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Basic Science Aspects

Nonhuman Primate (Rhesus Macaque) Models of Severe Pressure-Targeted Hemorrhagic and Polytraumatic Hemorrhagic Shock

Sheppard, Forest R.; Macko, Antoni R.; Glaser, Jacob J.; Vernon, Philip J.; Burdette, Alexander J.; Paredes, Ruth Madelaine; Koeller, Craig A.; Pusateri, Anthony E.; Tadaki, Douglas K.; Cardin, Sylvain

Author Information
doi: 10.1097/SHK.0000000000000910

Abstract

INTRODUCTION

Despite advancements in trauma care, injury patterns that include hemorrhagic shock with soft tissue and/or musculoskeletal injuries remain a leading cause of potentially survivable mortality in both military and civilian populations (1–7). The increasing use of explosive devices in combat has largely contributed to severe polytraumatic injuries as explosions accounted for 72% of those who died after combat injury during Operation Iraqi Freedom and Operation Enduring Freedom (8). Mortality is determined by injury severity, blood loss, and resulting pathophysiologic derangements such as metabolic acidosis, coagulopathy, and maladaptive immune responses. Novel life-saving therapeutic interventions are greatly needed. However, successful clinical translation will require evaluation in highly relevant animal models.

There is an ever-increasing interest in the development of human blood-derived therapeutics, such as lyophilized and spray-dried plasma, and platelet-derived hemostatic agents (PDHAs) that hold promise as resuscitative agents and adjuncts. It is prudent to preclinically evaluate the function, efficacy, and safety of these “new” agents in relevant animal models that recapitulate human responses to polytraumatic injury and hemorrhage (9, 10) and circumvent xenogeneic incompatibility issues (11–17). Specifically, Old World nonhuman primates (NHPs) are superior for such studies as compared with lower-order mammals as they exhibit responses to trauma that are overall most comparable to humans given their similarity to humans in terms of neuroendocrine, physiologic, coagulation, and inflammatory systems (9, 10); are available with multi-institutional familiarity and commercially available diagnostic reagents and assay kits; and, importantly, have compatibility with human-derived blood products and documented use of human-derived blood products with limited xenoincompatibility (11, 13, 15–17).

Experimental models of NHP traumatic hemorrhage exist; however, few incorporate a polytraumatic injury pattern. Those who do, have largely focused on the acute physiologic responses and have not fully characterized postinjury relevant physiologic, metabolic, immunologic, and coagulopathic responses to trauma. Among these, the majority of NHP models have been in baboons that are less accessible and more cost prohibitive for many institutions (18–21). Those who have used Old World NHPs are also limited and have relied on uncontrolled liver hemorrhage with blood loss, depth of shock, and inflammatory responses of minimal clinical translatability (14, 22, 23). Hence, there is a need for an NHP model that recapitulates clinically relevant polytraumatic injury patterns that will allow more clinically translatable assessments of physiologic, metabolic, immunologic, and coagulopathic responses to the injuries–shock and responses to treatment.

The development of clinically relevant, translatable, animal models of hemorrhage and polytraumatic injury has been challenging for multiple reasons, not the least of which are species-specific physiologic differences. The difficulties with relevant translation and preclinical testing are further exacerbated in animal models that carry the high likelihood, or certainhood, of xenogeneic reactions to potential therapeutics being tested. To address these challenges, we developed and evaluated five NHP models of polytraumatic hemorrhage as part of a model development program that utilized an adaptive study design approach, based on the hypothesis that additive tissue trauma and increasing shock duration would result in a model that has physiologic, immunologic, and coagulopathic responses that are clinically relevant and translatable. Here, we describe physiologic, metabolic, immunologic, and coagulopathic responses to severe pressure-targeted hemorrhagic shock (PTHS) of varying duration (30–305 min) in isolation and in combination with musculoskeletal and/or soft tissue injuries in Rhesus macaques (Macaca mulatta). The overall objective of this work was to develop an NHP model more readily translatable to human trauma patients for future therapeutic testing and evaluation.

MATERIALS AND METHODS

Ethical approval and accreditation

The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the 711th Human Performance Wing, Joint Base San Antonio-Fort Sam Houston, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 2011. All procedures were performed in facilities accredited by the Association for Assessment and Accreditation for Laboratory Animal Care International (AAALAC).

Preoperative preparation and vital signs monitoring

A total of 40 male Rhesus Macaques weighing 7–14 kg were utilized in the study. The animals were housed in compliance with the Secretary of the Navy Instruction (SECNAVINST) 3900.38C regulations with ad libitum access to feed and water. Feed was withheld 12 h before surgery.

Animals were premedicated with an analgesic (Buprenex 0.03 mg/kg; Reckitt & Colman Pharmaceuticals Inc, Richmond, Va), sedated with Telazol (3.0 mg/kg; Zoetis Inc, Kalamazoo, Mich), and weighed (Detecto Scale, Webb City, Mo). Airway intubation and control were achieved with 4–5.5 mm endotracheal (ET) tubes (Rusch – Teleflex, Research Triangle Park, NC). End-tidal carbon dioxide (EtCO2) confirmed proper placement of ET tube and chest auscultation confirmed ventilation of both lungs. Animals were placed on a Dräger Apollo Anesthesia Workstation (Draeger Medical Inc, Telford, Pa) with volume-controlled respiration (10 mL/kg) at 12–15 breaths per minute, FiO2 of 21% to25% and isoflurane (1.0%–2.0%) inhalational anesthesia.

