Page 72 - JSOM Spring 2020

Although not yet standard of care, extracorporeal membrane FIGURE 1 Altitude exposure profile of each day’s experiment.
oxygenation (ECMO) is already used to support severely in-
jured ARDS patients during AE and for that reason was part Pre flight – Vitals, Labs
of the model used in this study. 14–16 In this pilot study, we in-
vestigate the systemic expression of HMGB1 in a model of
ARDS due to bilateral PC treated with fluids, pressors, and 5,000 – 30 minutes
venovenous ECLS. We hypothesized that HMGB1 levels in- Vitals, Labs @ 15 min, 30 min
crease after trauma and during AE.
8,000 – 30 minutes
Methods Vitals, Labs @ 15 min, 30 min
This study was approved by a local institutional animal care
and use committee (Protocol No. BPTS 15-02) and carried out 30,000 – 15 minutes
in compliance with the Animal Welfare Act, principles of the
“Guide for the Care and Use of Laboratory Animals,” and all
5,000 – 15 minutes
local, state, and federal guidelines for the ethical use of animals. Vitals, Labs @ 15 min
Experimental Procedures
Anesthetized, female Yorkshire pigs (N = 15, 53.8 ± 1.4kg) Post flight – Vitals, Labs
received arterial and venous catheters, tracheostomy, and
Foley catheter placement. After baseline (BL) measurements,
animals were cannulated and venovenous ECLS was initiated hemoglobin (pfHb) was measured via the direct spectrophoto-
(CardioHelp; Maquet Gmbh, Gettinge Group, Rastatt, Ger- metric method as previously described. Plasma total protein
19
many) via Avalon 23Fr dual-lumen catheter (Getinge Group, concentration (PTPC) was measured with use of the Pierce
Rastatt, Germany). Blood flow was 1.2–2.2L/min, and sweep BCA protein assay kit (Thermo Scientific, Rockford, IL).
gas flow ranged from 4 to 8L/min. Continuous heparinization
was started at cannulation and titrated to 30%–50% higher Histological Lung Injury Severity Assessment and
than baseline ACT levels, as standard anticoagulation during Immunohistochemistry
ECLS therapy. Animals were then transported via a standard For postmortem injury severity assessment, 1.5 × 1.5-cm lung
NATO litter fitted with a next-generation medical equipment samples were excised from both lungs. Samples were fixed in
rail kit (MERK; Smeed Technologies, Cummings, GA) to an 10% normal buffered formalin for 48 hours, processed, em-
adjacent building housing the hypobaric chamber. The altitude bedded in paraffin, and then cut into 4µm sections. Slides were
simulation profile is depicted in Figure 1. The altitudes cho- deparaffinized in histological grade xylene and dehydrated
sen for testing correspond to both long-distance transport in through graded alcohols to water, followed by staining with
pressurized aircraft (8,000 ft) and potential altitudes of trans- hematoxylin and eosin (H&E) and immunohistochemistry
portation by drone (5,000 ft). In addition, we carried out a (IHC). Histological images were obtained with ×100 and ×200
30,000-ft step to study a rapid decompression scenario (e.g., magnification using an Axioskop microscope (Zeiss, Oberko-
during an aircraft losing cabin pressure). Altitude exposure oc- chen, Germany). Diffuse alveolar damage (DAD) scores were
curred in the same animals in healthy state on day 1 (to study the sum of the individual scores for fibrosis (%), alveolar in-
effects of ECLS without injury present) and in the injured state terstitial fibrosis (IF), alveolar space (AS), alveolar protein ag-
on day 2 (assuming ECLS would be used to treat trauma vic- gregation (PA) and type II epithelial cell proliferation (EC),
tims. The uninjured day 1 data served as control data for the each on a scale of 0–4. 20–22
injured day 2 experiments. Injury consisted of bilateral pulmo-
nary contusions using a modified captive-bolt stunner (Model IHC was performed to analyze HMGB1 and Toll-like recep-
ML; Karl Schermer, Packers Engineering, Omaha, NE) with tor 4 (TLR4) expression in lung tissue. Following fixation in
immediate chest-tube placement as previously described. 7,17,18 10% normal buffered formalin (NBF), tissue was incubated
Heparin administration for systemic ECLS anticoagulation with primary antibodies for HMGB1 (1:150, ab18256; Ab-
was discontinued ~8 hours prior to injury; thus, from mid- cam Inc.) with standard IHC avidin-biotin-peroxidase com-
night of day 1 and until the end of procedures on day 2, the plex technique (Elite ABC kits, Catalog No. PK-6100; Vector
animals received heparin-free ECLS for trauma because hepa- Laboratories, Burlingame, CA) with 3-diaminobenzidine tet-
rin would be contraindicated during trauma/hemorrhage. rahydrochloride (DAB).

Laboratory Measurements Statistical Methods
Arterial blood samples were collected into EDTA blood-col- Statistics were performed using SAS version 9.4 (Cary, NC).
lection vacutainers (Becton, Dickinson and Company). On All tests were two-sided with an α = .05 for significance.
day 1, blood was collected at the following time points: BL, First a Shapiro-Wilk test was conducted to test the distribu-
post-ECLS (PE), sea level, 5,000 ft, 8,000 ft, 30,000 ft, post- tion of the data for normality. If skewed, the data were then
flight, and 12 hours post-PE. On day 2, blood was collected log transformed or the nonparametric version of the test was
at the following time points: preinjury, postinjury (PI), sea used. Data were analyzed using a one-way mixed model with
level, 5,000 ft, 8,000 ft, 30,000 ft, and postflight. Samples repeated measures and a Dunnett adjustment to test for signif-
were centrifuged at 3000 rpm for 10 min at 4°C; plasma was icant change from baseline. Group differences were examined
stored at –80°C until analysis. Enzyme-linked immunosorbent using a two-way mixed model with repeated measures and a
assay (ELISA) was used to measure HMGB1 (ST51011; IBL Tukey adjustment. All data are expressed as means ± standard
International) in the blood at each time point. Plasma-free error of the mean.

66 | JSOM Volume 20, Edition 1 / Spring 2020