission reliably correlate with adverse neurological outcome and mortality risk after TBI .
Leukocyte infiltration of the CNS is a hallmark of neuroinflammation and a key mechanism contributing to the neuropathogenesis of secondary brain injury [25, 29]. Cerebral edema, raised intracranial pressure and neurotoxicity are mediated, at least in part, by intracerebral accumulation of blood-borne leukocytes [55, 56]. Trafficking of inflammatory cells from the bloodstream into the brain parenchyma depends upon the regulated expression of paired adhesion molecules on the surface of infiltrating leukocytes and cerebrovascular ECs [29, 30]. Results of this study show that leukocytes from TBI patients resuscitated with NS display rapid induction of constitutively expressed CD62L and activation of CD11b with translocation to the cell membrane from intracellular secretory granules . Consistent with these findings, studies in humans and animals demonstrate upregulated cell-surface CD62L and CD11b expression on leukocytes following polytrauma and isolated head-injury . CD11b expression is also closely associated with CD66b (co-localized in specific granules) and upon ligation forms a macromolecular complex that induces clustering of β2-integrins molecules, which potentiates avid binding to the endothelium with release of inflammatory mediators . Importantly, the current results, showing that HSD prevents TBI-induced upregulation of leukocyte adhesion molecules, confirm previous in vitro human cellular studies [59, 60], animal models [13, 61, 62] and clinical trials [12, 14] of shock/resuscitation, demonstrating that hypertonicity inhibits leukocyte adherence and activation via downregulation of selectins, integrins and immunoglobulin molecules, thus rendering cells incapable of rolling or adhering to the endothelium.
Experimental studies show widespread microvascular endothelial activation [20, 63] with upregulated adhesion molecule expression within 2-4 hours of neurological injury [24, 64]. E-selectin is not normally present on non-inflamed cerebrovascular ECs, but is dramatically induced at the transcriptional level by inflammatory stimuli and upregulated expression mediates initial adherence of PMNs and monocytes to brain microvasculature after traumatic/ischemic insults . Likewise, the constitutively low expression of ICAM-1 and VCAM-1 is rapidly upregulated on the cytokine-activated cerebrovascular ECs facilitating adhesion and transmigration of leukocytes into the CNS [35, 66, 67]. Immunohistochemical staining and microscopic analyses reveal early neutrophilic invasion of the brain parenchyma that peaks approximately 24 h after severe neurotrauma  and progresses to a predominance of monocyte/macrophage infiltrate by 72 h post-injury . The temporal profile of cell recruitment in these earlier reports is consistent with the peak kinetics of CD62L and CD11b molecule expression we observed on blood PMNs and monocytes. Inappropriate sequestration of adherent leukocytes in postcapillary venules may aggravate neurovascular damage by further impairing cerebral blood flow  and by intracerebral release neurotoxic mediators .
The damaging role of infiltrating leukocytes in cerebral injury is substantiated by studies in which PMN depletion or administration of anti-adhesion antibodies  reversed impaired tissue perfusion, BBB dysfunction and reduced infarct size . Furthermore, the extent of PMN accumulation is correlated with the degree of cerebral edema, injury severity, and poor outcome following TBI [21, 55]. Although direct examination of cerebral leukocyte invasion was not possible in the present study, our recent findings in the same cohort of TBI patients showing marked elevations of serum S100B and NSE as indicators of brain damage after resuscitation with NS , are strongly suggestive of neuroinflammation, BBB impairment and cellular infiltration that may be ameliorated by HSD [45, 46]. This assertion is also supported by intravital microscopy studies showing inhibition of leukocyte-EC interactions by hypertonic saline reduces microvascular permeability and tissue injury , and prevents BBB disruption allowing less intracerebral leukocyte sequestration in brain-injured animals compared to Ringers's [51, 71].
Many adhesion molecules exist as both transmembrane proteins and biologically active soluble forms arising from proteolytic cleavage of the extracellular region of the membrane-bound molecule . Increased levels of soluble adhesion molecules are reported in a variety of inflammatory conditions . Although their physiological role(s) and clinical relevance is not fully resolved, it is generally accepted that elevated concentrations of these endothelial derivatives are liberated by activation-induced shedding, or arise as a result of direct vascular damage . Our results showing enhanced serum concentrations of sE-selectin, sICAM-1 and sVCAM-1 are in accordance with the pattern of release of soluble selectins and immunoglobulin-type adhesion molecules characterized several experimental and clinical studies following brain injury [24, 74, 75]. High circulating concentrations of these molecules may serve as biomarkers for early diagnosis or prognosis of the development of the systemic inflammatory response syndrome, organ dysfunction and death after severe trauma or sepsis . Similarly, increased sICAM-1 levels are reported to correlate with the extent of brain damage and breakdown of the BBB leading to poor outcomes after TBI . Correspondingly, we found the highest levels of sE-selectin, sICAM and sVCAM in association with poor neurological outcome (i.e., GOS 1-3) in NS resuscitated patients. Notably, the only published study assessing the effects of hypertonic saline on the circulating profile of soluble adhesion molecules found lower sICAM-1 levels in the CSF of severe trauma patients compared to lactated Ringer's .