The right femoral vein and left femoral artery were cannulated with 14-gauge Abbocath-T IV Catheters (Hospira, Lake Forest, Ill) for intravenous resuscitation fluid infusion and controlled hemorrhage, respectively. The right femoral artery was cannulated with 18-gauge polyvinyl pressure tubing for continuous blood pressure monitoring. Noninvasive and invasive vitals sign monitoring utilized a Datex Ohmeda Cardiocap/5 patient monitor (General Electric Co, Fairfield, Conn). A three-lead electrocardiograph (ECG) was used to monitor heart rate (HR) and arrhythmia development throughout the study. Shock index (SI) was calculated by dividing HR by systolic blood pressure (SBP) (24).

Nontraditional vital signs collected throughout the study include EtCO2 and skeletal muscle tissue oxygenation (StO2). EtCO2 was measured via the Dräger Apollo Anesthesia Workstation (Draeger Medical Inc) and StO2 was measured utilizing an InSpectra Tissue Oxygen Monitor (model 650; Hutchinson Technology Inc, Hutchison, Minn) with the probe placed on the triceps brachii.

Core body temperature was monitored continuously via a 10-Fr rectal temperature probe connected to a Datex Ohmeda Cardiocap/5 (GE Healthcare Systems, Wauwatosa, Wis) patient monitor and maintained between 36.0 and 38.0°C before injury with blankets, heat packs, and Bair Hugger Model 750 (Arizant Inc, Eden Prairie, Minn) as required. Following injury/hemorrhage, heat support was only applied if the core body temperature decreased below 35.0°C and was discontinued when the core body temperature reached 37.0°C.

Experimental shock and trauma protocol

The experimental outline is in Figure 1. After instrumentation, animals were allowed to stabilize for 10 min before proceeding with hemorrhage (PTHS) ± additional injuries. Initial experimental modeling entailed the determination of the effect of duration of PTHS (i.e., 30 vs. 60 min). Subsequent experiments were undertaken to characterize the effects of PTHS combined with musculoskeletal and/or soft tissue injuries. Interim analysis revealed that a more severe injury was required to elicit physiologic, metabolic, coagulopathic, and immunologic outcomes consistent with clinical descriptions of severe trauma. Therefore, the adaptive design culminated in a combination of musculoskeletal and soft tissue injuries with a shock duration that was not time dependent but dependent upon detected loss of blood pressure maintenance (i.e., compensation).

F1
Fig. 1:
Experimental protocol.

PTHS for 30 and 60 min

Anesthetized NHPs were randomized to one of two duration-controlled PTHS groups (n = 8 each): 30-min PTHS (PTHS-30) or 60 min PTHS (PTHS-60). Hemorrhage was initiated by opening a stop-cock in-line with the femoral arterial catheter which remained open until mean arterial pressure (MAP) reached 20 mmHg, at which time the shock period began (T = 0 min). MAP was maintained between 20 and 24 mmHg throughout the PTHS period by additional controlled hemorrhage when the MAP exceeded 24 mmHg. Neither blood nor fluids were administered during this period. Hemorrhaged blood was collected in an anticoagulant citrated dextrose solution A (ACD-A) primed bag on a rocking scale. The whole blood to ACD-A ratio was kept at 10:1 by volume, by supplementing additional ACD-A as needed throughout the shock period. Animals remained in shock for a duration according to their assigned group, after which they were resuscitated as described below.

PTHS for 60 min with soft tissue and musculoskeletal injuries

Based on the findings of initial model development incorporating 30- and 60-mine shock periods, PTHS-60 was selected for further model development incorporating polytraumatic injury. Anesthetized Rhesus Macaques were randomized to one of two polytrauma groups (n = 8 each): PTHS-60 with soft tissue injury (PTHS-60+ST) or PTHS-60 with ST and femur fracture (PTHS-60+ST+FF). The ST and FF injuries were performed as described below and completed within a 10-min injury window immediately preceding induction of PTHS.

The ST injury consisted of a 15-cm laparotomy incision spanning from the xiphoid process toward the umbilicus along the linea alba. The incision was left open, but covered with a laparotomy pad until closed during the resuscitation period. The musculoskeletal injury consisted of a left femur fracture. The fracture was created on the left leg with a longitudinal 5 cm skin incision spanning the anterior mid- to distal-femur followed by blunt dissection to separate muscle and muscle insertions from a 4-cm span over the diaphysis. With a malleable retractor placed between the femur and underlying muscle, an oscillating power bone saw was used to rapidly transect the femur, the wound was packed with preweighed gauze and temporarily closed with staples.

At the completion of the injury window, PTHS was initiated and maintained as described previously. At the end of the shock period (EOS), fluid resuscitation commenced and injuries were repaired as described below.

