Lyophilized plasma attenuates vascular permeability, inflammation and lung injury in hemorrhagic shock

<div><p>In severe trauma and hemorrhage the early and empiric use of fresh frozen plasma (FFP) is associated with decreased morbidity and mortality. However, utilization of FFP comes with the significant burden of shipping and storage of frozen blood products. Dried or lyophilized plasma (LP) can be stored at room temperature, transported easily, reconstituted rapidly with ready availability in remote and austere environments. We have previously demonstrated that FFP mitigates the endothelial injury that ensues after hemorrhagic shock (HS). In the current study, we sought to determine whether LP has similar properties to FFP in its ability to modulate endothelial dysfunction in vitro and in vivo. Single donor LP was compared to single donor FFP using the following measures of endothelial cell (EC) function <i>in vitro</i>: permeability and transendothelial monolayer resistance; adherens junction preservation; and leukocyte-EC adhesion. <i>In vivo</i>, using a model of murine HS, LP and FFP were compared in measures of HS- induced pulmonary vascular inflammation and edema. Both <i>in vitro</i> and <i>in vivo</i> in all measures of EC function, LP demonstrated similar effects to FFP. Both FFP and LP similarly reduced EC permeability, increased transendothelial resistance, decreased leukocyte-EC binding and persevered adherens junctions. <i>In vivo</i>, LP and FFP both comparably reduced pulmonary injury, inflammation and vascular leak. Both FFP and LP have similar potent protective effects on the vascular endothelium in vitro and in lung function in vivo following hemorrhagic shock. These data support the further development of LP as an effective plasma product for human use after trauma and hemorrhagic shock.</p></div

Schematic of hemorrhagic shock experiment.

<p>Animals, excepting shams, under went hemorrhagic shock for ninety minutes before being either being resuscitated with LR, FFP or LP, or receiving no intervention.</p

LP and FFP increase TEER of EC monolayers.

<p>(A) Mean average ECIS generated traces of the TEER of HUVECs treated with 10% media (control), LR, FFP or LP. (B) Area under the curve analysis for thirty minutes after the addition of treatment. * = (p<0.05) compared to control via post hoc turkey tests of an unpaired one-way ANOVA.</p

FFP and LP prevent VEGF disruption of adherens junctions.

<p>Cells were pretreated with LR, FFP, LP or not treated for 1 hour before VEGF was added. Cells were fixed and stain for (A) Dapi (blue), β-Catenin (red) and VE-Cadherin (green). In (B) it is qualitatively observed that FFP and LP preserve the overlap of VE-Cadherin and β-Catenin (yellow) compare to untreated and LR controls.</p

LP and FFP attenuate EC permeability on Transwells.

<p>Transwell permeability of treated wells to FITC-dextrans (40kD) after treatment with 10%(A) and 30%(B) of the fluids tested respectively. FFP and LP are significantly less than control and LR at time points 30 minutes and beyond while FFP and LP are significantly different on at the 90 and 120 time points, p < 0.05 via post hoc turkey tests of an unpaired Two-way ANOVA. Controls and FFP were featured in Wataha et al. (2013). [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192363#pone.0192363.ref016" target="_blank">16</a>].</p

LP and FFP reduce that amount of leukocyte binding in vitro.

<p>Fluorescently labeled U397 cells were added to wells of HUVEC cells treated with 10(A) and 30(B) percent LR, FFP, LP or no treatment control. Unbound cells were removed after a period of one hour and the remaining cells quantified by fluorescence. All wells were normalized to control. * = (p<0.05) compared to control via post hoc turkey tests of an unpaired one-way ANOVA. N.S. clarifies that the indicated groups are Not Significantly different. Controls and FFP were featured in Wataha et al. (2013). [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192363#pone.0192363.ref016" target="_blank">16</a>].</p

Measures of permeability and inflammation in the lungs after hemorrhagic shock.

<p>(A) Resuscitation with FFP and LP reduce the number of cells positive for MPO, a marker of inflammation compared to LR and shock alone. (B) Treatment with FFP or LP reduces the amount of Evans blue permeability to a point statistically indistinguishable from sham treatment after 3 days. (C) Both plasma groups also attenuate edema compared to animals treated with LR but not compared to HS alone. Bars indicate significant differences (p < 0.05) via post hoc tukey tests based on a one-way ANOVA.</p

LP and FFP reduce the damage done to the lungs by a model hemorrhagic shock.

<p>Representational images of lungs stained with H&E from Sham (A), Shock (B), Shock+ LR (C), Shock + FFP (D) and Shock + LP (E). Histopathology scores average across all animals (F). Bars indicate significant differences (p < 0.05) via post hoc tukey tests based on a one-way ANOVA. N.S. clarifies that the indicated groups are Not Significantly different.</p

Post-injury administration of SB-216763 improves motor function.

<p><b>A</b>) Schematic of the drug injection and behavioral testing paradigm. Rats (n = 10/group) were injected (i.p.) for the first 5 days post-injury with either 5.0 mg/kg SB-216763 or vehicle. SB-216763-treated rats had <b>B</b>) comparable vestibulomotor deficits, but made significantly fewer <b>C</b>) ipsilateral and <b>D</b>) contralateral foot faults. <b>E</b>) Body weight was unaffected by SB-216763 administration. Data are presented as the mean ± SEM. ‡, P<0.05 by two-way repeated measures ANOVA.</p

Post-TBI lithium administration has no effect on motor function, but exacerbates post-injury weight loss.

<p><b>A</b>) Schematic of the injection and behavioral testing paradigm. Injured rats receiving 1 mEq/kg lithium (n = 10) performed similarly to vehicle-injected animals (n = 10) when tested for their <b>B</b>) balance beam performance, and <b>C</b>) ipsilateral and <b>D</b>) contralateral foot faults. <b>E</b>) Lithium exacerbated post-injury weight loss. Data are presented as the mean ± SEM. ≠, significant difference by repeated measures two-way ANOVA. *, P≤0.05.</p