Introduction

Almost all trauma deaths happen in the first few hours after injury, often before the injured patient gets to a medical facility. Hemorrhagic shock is invariably the second-leading cause of early death among trauma patients, with most traumatic brain injuries more deadly. However, hemorrhage and hemorrhagic shock are more manageable for treatments to reduce morbidity and mortality secondary to hemorrhage  [1,2]. Ischemia/reperfusion injury following hemorrhagic shock resuscitation could be amplified by traumatic brain injury (TBI)-induced cut-off of cerebral autoregulation [3]. In the pre-hospital trauma situation, the golden-hour rule always evokes providers. Therefore, prompt transportation may be beneficial for patients with neurotrauma and penetrating injury with unstable hemodynamic features [4]. Most injuries in trauma patients require major surgery, as it is reiterated a decade and a half ago. Major surgery causes critical inflammatory, immune, neuroendocrine, and metabolic changes, broadly accounting for the “stress response” [5]. Complement activation is a component of the stress response to traumatic injury, crisscrossing and amplifying the coagulation activation pathways [6]. We showed that rhC1-inhibitor reduced organ injury and extended survival in porcine combat simulated injury  [7]. Human plasma-derived C1-inhibitor (pdC1-INH) indications are used to prevent and treat hereditary angioedema (HAE) [8,9]. We assumed that this off-label use would be effective in the early treatment of traumatic hemorrhage. This study aimed to 1) assess systemic complement activation in rats subjected to the blast wave and controlled bleeding, 2) assess pharmacokinetic parameters of pdC1-INH in rats, and 3) test the therapeutic effects of pdC1-INH on traumatic hemorrhage.

Materials and Methods

Animal Study

The study complied with the Animal Welfare Act, the corresponding Animal Welfare regulations, the principles of the Guide for the Care and Use of Laboratory Animals, and National Research Council. The facility’s Institutional Animal Care and Use Committee approved all research conducted in this study. The facility where this research was conducted is fully accredited by AAALAC International.

General Procedures, Surgical Preparations, and Experimental Design

As the timeline shown in Figure 1, male Sprague-Dawley rats (350-475g; 10-12 weeks of age; Charles River Laboratories, Wilmington, MA) were randomly assigned to two groups: rats subjected to the blast wave and hemorrhagic shock (Injury + placebo/saline, n = 10) and injured rats treated with pdC1-INH (Injury + pdC1-INH, n = 10). The rats underwent anesthesia (1-3 % isoflurane) with suspension and the cannulation of the left carotid artery and jugular vein by cut-down. After recovery over 5-7 days, the rats were anesthetized (60/5 mg/kg of ketamine/ xylazine, intraperitoneally) and exposed to the moderate blast wave (Applied Research Associates, Littleton, CO). A rat was placed in a prone position, allowing the head to face the blast front, on a holder of a flat plastic mesh suspended between two stainless steel rods running horizontally alongside the animal. A second plastic mesh was placed over the top of the rat and secured to the rods to prevent the rat from falling off the holder. The holder was attached to a swing mechanism to let the rat partially recoil against the blast. The animal was transferred to the surgical room, anesthesia continued maintained with isoflurane1-2.5%), and 15 minutes after the blast overpressure and immediately before controlled hemorrhage [52% of the Estimated Total Blood Volume (ETBV)] was started, an intravenous bolus of pdC1-INH (200IU/kg in 0.5ml saline, C1-esterase inhibitor (human), Berinert, CSL Behring GmbH, Marburg, Germany) was injected. The hemorrhage was completed in 15 minutes, followed by a 30-minute shock phase, which ended with the infusion of Plasma-Lyte A (PLA) at two times of Shed Blood Volume (SBV) via the jugular vein line at a rate of 0.5 ml/min to create hypotensive resuscitation. Three hours after the shock, animals were allowed to recover from anesthesia, return to their cages, and be observed for 25 hours. The arterial blood pressures of animals were consistently monitored and recorded with the BIOPAC MP160 Data Acquisition and Analysis Systems via the carotid arterial catheter. 

Biological Samples

At the end of the total procedure and observation time of 25 hours, the animals were euthanized with Fatal Plus at 150 mg/kg. Necropsies and tissue harvests were carried out. Tissue samples were fixed with 10% formalin for subsequent histological analyses. Blood samplings were performed at 7 time points as shown in Figure 1. Serums and plasmas were prepared by centrifuging at 4000 rpm for 10 minutes. Aliquots of serum and plasma samples were stored at – 80°C for subsequent analyses. 

Assays

Pharmacokinetic Measurement: The plasma level of functional C1-INH was determined using the MicroVue^TM C1-Inhibitor Plus EIA kit (Quidel, San Diego, CA) following the manufacturer’s instructions. The maximum concentration (C_max) above the baseline was derived directly from the concentration-time profiles. The pharmacokinetic parameters of functional C1-INH were determined using a 1-compartment pharmacokinetic model. Calculations were performed using linear model (first-order kinetics) estimation.

Blood chemistry: Blood lactate, base excess/deficit, and potassium were analyzed by i-STAT using CG4+ and CG8+ cartridges (Abbott Laboratories, Green Oaks, IL),

Hemolytic Complement Activity Assay: Serum complement hemolytic 50% activity (CH50) assay was performed to determine the function of the complement classical pathway, as previously described  [10]. Briefly, antibody-sensitized chicken red blood cells (Colorado Serum Company, Denver, CO) were incubated for 30 min at 37 ^◦C with serial dilutions of rat serum samples in gelatin-veronal buffer (Complement Technology, Tyler, TX). After centrifugation, the supernatant was transferred to a new plate, and the absorbance of the supernatant was determined at 405 nm by SpectraMax microplate reader (Molecular Devices San Jose, CA, USA). The fold serum dilution inducing 50% of complement hemolytic activity was determined and presented as the CH50 value.