PTHS to decompensation with soft tissue and musculoskeletal injuries

Ultimately, as model development progressed, the fifth model developed utilized a nontime-controlled duration of PTHS (n = 8). In this fifth model, a MAP of 20–24 mmHg was maintained until the animals no longer exhibited cardiovascular compensatory responsiveness to maintain or elevate MAP (i.e., decompensation; PTHS-D). The threshold used to define decompensation was a spontaneous decline in an animal's MAP to a value 25% lower than the average MAP during that animal's first 60 min of PTHS (e.g., if the MAP average was 23 mmHg for the first 60 min, then after the 60 min time point, when the animal achieved a spontaneous MAP of 18 mmHg it was deemed “decompensated,” the shock period ended, and fluid resuscitation initiated).

Resuscitation and injury repair

At EOS, the volume of hemorrhaged blood was determined by: [weight of blood in the collection bag (g) + weight of blood from femur fracture site] × density of Rhesus macaque whole blood (1.05 g/mL) (25). Percent blood loss was calculated by dividing the volume of hemorrhage blood by the estimated total blood volume (eTBV = animal body weight (kg) × 54 mL/kg) (26). Resuscitation commenced at the end of the designated shock period, with a standardized 2-h crystalloid/whole blood protocol consisting of 1/2X shed blood volume (SBV) as whole blood and 3X SBV as normal saline (NS). Immediately before transfusion of autologous ACD-A-treated whole blood, 150 mg of calcium gluconate in a 1.5-mL solution (Vedco Inc, Saint Joseph, Miss) was infused intravenously to counteract the effects of the citrated anticoagulant (Fig. 1).

In the PTHS-D model it was realized very early that the animals were near extremis at the time of EOS. To facilitate a survivable model, the inclusion of up to 5 min of closed chest compressions and up to two intravenous doses of 2 mg epinephrine each were added to the protocol during the resuscitation phase.

The midline laparotomy was closed in two layers (fascia and skin) with continuous absorbable sutures. Before femur repair, the gauze was removed and weighed to quantify the volume of blood lost at the femur fracture site [Gauze weight (g) difference × density of Rhesus macaque whole blood (1.05 g/mL)] (25). The femur was repaired by internal fixation utilizing steel plates and screws (Keebomed Inc, Mount Prospect, Ill), and the incision was closed in two layers (fascia and skin) with continuous absorbable sutures.

Maintenance fluid and postoperative care

At the end of resuscitation (EOR), the animals remained under anesthesia for observation until T = 360 min (360 min after start of shock [T = 0]). During this time, maintenance fluids were administered in the following manner: 0.9% NS infused at 4 mL/kg/h for the first 10 kg of body weight and at 2 mL/kg/h for the second 10 kg of body weight (e.g., 11 kg animal would receive 42 mL/h of NS). At T = 330 min, Buprenorphine SR (0.2 mg/kg; ZOOPHARM, Windsor, Colo) was given subcutaneously for postoperative analgesia. Following T = 360 min, anesthesia was discontinued and the animals were returned to their cages for postoperative observations until 24 h postshock. During postoperative recovery, animals were continuously observed until they were conscious, alert, and able to move about the cage. If an animal was not able to breathe spontaneously for more than 2 h following the termination of anesthesia, unable to be extubated 2 h after spontaneous breathing returned or unable to sit upright 2 h after extubation, the animal was euthanized. In addition, after recovery, animals were routinely observed until the 24 h mark. If signs of distress (e.g., increasing respiratory rate and dyspnea that could not be alleviated without reanesthetizing the animal, decreased neurological status, star gazing, unresponsive to noise, touch or light or uncontrollable pain) were observed during postoperative recovery, animals were humanely euthanized; otherwise, at 24 h postshock, animals were sedated, and humanely euthanized for subsequent necropsy evaluation and tissue collection for histopathologic analysis.

Measurements and laboratory assays

Arterial blood samples (10 mL each) were collected at scheduled intervals throughout the study: baseline (BSLN), EOS, EOR, T = 360 and at T = 1,440 min (24 h) or time of death or euthanasia (if occurring before T = 1,440). At each time point, physiological, metabolic, coagulopathic, and immunological parameters were analyzed. Arterial blood gas parameters were assessed utilizing a GEM Premier 4000 (Instrumentation Laboratory, Bedford, Mass). Complete blood cell counts and serum chemistries were evaluated on a HemaTrue analyzer (HESKA, Loveland, Colo). Systemic inflammatory cytokines and chemokines in serum were analyzed including interleukins (IL) IL-6, IL-8, IL-10, IL-15, and monocyte chemotactic factor-1 (MCP-1), which were quantified utilizing the Milliplex MAP Non-Human Primate Cytokine Panel (Millipore, Billerica, Mass) performed on a Luminex IS100 (Luminex Corporation, Austin, Tex). Additional plasma and serum samples for possible future analyses were collected and stored for later, potential, additional analyses.

Whole blood viscoelastic clotting properties were evaluated by rotational thromboelastrometry (ROTEM Delta system; TEM Systems Inc, Durham, NC). ROTEM analyses included evaluation of extrinsic coagulation pathway function in the absence and presence of the platelet inhibitor, cytochalasin D (ExTEM and FibTEM, respectively). Clotting time (CT), alpha-angle (α), and maximum clot firmness (MCF) were evaluated for both of the ROTEM tests. Platelet aggregation was performed on whole blood by impedance aggregometry (Multiplate; Verum Diagnostica GmbH, Munich, Germany), using the agonists: adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA). Agonist responses were reported as area under the aggregation curve in units (U) over a 6-min measurement period and as non-normalized and normalized to platelet count.

