Animal Use
Male rats in the weight range of 370–430 grams (Sprague-Dawley from Charles Rivers, www.criver.com) were used. The light/dark cycle was 12 hours of light and 12 hours of darkness. Food (Laboratory Rodent Diet 5001 (www.LabDiet.com)) and water were given ad libitum. Animal experiments, including all surgical procedures, were performed under anesthesia using 1.5–2.5% Isoflurane (Forane, Baxter, US) with 100% oxygen.
Rat model of polytrauma with hemorrhagic shock: This animal model was performed in Sprague-Dawley rats and previously demonstrated to recapitulate the pathologic changes of trauma and hemorrhagic shock in humans (57–59). Briefly, polytrauma was performed in anesthetized rats, including sequential injuries comprised of laparotomy, crush injury on the small intestine and liver, bone fracture at the right femur, and crush injury on skeletal muscle. A fixed-volume/pressure-controlled hemorrhage was started immediately after tissue injury by repeatedly withdrawing blood via the femoral vein to maintain mean arterial blood pressure at 40 mmHg until 40% of the estimated blood volume (EBV: 6% of body weight + 0.77 (60)) was removed. The 500 µl MK-8617 (MK, 1mg/ml in 5% DMSO, 10% PEG-400, and 85% normal saline) or vehicle (dissolvent only) was either given by gavage prior to trauma (n = 4 per group) or injected intravenously at 20 min after starting the trauma (n = 7 per group). Blood samples were taken at baseline, 60 min (only in intravenous administration groups), 120 min, and 240 min after trauma (prior to euthanasia), and the volume of the basal- and 120-min-blood samples was counted as a part of the total hemorrhage. Surviving animals were euthanized at 240 min post-trauma. The animals in sham control (n = 4–6) underwent the same procedure of cannulation and blood sampling except for hemorrhage and trauma.
Rat model of lethal decompensated hemorrhagic shock
The fixed-volume hemorrhage (65% of EBV) was performed in anesthetized rats. Briefly, the initial 40% of total calculated hemorrhage was removed within the first 2 min from the femoral artery, followed by repeatedly withdrawing 1.5 ml of the blood every 2 min until 10 min from the beginning hemorrhage and 1 ml of blood every 3 min thereafter through the femoral vein until the targeted volume of hemorrhage was achieved. Blood samples taken at baseline (1 ml) and 60 min (0.5 ml) were counted as a part of the total amount of hemorrhage. At 20 min immediately prior to completion of hemorrhage, the animals were treated with 500 µl MK-8617 at 1 mg/ml (0.5 mg, MK-L), 2 mg/ml (1 mg, MK-H), or vehicle (n = 16 each) intravenously. At one hour after hemorrhage, the surviving animals were resuscitated by a limited volume of whole blood (20% EBV, equivalent to 2 units of whole blood in humans) using collected shed blood from the first 2 min of hemorrhage and then euthanized at 4 hr (3 hr after resuscitation).
Measurements
The hemodynamics (mean arterial blood pressure (MAP) and heart rate (HR)) were recorded through femoral artery cannulation by PowerLab (ADInstruments, Colorado Spring, CO). The blood samples were used to measure blood biochemistry using CG4 and CHEM8 cartridges by iSTAT (Abbott, Washington, DC), complete blood count (WBC, RBC, platelet, and hematocrit) by ADVIA 120 Hematology System (SIEMENS Healthineers, Erlangen, Germany), and hemostasis (prothrombin time (PT), fibrinogen) by Start-4 (Diagnostic Stago Inc., Parsippany, NJ). The tissues were taken immediately after euthanasia to extract RNA to measure gene expression of HIF-1α, Glut1 (glucose transporter protein type 1), VEGFa (vascular endothelial growth factor A), Hexokinase, and EPO (erythropoietin) by PCR using a thermal cycler (Bio-Rad, Hercules, CA); nuclear proteins were also extracted to measure tissue levels of HIF-1α The lung tissue taken at 4 hr was weighed as wet weight, and its dry weight was measured after drying in the oven at 60°C for 10–14 days until there was no weight change for 3 consecutive days. The wet/dry weight ratio was then calculated.