Soluble adhesion molecules are suggested to serve a functional role, either by inhibiting ongoing immunoinflammatory responses by competitive binding or conversely by inducing a response in ligand-bearing cells . For instance, elevated sE-selectin and sICAM-1 have been shown to activate leukocytes following neurological insult , whereas high sL-selectin levels are closely associated with decreased cellular interactions and reduced microvascular damage in critically injured patients . Our findings of initially low sL-selectin levels in TBI patients are consistent with earlier studies showing an inverse relationship between sL-selectin concentrations and increased risk of organ failure or death following isolated head injuries . Furthermore, the current results showing reduced surface expression of CD62L in HSD-treated patients, accompanied by corresponding increases in sL-selectin and better patient outcome, are in accordance with our previous findings in shock patients , and supports the theory that sL-selectin shedding represents an endogenous regulatory mechanism to limit leukocyte-mediated injury .
Cytokines are critical mediators of neuroinflammation after TBI [5, 23], regulating a wide-variety of cellular functions through autocrine and paracrine signaling networks that initiate and perpetuate inflammatory reactions . Severe TBI is associated with rapid and substantial increases in the synthesis and release of various pro/antiinflammatory cytokines into CSF and blood . Experimental models of closed-head injury  and clinical studies [81, 82] of TBI demonstrate early induction of TNF-α and IL-1β that peaks within 3-8 h of injury, followed by more sustained elevations of IL-6 and IL-10 [5, 83]. In this study, we focused on the prototypical pro- and antiinflammatory cytokines, TNF-α and IL-10, respectively, since large increases in both correlate with head injury severity and are indicative of poor clinical outcome . Like earlier experimental and clinical studies, our findings indicate peak serum concentrations of TNF-α and IL-10 are detectable within the first 3 h of injury, but remain above control values for up to 48 h. These cytokines are produced systemically and in high concentrations by resident microglia and infiltrating monocyte/macrophages in the acute phase of injury [22, 69]. TNF-α induces capillary leak and edema formation , causing enhanced BBB permeability  and upregulated expression of adhesion molecules such as ICAM-1 and VCAM-1 on the surface of brain ECs and glial cells [24, 85], exacerbating intracerebral leukocyte infiltration and microcirculatory dysfunction [23, 41]. Inflammatory cytokines also stimulate astrocyte reactivity, contributing to increased neuroinflammation and development of secondary injury following neurotrauma [22, 86]. Correspondingly, recent in vivo and in vitro animal studies have shown that both central and peripheral osmotic stimulation with hypertonic saline attenuates the brain's innate immune response to injury, reducing microglial activation, astrocytosis, cytokine production and associated neural tissue loss [15, 16].
A remarkable finding of the current study was that HSD resuscitation halved the multifold increases in both TNF-α and IL-10 observed upon admission, but levels remained elevated for at least 48-h in comparison to control values. Although, this is the first published report of the effects of HSD on cytokine production after TBI, our results are consistent with several earlier studies in humans and animals demonstrating a more balanced pro versus antiinflammatory cytokine profile following hypertonic resuscitation of hemorrhagic shock or sepsis . Previous studies examining TNF-α gene transcription and protein expression in response to hypertonicity have consistently shown inhibition in animal models of posttraumatic shock/resuscitation [87, 88], in vitro exposure of human peripheral blood cells to hypertonic media [89–91], and ex vivo intracellular production by blood monocytes of resuscitated trauma patients . Reports of endogenous IL-10 responses to hypertonicity are less consistent, exhibiting differential expression according to the dose administered, timing of treatment, type of injury, and tissue sample specificity. For example, we have demonstrated previously that in-hospital resuscitation with HSD enhances spontaneous and LPS-stimulated IL-10 expression in blood monocytes of shock patients , which corresponds with increased production by tissue macrophages in rodent models of shock/resuscitation [92–94]. By contrast, our present findings show that pre-hospital HSD treatment of TBI induced a roughly 50% reduction in circulating IL-10 levels lasting for at least 24 h, compared to NS. Similarly, clinically relevant doses of hypertonic saline in vitro were found to suppress IL-10 production in isolated LPS-stimulated human γδT cells  and to decrease circulating IL-10 concentrations in rats sustaining shock/resuscitation .