Pathology

Immediately following time of death, necropsy was performed to include gross evaluation of brain, heart, lungs, liver, kidney, spleen, intestines, and skeletal muscle, as well as major blood vessels and vascular organs. Following necropsy, representative sections of brain, heart, lungs, liver, kidney and spleen were collected for subsequent histopathologic evaluation. Tissue sections were fixed and stained with hematoxylin and eosin, and then examined by a veterinarian pathologist who was blinded to the injury groups, utilizing an Olympus BX51 upright brightfield microscope (Olympus Scientific Solutions, Waltham, Mass). Only the liver and kidney tissues showed morphological changes. Liver and kidney histological scoring was performed for the presence or absence of centrilobular and tubular necrosis.

Statistical analysis

Animals were randomly assigned to injury groups. All data are presented as mean ± SEM. Multiple time point analysis was analyzed by two-way ANOVA with repeated measures using post hoc Bonferroni correction. Aggregometry data were analyzed using the Kruskal–Wallis nonparametric test for group comparisons, while the Friedmans's one-way ANOVA was used for baseline comparison. Blood loss was analyzed by one-way ANOVA with Bonferroni correction. P < 0.05 was considered to be statistically significant.

RESULTS

Blood loss and survival

The average time from the start-of-hemorrhage to start-of-shock (T = 0 min) was 2.1 ± 0.2 min with an average initial blood loss [i.e., blood loss from start of hemorrhage to MAP = 20 mmHg (T = 0 min)] of 27 ± 3% for all groups. Total blood loss at EOS was 40 ± 2% in PTHS-30, 59 ± 3% in PTHS-60, 51 ± 3% in PTHS-60+ST, 49 ± 2% in PTHS-60+ST+FF, and 54 ± 6% in PTHS-D, with the PTHS-30 group having significantly lower blood loss than all other models (P < 0.05).

Animals in the PTHS-30, PTHS-60, PTHS-60+ST, and PTHS-60+ST+FF groups survived to the end of the observation period (T = 1,440 min) except one animal each in the PTHS-60 and PTHS-60+ST+FF groups, which were euthanized at T = 1,333 and 775 min according to protocol criteria, respectively. In the PTHS-D group, all animals survived to T = 360 min; however, seven of the eight animals were euthanized before the 24 h time point based upon euthanasia criteria. Six of the eight animals did not meet the recovery criteria of consciousness, alertness, and mobility. The seventh animal recovered and, before the 24 h time point, was euthanized according to protocol criteria.

Based on early and disparate times of death beyond T = 360 and before T = 24 h, direct comparison across all models at matched time points was examined from T = 0 to T = 360 for physiology, arterial blood gases, complete cell counts, serum chemistries, cytokines, chemokines, and coagulation parameters.

Physiologic characterization

Systemic hemodynamics were equivalent among groups at baseline (Fig. 2). Regardless of group, MAP and SBP decreased uniformly in response to hemorrhage. At EOS, all groups except PTHS-30 demonstrated an equivalent tachycardia response which remained elevated in the PTHS-60+ST+FF and PTHS-D groups out to T = 360. Compared to their respective baselines, MAP, SBP, and HR were normalized in all groups at EOR except the PTHS-D group, in which MAP never fully returned to baseline levels (P < 0.05).

F2
Fig. 2:
Physiological parameters across groups.

Tissue perfusion indices declined after hemorrhage and were equivalent among groups at all time points, except for the PTHS-30 group (Fig. 2). Across all groups, baseline EtCO2 and StO2 were 39.4 ± 0.4 mmHg and 91 ± 1%, respectively. At EOS, all groups demonstrated equivalent and significant reductions in EtCO2 by 11 ± 1 mmHg. StO2 levels were significantly reduced by 35 ± 4% on average for all groups except the PTHS-30 group, which decreased by 14 ± 1.84%. Resuscitation normalized both EtCO2 and StO2 in all groups.

Arterial blood gases

Base excess was significantly reduced compared to baseline at EOS, EOR, and T = 360 for all groups (Fig. 3). In the PTHS-D group, base excess was significantly lower than all other groups at EOS and EOR. By T = 360, base excess in the PTHS-D group was significantly lower than the PTHS-30, PTHS-60+ST, and PTHS-60+ST+FF groups, which did not differ. All groups demonstrated an increase in lactate levels by EOS, with the PTHS-D group demonstrating significantly higher levels of lactate than all other groups (P < 0.05). At EOR, the PTHS-30, PTHS-60+ST, and PTHS-60+ST+FF groups had lactate levels that were not different from baseline values and that were significantly lower than the PTHS-D group; furthermore, the PTHS-D group continued to have lactate levels significantly higher than baseline (P < 0.05) (Fig. 3).

F3
Fig. 3:
Arterial blood gas analysis.

Bicarbonate (HCO3) levels were significantly reduced in all groups at EOS, EOR, and T = 360, as compared to baseline. Bicarbonate levels in the PTHS-D group were significantly lower than all other groups at EOS and EOR. By T = 360, the bicarbonate levels in the PTHS-D group were only significantly lower than the PTHS-30, PTHS-60+ST, and PTHS-60+ST+FF groups (Fig. 3).