Reverse transcription polymerase chain reaction (RT-PCR)
RNA was extracted from tissues as described previously (61). cDNA was synthesized with M-MLV reverse transcriptase and random primers (Promega, Madison, WI) from 1 µg of total RNA. Amplification reaction mixtures (16 µl) consisted of 1x iTaq Universal SYBR Green Supermix (Bio-Rad), 10 pmol each of forward and reverse primers, and 40 ng of cDNA in duplicate or triplicate technical replicates. Thermocycling parameters were 95°C for 2 min followed by 40 cycles of 95°C for 15 s and 60°C for 30 s in a CFX96 thermocycler (Bio-Rad). Raw fluorescence values were analyzed and amplicon efficiencies calculated using LinRegPCR (v2021.1) (62, 63) with the PavrgECt method for efficiency averaging (64). Target gene transcript abundance was normalized to the mean of two or three of the most stably expressed reference genes which were identified using the geNorm and Normfinder algorithms (65, 66). Rpl19, Polr2f, and B2m served as reference genes, for lung, muscle, and kidney tissues, while B2m and Polr2f served as reference genes for liver samples. Relative gene expression was calculated as described previously (67).
ELISA
Tissue samples were weighed to 10 mg, minced with a razor blade, homogenized with a Kimble Pellet Pestle Cordless Motor (Millville, NJ), and sonicated using an Omni Ruptor 250 Ultrasonic Homogenizer (Vernon Hills, IL). Nuclear protein was extracted from rat lung, kidney, and liver tissue samples using a Nuclear Extraction Kit (Abcam, Waltham, MA). For HIF-1α analysis, tissue samples were normalized by protein content such that when loading 100 µL of sample per well, 1 µg of equivalent nuclear protein was loaded. HIF-1α levels were measured using a sandwich ELISA kit (LSBio, Seattle, WA) following manufacturer protocols.
Study Approval
This animal study was conducted under the protocol approved by the Institutional Animal Care and Use Committee of the U.S. Army Institute of Surgical Research and in compliance with the Animal Welfare Act, the implemented Animal Welfare Regulations, and the principles of the “Guide for the Care and Use of Laboratory Animals”. The current study was reported following the guidelines of ARRIVE (Animals in Research Reporting In Vivo Experiments, PLoS Bio 8(6), e1000412,2010).
Data analysis
A sample size of 4 rats per group was used to perform a pilot proof-of-concept study to determine whether administration of MK-8617 prior to trauma mitigated lactate after polytrauma and hemorrhage. From that pilot study, the lactate at 4 hr was 6.60 ± 3.83 mmol/l and 2.37 ± 0.73 mmol/l in rats treated with vehicle and MK-8617, respectively. Using a two-sample t-test with a pooled standard deviation of 2.76, a sample size of 7 animals was determined for post-trauma intravenous administration of MK-8617 or vehicle in the polytrauma and hemorrhage model to meet our expectations of 2-sided test 95% confidence intervals and 80% power in reducing the lactate level at 4 hr by treatment with MK-8617 versus vehicle. For the lethal hemorrhagic shock model, the primary goal was to determine whether administration of PHDi extended survivability from a range of 0–20% to a range of 90–100% at 60 min after hemorrhage. A sample size of 13 rats per treatment group was initially estimated to provide 80% power to observe a statistical improvement in survivability from 20–80% between the vehicle and MK-8617 (low dose) groups. Based on the preliminary data from the lower dose of MK-8617 (0.5 mg) compared to the vehicle group (n = 13 per group) with survival proportions of 0.15 and 0.62 in 60-minute follow-up and 0.08 and 0.46 in 240-minute follow-up, the sample sizes calculated were 14 and 16 animals for 60- and 240-minute follow-up, respectively. Based on these parameters, a sample size of 16 animals per treatment group (vehicle, low-dose MK-8617, and high-dose MK-8617) was determined to power sufficiently for the expectations of 95% confidence intervals and 80% power. A randomization process was used to allocate animals to the treatment groups. The data analysis was performed by GraphPad Prism 9 (GraphPad Software, San Diego, CA). The two-way repeated ANOVA (parametric) was used to test mean differences among the groups or within each group for the continuous variables over multiple study time points, followed by a pairwise comparison with the Tukey method if applicable. The time to death was recorded, and survivability was analyzed by both chi-square or Fisher exact tests and Kaplan-Meier plots with log-rank tests. Data are presented as means ± standard deviation, and statistical significance is accepted at p < 0.05.