Measurement of complement protein, plasminogen, MCP-1, HMGB1 and MPO Levels: Plasma levels of C1q (HycultBiotech, Plymouth Meeting, PA), C3 (Abcam, Cambridge, MA), plasminogen C3 (Abcam, Cambridge, MA), MCP-1 C3 (Abcam, Cambridge, MA), HMGB1 (IBL International Morrisville, NC), and myeloperoxidase (MPO) (HycultBiotech, Plymouth Meeting, PA) were measured using ELISA kits according to the manufacturer’s instructions.

Histological Examination and Tissue Injury Scoring: Ten percent of formalin-fixed tissues were embedded in paraffin. Coronal sections were then stained with hematoxylin-eosin (H&E). Five random histologic images were recorded at ×2, ×100 and ×400 magnifications under a slide scanner (Axio Scan.Z1, Carl Zeiss, Thornwood, NY), evaluated, and scored by a pathologist blinded to the treatment group using the tissue scoring protocols as previously described [10,11].

Statistical Analysis

All data were presented as means ± SEM. The responses in inflammation and organ injury parameters were compared between groups using Mann-Whitney U test or unpaired t-test with Welch’s correction. Two-way ANOVA was performed to compare the animal groups on particular variables. Bonferroni post hoc tests were conducted only if the F value in ANOVA achieved p < 0.05. Survival was analyzed by the Kaplan-Meier method. Statistical significance was accepted at p < 0.05.

Results

Blast Wave Parameters, Mean Arterial Pressure, and Blood Chemistry after Combined Injury

As shown in Table 1, the shock tube produced typical open-field blast parameters. There was no significant difference in blast wave parameters between injured/untreated and injured/treated groups. The mean arterial pressure (MAP) was significantly decreased in 30 minutes and the first 11 hours after the blast and hemorrhage

(Figure 2). Injured/treated animals tended to increase MAP in the first 40 min of the resuscitation phase compared with injured/untreated rats but did not reach a significant difference. Base excess/base deficit significantly decreased (Figure 3A), unlike plasma lactate (Figure 3B) and potassium (Figure 3C) concentrations, which significantly increased immediately after hemorrhagic shock. However, there was no significant difference in these parameters between the injured and treated rats.

Table 1: Blast wave parameters.

Figure 2: Effects of pdC1-INH on mean arterial pressure (MAP) in a rat blast injury and hemorrhage model. Changes in MAP were monitored via the carotid artery using the BIOPAC system. During the shock and fluid resuscitation period, the MAP was recorded every 5 min. Data are expressed as mean ± SEM. n = 10.

Figure 3: Effects of pdC1-INH on blood chemistry changes in a rat blast injury and hemorrhage model. Rat blood levels of BE/BD (A), lactate (B), and potassium (C) were measured by i-Stat. Data are expressed as mean ± SEM. n = 10. BE, base access; BD, base deficit.

Effect of pdC1-INH on Systemic Inflammatory Response

The HMGB1 plasma concentrations significantly increased 3 hours after hemorrhagic shock, but no significant difference existed between the treated and non-treated rats (Figure 4A). Plasma myeloperoxidase (MPO; Figure 4B) and monocyte chemoattractant protein [MCP-1, also referred to as the chemokine ligand 2 (CCL2); Figure 4C] concentrations were not affected by the combined injury and no significant difference in these parameters between treated and non-treated rats.

Figure 4: Effects of pdC1-INH on systemic inflammatory response in a rat blast injury and hemorrhage model. Rat blood levels of HMGB1 (A), MPO (B), and MCP-1 (C) were measured by ELISA. Data are expressed as mean ± SEM. n = 10. HMGB1, high mobility group box 1 protein; MCP-1, monocyte chemoattractant protein-1; MPO, myeloperoxidase.

Effect of pdC1-INH on Survival

As shown in Figure 5, the injured/untreated rats had a 70% mortality rate, and the treatment with an IV bolus of pdC1-INH 15 minutes after the blast but immediately before hemorrhage did not improve survival compared to the vehicle control.

Figure 5: Effect of pdC1-INH on injury-induced mortality. All rats were subjected to blast and severe hemorrhage, treated with vehicle control (saline, Injury) or pdC1-INH, and monitored for survival up to 25 h after blast injury. The survival distribution of these two groups was determined using the log-rank Mantel-Cox test.

Effect of pdC1-INH on Organ Histopathological Changes

Exposure to the blast wave and hemorrhagic shock elicited pathological alterations characterized by neuroinflammation, neuronal apoptosis, neuronal death, and cerebral cytotoxic and vasogenic edema in the cortex (Figure 6A & B) and hippocampus (Figure 6C & D). The injured rats treated with pdC1-INH significantly improved the cerebral pathological changes (Figure 6A-D). Figure 6E & F illustrate hepatic ischemic infarct injury around the portal vein featured by foci of moderate coagulative necrosis with pyknosis, karyolysis, nuclear absence, significant neutrophil infiltration, and the sharp border between normal and ischemic tissue in injured/ untreated animals, while pdC1-INH significantly attenuated the liver injury. Traumatic hemorrhage-induced severe pulmonary damage manifested as a septal thickening, inflammatory cell infiltration, alveolar hemorrhage, and edema (Figure 6G & H). The lung injury score showed no significant improvement in the tissue alterations in the injured animals treated with pdC1-INH (Figure 6H).