The pH demonstrated significant decreases from BSLN at EOR and T = 360 for the PTHS-D, PTHS-60+ST+FF, and PTHS-60 groups. Consistent with previous reports, the pH at BSLN in rhesus macaques trended higher than would be consistent in human patients, alongh with a smaller pH change in magnitude in comparison to base deficit, lactate, and bicarbonate levels (14).

Complete blood cell counts

Compared to baseline, all groups demonstrated a significant reduction in hematocrit and hemoglobin beginning at EOS and persisting through T = 360 (Fig. 4). The PTHS-D group had significantly lower levels of hematocrit than the PTHS-30 group at EOR and T = 360 and significantly lower levels of hemoglobin than the PTHS-30 group at EOS, EOR, and T = 360. Both the PTHS-60 and PTHS-60+ST+FF groups had significantly lower levels of hematocrit than the PTHS-30 group at T = 360, while only the PTHS-60 group had significantly lower levels of hemoglobin than the PTHS-30 group at T = 360 (Fig. 4).

F4
Fig. 4:
Complete blood cell counts.

Platelet count was significantly reduced compared to baseline values starting at EOR for all groups except the PTHS-60 group, which was significantly reduced starting at EOS (Fig. 4). Significant differences between groups were seen at EOR and T = 360 for the PTHS-30 and PTHS-D groups. Compared to baseline, a significant elevation in white blood cell (WBC) count was seen in the PTHS-60 group at EOR. The PTHS-60+ST group had a significant increase in WBCs by T = 360. At EOR and T = 360, WBC counts exhibited a significant elevation in the PTHS-60+ST+FF and PTHS-D groups (Fig. 4). At EOS, WBC counts were significantly different only between the PTHS-60 and PTHS-D groups.

Serum cytokine and chemokine profiles

Significant elevations in IL-6 compared to baseline were identified beginning at EOS, most notably a 45-fold increase in the PTHS-D group. All groups demonstrated significant increases in IL-6 at EOR and T = 360 compared to baseline values, with the greatest EOR and T = 360 elevations occurring in the PTHS-D (102- and 143-fold increases, respectively) group. A significant increase in the level of IL-10 was observed at EOS for the PTHS-D group. This continued increasing to EOR in the PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D groups. IL-10 levels returned to BSLN levels at T = 360, except for the PTHS-D group, which remained significantly elevated above baseline at T = 360. Increased levels of IL-15 were observed starting at EOS compared to baseline (up to 3.6-fold in PTHS-D) and remained elevated until T = 360 min in the PTHS-60+ST+FF and PTHS-D groups. Similarly, levels of MCP-1 also increased significantly at EOS compared to baseline for the PTHS-D group, and remained significantly elevated out to T = 360. Although MCP-1 increased at EOS in the PTHS-60+ST+FF group, it was not significant. Compared to their baseline values, all groups demonstrated significant reductions in IL-8 from EOR to T = 360 min. This reduction of IL-8 in response to injury is different from the described human response, but consistent with previous reports in Rhesus Macaques (14) (Fig. 5).

F5
Fig. 5:
Serum cytokines and Chemokines.

Group comparison revealed significant differences between the PTHS-D group compared to the PTHS-30 group in levels of IL-6 (increased in PTHS-D) and IL-8 (decreased in PTHS-D) at EOS, EOR, and T = 360, as well as for IL-10 levels at EOR which were increased in the PTHS-D group. Also, levels of IL-8 at EOR were significantly decreased in the PTHS-D group compared to the PTHS-30 and PTHS-60 groups, and the PTHS-60+ST group compared to PTHS-30 group. Levels of IL-6 were significantly increased in the PTHS-D group compared to the PTHS-60 group at all time points. IL-6 levels were also significantly increased in the PTHS-D group compared to the PTHS-60+ST group at EOR and at EOR and T = 360 in the PTHS-60+ST+FF group compared to the PTHS-60 group. IL-10 levels were significantly increased in the PTHS-60+ST+FF group compared to the PTHS-30 group and compared to the PTHS-60 group at EOR and compared to the PTHS-60+ST group at T = 360. In addition, increased levels of IL-10 (at EOR) and MCP-1 (at EOS) were observed in the PTHS-D group compared to the PTHS-60 group. MCP-1 levels were increased in the PTHS-60+ST+FF group compared to the PTHS-60 group at EOS and EOR. No significant differences between groups were observed for IL-15 at any of the time points (Fig. 5).

Rotational thromboelastometry

Compared to baseline values, EXTEM CT was significantly reduced at EOS through T = 360 in the PTHS-60 group. EXTEM CFT was significantly reduced in the PTHS-60 group at EOS, but increased significantly in the PTHS-60+SF+FF group at EOR. Clot formation kinetics were significantly increased as shown by EXTEM alpha angle in the PTHS-60 group at EOR through T = 360, and MCF was significantly reduced at EOR in the PTHS-30 group (Supplementary Table 1, https://links.lww.com/SHK/A592). Between groups, EXTEM CT was significantly longer at baseline in the PTHS-60 group compared to the PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D groups. For CFT, baseline values were significantly greater in the PTHS-30 and PTHS-60 groups compared to the PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D groups.