The ability of HSD to attenuate, but not abrogate, cytokine responses after TBI may be critical in light of their proposed duality in mediating both protective and injurious roles . It has been suggested that high levels of TNF-α exert deleterious effects on the progression of tissue damage during the acute stages of CNS injury, but play a reparative role at lower levels . Also, the relatively low IL-10 concentrations seen in HSD patients are consistent with the premise that moderate sustained levels of this potent antiinflammatory/immunosuppressive cytokine may be neuroprotective in early pathogenesis and subsequent termination of neuroinflammation, but detrimental at high levels [84, 95]. In fact, it has been reported that up to 50% of isolated head-injury patients who survive an initial neurological insult subsequently die as a result of infection or non-neurological organ dysfunction . Paralysis of cell-mediated immunity following severe TBI, partially induced by enhanced sympathetic-mediated IL-10 release, appears to be responsible for systemic immunosuppression with increased susceptibility to infectious complications . This is consistent with our data showing that NS treated patients with poor outcome according to GOS score also exhibit the highest levels of TNF-α and IL-10. These findings support the idea that HSD leads to a more homeostatic cytokine profile that may alter secondary injury processes without compromising neurologic recovery.
The posttraumatic inflammatory response impinges upon hemostatic regulatory mechanisms at multiple levels, including effects on procoagulant, anticoagulant and fibrinolytic systems . Patients sustaining severe injury are at risk for coagulopathy due to concurrent hypothermia, blood loss, acidosis, and hemodilution . Coagulation disorders are a frequent complication in patients with head injuries , which can be either hypercoagulable or hyperfibrinolytic and represent a powerful, independent predictor of prognosis . TBI-associated hypercoagulability is associated with injury-mediated disruption of the cerebrovasculature with exposure of abundant subendothelial TF to circulating factor VII initiating the extrinsic pathway . Intravenous fluids contribute to dilutional coagulopathy and also show intrinsic effects on the hemostatic system , but relatively few studies refer to the functional consequences after hypertonic resuscitation  and none evaluating the effects of HS/D on coagulation in TBI patients. Available in vitro and in vivo laboratory data suggest that hypertonic fluids exerts dose-dependent anticoagulant activity in animals or in serially diluted normal human plasma, as determined by routine clot-based assays and thromboelastography [101–104]. These studies provide evidence of direct anticoagulant effects of hypertonicity on plasma clotting factors and platelet activity and/or through interaction with inflammatory pathways [104, 105].
Patients in the current study did not exhibit coagulopathy, as defined by standard clinical tests (i.e., INR > 1.3 or aPTT > 34 s), but significant differences were evident in circulating concentrations of coagulation cofactors between treatment groups. Based on the measured plasma sTF, sTM and D-D levels, our results show that compared to control, NS-resuscitated TBI patients exhibit activation of coagulation with concurrent down-regulation of anticoagulant systems and fibrinolysis. Furthermore, a key finding of this study was that HSD reduced TBI-induced increases in sTF and D-D levels by half, while maintaining sTM levels near control values. This suggests that hypertonic resuscitation may act to improve coagulofibrinolytic balance after TBI. Previous studies have shown liberation of a soluble TF from both vascular and extravascular sources following traumatic injury . sTF is released into the circulation within hours of head injury , and inducible TF expression is rapidly upregulated on the surface of circulating monocytes after injury . Although the source of the early increase in sTF is not clear, it likely the result of cleavage from TF-bearing monocytes and/or release from dysfunctional brain ECs in response to inflammatory cytokines [18, 39]. Elevated systemic and intracerebral TNF-α release after TBI has been shown to have procoagulant effects, stimulating synthesis and release of sTF while suppressing cell-surface TM levels and the protein C pathway . Normally, TM acts to control hemostasis through high-affinity binding of thrombin, which activates anticoagulant and antiinflammatory protein C pathways [40, 97]. Elevated levels of sTM, as in the current study, are thought to reflect increased surface expression and shedding of transmembrane TM, which plays a role in regulating not only hemostasis, but also inflammation, thus providing a close link between these processes . Although the biological relevance of changes in sTM in brain injury is not well characterized , emerging data from clinical and animal studies suggest that increases in endogenous sTM or exogenous administration of TM may have potent antithrombotic/antiinflammatory properties in inflammatory disease .
In conclusion, this study of severe head injury patients demonstrates that prehospital treatment with HSD attenuates inflammatory and coagulation cascades by modulating leukocyte and endothelial cell adhesion/activation molecule expression, pro/antiinflammatory cytokine release and pro/anticoagulant responses. These findings provide direct evidence that initial resuscitation with HSD imparts functional changes to inflammatory cells after TBI, which may inhibit the capacity for infiltration of damaged CNS tissues, thereby reducing potential neuroinflammatory events associated with secondary brain injury. Our results also suggest that by downregulating inflammatory mediator production, HSD may help prevent procoagulant TF-dependent derangements of hemostasis and fibrinolysis, which may otherwise contribute to intravascular thrombosis.