Enoxaparin ameliorates post–traumatic brain injury edema and neurologic recovery, reducing cerebral leukocyte endothelial interactions and vessel permeability in vivo

Abstract

BACKGROUND Traumatic brain injury (TBI) confers a high risk of venous thrombosis, but early prevention with heparinoids is often withheld, fearing cerebral hematoma expansion. Yet, studies have shown heparinoids not only to be safe but also to limit brain edema and contusion size after TBI. Human TBI data also suggest faster radiologic and clinical neurologic recovery with earlier heparinoid administration. We hypothesized that enoxaparin (ENX) after TBI blunts in vivo leukocyte (LEU) mobilization to injured brain and cerebral edema, while improving neurologic recovery without increasing the size of the cerebral hemorrhagic contusion. METHODS CD1 male mice underwent either TBI by controlled cortical impact (CCI, 1-mm depth, 6 m/s) or sham craniotomy. ENX (1 mg/kg) or vehicle (VEH, 0.9% saline, 1 mL/kg) was administered at 2, 8, 14, 23, and 32 hours after TBI. At 48 hours, intravital microscopy was used to visualize live LEUs interacting with endothelium and microvascular leakage of fluorescein isothiocyanate–albumin. Neurologic function (Neurological Severity Score, NSS), activated clotting time, hemorrhagic contusion size, as well as brain and lung wet-to-dry ratios were evaluated post mortem. Analysis of variance with Bonferroni correction was used for statistical comparisons between groups. RESULTS Compared with VEH, ENX significantly reduced in vivo LEU rolling on endothelium (72.7 ± 28.3 LEU/100 μm/min vs. 30.6 ± 18.3 LEU/100 μm/min, p = 0.02) and cerebrovascular albumin leakage (34.5% ± 8.1% vs. 23.8% ± 5.5%, p = 0.047). CCI significantly increased ipsilateral cerebral hemisphere edema, but ENX treatment reduced post-CCI edema to near control levels (81.5% ± 1.5% vs. 77.6% ± 0.6%, p < 0.01). Compared with VEH, ENX reduced body weight loss at 24 hours (8.7% ± 1.2% vs. 5.8% ± 1.1%, p < 0.01) and improved NSS at 24 hours (14.5 ± 0.5 vs. 16.2 ± 0.4, p < 0.01) and 48 hours (15.1 ± 0.4 vs. 16.7 ± 0.5, p < 0.01) after injury. There were no significant differences in activated clotting time, hemorrhagic contusion size, and lung water content between the groups. CONCLUSION ENX reduces LEU recruitment to injured brain, diminishing visible microvascular permeability and edema. ENX may also accelerate neurologic recovery without increasing cerebral contusion size. Further study in humans is necessary to determine safety, appropriate dosage, and timing of ENX administration early after TBI.

Does enoxaparin interfere with HMGB1 signaling after TBI? A potential mechanism for reduced cerebral edema and neurologic recovery

“…While LEU rolling is an important step in LEU/EC interactions leading to transmigration into the tissue phase, in the current study, the number of sticking or adherent LEUs was extremely low as we could only safely record 30 seconds of fluorescent exposure at a time to prevent fluorescent light effects on the pial surface. 21,26,47 We can only postulate that differences could not be ascertained with such low frequency of adhesion. It is important to note that other plausible explanations may also exist linking heparinoids to reduced LEU rolling.…”

“…44 Our group investigated the role of cerebral LEU infiltration in this setting and showed how ENX administration after TBI diminished live LEUs rolling on pial endothelium, and that this was associated with concurrent reductions in microvascular leakage, also attenuating brain edema and accelerating neurologic recovery. 21 While the modulation of the HMGB1 signaling pathway by heparins has been demonstrated in different animal models replicating sepsis, 23,45 ischemia reperfusion, 24 and lung injury, 22,46 little is known of their interrelation following TBI. In the current study, we investigated the role of the HMGB1 signaling pathway in this setting and showed how HMGB1 blockade alone almost identically mirrored ENX effects, reducing penumbral LEU recruitment, cerebral edema (post mortem), and neurologic recovery.…”

“…5,12 Second, it would have been useful to include an uninjured, untreated negative (sham) control group to further characterize normalization of observed parameters. Yet, we included this group in a previous study, 21 and to keep the study simpler to interpret, we restricted it to four experimental groups and chose not to add this group in this report. Third, it would have been beneficial to also measure circulating and tissue HMGB1 levels before and after MAb blockade or ENX treatment but because of the already lengthy time frame of each animal experiment and insufficient tissue available for this analysis, we were unable to do so.…”

“…This was done to reflect previously published models of CNS rodent studies and was not associated with increased bleeding complications. 21,44,50 …”

“…In the CNS, heparins were shown to blunt cerebrovascular LEU and EC activation following injury and reduce cerebral edema while accelerating neurologic recovery. 18–21 The mechanism by which heparinoids exert these effects on inflammation remains unclear but has been investigated in different injury models. In a stretch-induced lung injury model, ENX administration was found to inhibit HMGB1 production, pulmonary PMN infiltration, oxygen free radical release, and microvascular permeability in injured lung tissue.…”