For FIBTEM measurements, compared to baseline values, MCF was significantly increased at T = 360 in the PTHS-60 group and by EOR in the PTHS-60+ST+FF group. Among groups, MCF was significantly different at EOR for the PTHS-30 vs. PTHS-D groups and at T = 360 for the PTHS-30 vs. PTHS-60 and PTHS-D groups (Supplementary Table 1, https://links.lww.com/SHK/A592).

Platelet aggregometry

Compared to baseline values, only the PTHS-60+ST group demonstrated significantly reduced platelet aggregation for normalized and non-normalized platelet aggregation response to ADP at EOS, while the PTHS-60, PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D groups all displayed significantly reduced platelet aggregation at EOR, but once normalized to platelet count, only the PTHS-60+ST and PTHS-D group's aggregation remained significantly reduced. Statistically significant differences in platelet dysfunction between injury groups were seen in platelet aggregation only in response to ADP in both normalized and non-normalized data. Normalized platelet aggregation in response to ADP demonstrated decreased aggregation in the PTHS-D group compared to the PTHS-30 group at EOR, the PTHS-60 group at EOS and EOR, and the PTHS-60+ST group at T = 360 (Fig. 6). In non-normalized platelet aggregation, the PTHS-D group had significantly reduced aggregation compared to the PTHS-30 group at EOS and EOR, the PTHS-60 group at EOR, and the PTHS-60+ST group at T = 360 (Fig. 6).

F6
Fig. 6:
Platelet aggregometry.

Compared to baseline, all treatment groups displayed reduced platelet aggregation in response to COL at EOR and at T = 360, with the exception of the PTHS-60+ST+FF group. Once normalized, a significant reduction in aggregation was only observed in the PTHS-60+ST and PTHS-D groups at EOR, and at T = 360 for the PTHS-60+ST group (Fig. 6). In response to AA, all treatment groups had significantly reduced aggregation at EOR for both normalized and non-normalized platelet aggregation compared to BSLN (Fig. 6). At T = 360, reduced aggregation was significant for normalized and non-normalized platelet aggregation in the PTHS-30 and PTHS-60+ST groups, and for non-normalized platelet aggregation in the PTHS-60 group (Fig. 6).

Serum blood chemistry for organ damage

Kidney function was assessed by blood urea nitrogen (BUN) and creatinine (CREA) (Fig. 7). Compared to baseline values, BUN was significantly elevated at EOS in all groups and at T = 360 for all groups except the PTHS-30 group. CREA was significantly elevated from baseline at EOS for all groups and at T = 360 for the PTHS-D group. Liver function was assessed by albumin (ALB), alanine aminotransferase (ALT), and aspartate transaminase (AST). ALB was significantly decreased from baseline values starting at EOS and continuing through T = 360 for all groups. ALT levels were significantly increased from EOS through T = 360 for the PTHS-D group and at T = 360 for the PTHS-60 group. The levels of AST were significantly elevated from EOS through T = 360 for the PTHS-D group, while all other groups had significantly increased levels of AST by T = 360 (Fig. 7).

F7
Fig. 7:
Blood chemistries.

Hyperglycemia was assessed by blood glucose levels. Compared to baseline, all groups had significantly elevated levels of glucose at EOS except for the PTHS-D group, which had a significantly lower amount of glucose at EOR compared to baseline. Overall, the PTHS-D group consistently had higher levels of AST, ALT and CREA, and lower levels of ALB over time compared to the other groups (Fig. 7).

Pathology

Gross histopathologic lesions were observed for the liver and kidney. Damage to the kidney was observed as tubular necrosis which became more prominent with the addition of the femur fracture to the laparotomy and hemorrhage (Table 1; Fig. 8). No tubular necrosis was seen in the PTHS-30 group. In the PTHS-60 group, tubular necrosis was observed in 25% of the NHPs. In the PTHS-60+ST+FF and PTHS-D groups, 50% of the NHPs had tubular necrosis. Liver damage was observed as centrilobular necrosis which became more prominent with increasing hemorrhage duration as observed in the PTHS-D group. The PTHS-30 group had centrilobular necrosis detected in 12.5% of the NHPs. The PTHS-60 group had centrilobular necrosis in 37.5% of the NHPs, while the PTHS-60+ST group had centrilobular necrosis in 25% of the NHPs. In the PTHS-60+ST+FF group, 37.5% of the NHPs had centrilobular necrosis. The highest incidence of centrilobular necrosis occurred in the PTHS-D group, in which all NHPs had centrilobular necrosis (Table 1; Fig. 8). Splenic contraction as observed by decreased red pulp was observed in 100% of the NHPs in the PTHS-30, PTHS-60, and PTHS-D groups. In the PTHS-60+ST and PTHS-60+ST+FF groups, splenic contraction occurred in 75 and 50% of the NHPs, respectively (Table 1; Fig. 8).

T1
Table 1:
Histological scoring of organ damage
F8
Fig. 8:
Representative images of liver (centrilobular necrosis) and kidney (tubular necrosis) damage from each group of NHPs.

DISCUSSION

The objective of the study was to generate and characterize robust, reproducible, and clinically relevant models of traumatic hemorrhage. This includes recapitulation of severe metabolic acidosis, trauma-induced coagulopathy, and maladaptive immunologic responses clinically associated with human injury. We anticipate such models will be utilized for evaluation of human blood-derived therapeutics, drug therapies, and for further investigations of the responses to injury and shock. In the present study, we evaluated the effects on increasing durations of hemorrhagic shock with or without soft tissue and long bone injuries in five distinct NHP models.

Rhesus Macaques were selected as they are void of identified adverse xenogeneic reactions with human-derived therapeutic products previously observed in lower-order species (11, 13, 17, 27). Hemorrhage models are classified as either controlled (fixed volume or pressure) or uncontrolled (e.g., via organ or vessel transection). Uncontrolled hemorrhage (UH) models are considered to be the most clinically relevant mechanism of injury; however, consistent with previous reports, we observed a high degree of variability with regard to metabolic, inflammatory, and coagulation parameters in a previous NHP model of UH (9, 14). For this study, we chose a PTHS model which maintains a specific level of hypotension despite recruitment of the individual's compensatory mechanisms.

Although a PTHS approach reduces variability, it is discordant with clinically relevant mechanisms of injury as the hemorrhage is induced via a pump set at a fixed rate. The translational research value is, however, high due to the controlled injury mechanism and physiologic, metabolic, and immunologic responses which are all consistent to those seen in the clinic. A clinical study with this level of physiologic fidelity would be impossible to conduct. We implemented an arterial free bleed so that hemorrhage would be facilitated by physiologic pressures and rates. Although the pressure-target range implemented in the hemorrhagic shock protocol (i.e., MAP maintained between 20 and 24 mmHg) is relatively severe in comparison to other reported animal models, this target pressure range was selected based on our previous experience with UH in NHPs (14). In a previous study by our group, MAP exhibited a rapid decline in response to UH to a nadir of 22 ± 1 mmHg within 3.8 ± 0.4 min of injury and averaged 32 ± 2 mmHg at 30 min after injury (17). Consistent with the UH model, arterial free bleed achieved the pressure target within 3 min. We evaluated outcomes following the PTHS-30 and PTHS-60 groups to downselect a shock duration for subsequent incorporation of polytraumatic injuries into subsequent models. Given our previous experience with NHP modeling, and previously published results by other groups, we expected that both the PTHS-30 and PTHS-60 groups would elicit severe insults and possibly some mortality due to the hemorrhagic shock alone. However, our findings demonstrate minimal mortality and mild-to-moderate perturbations in physiologic, metabolic, coagulation, and immunologic outcomes. We therefore down-selected the PTHS-60 methodology for subsequent polytrauma model development. The observation of minimal mortality and mild physiologic derangements even in the PTHS-60+ST+FF group prompted extending the PTHS period to the point of cardiovascular decompensation.

The physiologic and metabolic derangements observed in the current models demonstrate stepwise increases with increased and more clinically relevant severity as compared to our group's previous NHP UH model. In all PTHS-60 groups, a tachycardic response was consistently observed at EOS, which is consistent with compensatory recruitment characteristic of a shock-state in trauma patients (28). The PTHS-60 groups revealed consistent elevations in lactate at EOS to values not uncommon to those observed clinically, though analyzed collectively (PTHS-60, PTHS-60+ST, PTHS+ST+FF) the lactate levels of the PTHS-60 groups were slightly lower than those considered “high” (i.e., >4 mmol/L) (29, 30). In addition, clinical studies have classified shock severity by stratifying trauma patients according to BD at the time of admission (mild [BD < 6 mmol/L], moderate [BD = 6–9 mmol/L], and severe [BD>10 mmol/L] shock), and revealed an increase in mortality with increasing BD severity (31). According to these classifications, 11 animals in this model development study would be classified as moderate: 2PTHS-60 animals, 2 PTHS-60+ST animals, 1 PTHS-60+ST+FF animal, and 6 (75%) of the PTHS-D animals. In addition, three animals would be classified as severe shock: one PTHS-60+ST+FF animal and two (25%) PTHS-D animals. Within the PTHS-60 and PTHS-60+ST+FF groups, the two nonsurvivors were in moderate and severe shock, while all animals in the PTHS-D group were in moderate-to-severe shock with seven out of eight animals being nonsurvivors. When considered as a proportion of the overall number of animals among the groups, the relationship of shock severity and mortality is consistent with the mortality rate observed in the clinical reports.

Traumatic hemorrhage has long been known to elicit a systemic inflammatory response, the severity of which is moderated by extent of injury and depth of shock. Circulating cytokine and chemokine levels followed similar trends, though more severe than previously observed in NHPs subjected to hemorrhagic shock (14, 23). All PTHS-60 groups and the PTHS-D group elicited elevations in the proinflammatory cytokine IL-6, consistent with clinical reports of IL-6 elevations in states of systemic and vascular inflammation (32, 33). A trend of increased levels of the proinflammatory chemokine MCP-1 was observed across groups with the greatest apparent increase being in the PTHS-D animals, which correlates with increasing injury severity. Similarly, studies in trauma patients have also revealed a correlation between high levels of IL-6 and MCP-1 levels and worse patient outcomes (34). Contrary to clinical data, yet consistent with our previous observations, the proinflammatory cytokine IL-8 was significantly reduced in response to trauma (14). Elevation of IL-8, a potent neutrophil chemotactic factor, has been associated with the development of multiple organ dysfunction in trauma patients (35, 36). The decreased systemic levels of IL-8 may be due to a species-specific phenotypic response to hemorrhagic shock. The relevance of IL-15 in the setting of trauma is relatively unknown, but previous studies have demonstrated a role for IL-15 in the maturation and proliferation of natural killer cells (37). Furthermore, the elevation of IL-15 observed in this study is consistent with our previous NHP studies and elevations seen in burn patients (38).

Elevation of the anti-inflammatory cytokine IL-10 is commonly observed in trauma patients (33). Similarly, the PTHS-60+ST+FF and PTHS-D groups exhibited a 2-fold increase in the anti-inflammatory cytokine IL-10 as well. Interestingly, this was not observed in the PTHS-30 and other PTHS-60 groups. Collectively, these data demonstrate differential effects of isolated hemorrhage, duration of hemorrhage, and polytraumatic injuries on the systemic inflammatory response, and suggest that the models developed elicited a maladaptive immune response. However, the systemic inflammatory assessment performed was not comprehensive and subsequent investigations should include more direct assessment of innate and adaptive immune cell activation and tissue-specific responses.

Both multisystem injury and depth of shock have been associated with the development of trauma-induced coagulopathy (TIC). TIC results in a disruption of hemostasis which can further complicate interventions to treating hemorrhage with low platelet counts and/or platelet dysfunction being some of the principal components involved (39–41). In all groups, low platelet counts and platelet dysfunction were observed that increased in severity correlating to model severity in this study, with the polytraumatic groups showing the most severe platelet dysfunction and lowest platelet count. The most prominent drop in platelet aggregation in response to agonists was observed at EOR with the response to collagen and arachidonic acid being the most affected even after adjusting for platelet count. Such platelet dysfunction was similar to that reported in the clinical trauma literature (41–43).

The coagulopathy observed in these models, based on whole blood viscoelastic properties, was not as severe as expected, or potentially desired; however, the influence of the whole blood-based resuscitation may well have been a confounder. The majority of literature on trauma-induced coagulopathies rely on diagnostics potentially influenced by antecedent non-whole blood-based prehospital fluid administration, with crystalloid and its recognized negative coagulation effects (44, 45). Further exploration of potential derangements in plasmatic coagulation and platelet function is warranted.

The most significant histopathologic finding across all groups was necrosis of centriolobular hepatocytes. The severity of hepatocyte degeneration was classified as mild/moderate with cell swelling, expansion of sinusoids, and disorganized hepatic cords, or as severe with the concurrent presence of hepatic lobule degeneration. Hepatocyte necrosis was classified as mild/moderate with the evidence of necrotic cells surrounding hepatic veins and marginal zones. These histopathologic findings correlated with elevated levels of ALT and AST at time of death. Furthermore, these patterns of hepatic degeneration and necrosis observed are consistent with lesions observed in humans diagnosed with hypoxic hepatitis (46).

These models, in particular the PTHS-60+ST+FF and the PTHS-D models, demonstrated physiologic, metabolic, coagulopathic, and inflammatory derangements with the most robust derangements observed in the PTHS-D model. These derangements are representative of those observed in human trauma patients, for example with regard to blood loss and acid-base disturbances. Importantly, the models elicited consistent, reproducible derangements. Although the severity of outcomes observed in the PTHS-60 groups are clinically relevant, they were not as severe in comparison to values published in clinical trauma literature. Interestingly, despite 10% less blood loss in the PTHS-60+ST+FF group, outcomes were similar to those in the PTHS-60 group. Extending the PTHS duration in a time-independent fashion (PTHS-D) to the point of cardiovascular decompensation in combination with polytraumatic injury was required to elicit physiologic, metabolic, coagulopathic, and immunologic derangements most translatable to severely injured trauma patients.

In summary, the polytrauma models developed in this series of experiments induce a spectrum of clinically relevant and highly reproducible injury patterns with consistent outcomes varying in severity with reduced risk of xenogeneic confounders present in lower species models. These models, with the PTHS-D model being the most translatable to a severely injury patient, should facilitate higher fidelity preclinical therapeutic studies, serve as a foundation for potential variants to include solid organ and UH aspects, and optimally improve the delivery of potentially efficacious therapeutics and treatments to clinical practice.

Acknowledgments

The authors thank the contributions of Darren Fryer, Savannah Green, Kassandra Ozuna, and Leasha Schaub of the NAMRU-SA Expeditionary and Trauma Medicine Department, as well as Carrie Crane and her NAMRU-SA Veterinary Sciences Department. In addition, the authors are indebted to the steadfast support and vision of CAPT's Rita G. Simmons and Elizabeth Montcalm-Smith, the essential collaboration(s) with Dr Andrew Cap and the USAISR, and acknowledge LTC Christine Christensen and MAJ James Johnson of the Air Force for performing and scoring the histology and taking images. In addition, we wish to acknowledge the continued support of the Office of Naval Research, Navy Advanced Medical Development and the Hemorrhage and Resuscitation Portfolio of the USAMRMC Joint Program Committee 6 and the continued support and collaboration of the USAISR and specifically Dr Andrew Cap.

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Keywords:

Hypotension; injury; Macaca mulatta; musculoskeletal; resuscitation; shock; soft tissue; trauma

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