The impact of septic stimuli on the systemic inflammatory response and physiologic insult in a preclinical non-human primate model of polytraumatic injury
© The Author(s). 2018
Received: 22 December 2017
Accepted: 13 May 2018
Published: 24 May 2018
Established animal trauma models are limited in recapitulating the pathophysiology of human traumatic injury. Herein, we characterize the physiologic insult and inflammatory response in two clinically relevant non-human primate (NHP) trauma models.
Mauritian Cynomolgus Macaques underwent either a laparoscopic closed abdomen liver injury (laparoscopic 60% left-lobe hepatectomy) in an established uncontrolled severe hemorrhage model (THM), or a polytrauma hemorrhage model (PHM) involving combined liver and bowel injury, uncontrolled severe hemorrhage as well as an open full-thickness cutaneous flank wound. Fixed volume resuscitation strategies were employed in the THM and goal directed resuscitation was used in the PHM. Complete peripheral blood and critical clinical chemistry parameters, serum biomarkers of systemic inflammation, tissue perfusion parameters, as well as survival, were compared between the models throughout the 2-week study period.
NHPs in both the THM (n = 7) and the PHM (n = 21) demonstrated tissue hypoperfusion (peak lactate 6.3 ± 0.71 mmol/L) with end organ injury (peak creatinine 3.08 ± 0.69 mg/dL) from a similar liver injury (60% left hemi-hepatectomy), though the PHM NHPs had a significantly higher blood loss (68.1% ± 12.7% vs. 34.3% ± 2.3%, p = 0.02), lower platelet counts (59 ± 25 vs. 205 ± 46 K/uL, p = 0.03) and a trend towards higher mortality (90.5% vs. 33.3%, p = 0.09). The inflammatory response was robust in both models with peak cytokine (IL-6 > 6000-fold above baseline) and peak leukocyte values (WBC 27 K/uL) typically occurring around t = 240 min from the time of hepatic injury. A more robust systemic inflammatory response was appreciated in the PHM resulting in marked elevations in peak serum IL-6 (7887 ± 2521 pg/mL vs.1076 ± 4833 pg/mL, p = 0.02), IL-1ra (34,499 ± 5987 pg/mL vs. 2511 ± 1228 pg/mL, p < 0.00), and IL-10 (13,411 pg/mL ± 5598 pg/mL vs. 617 pg/mL ± 252 pg/mL, p = 0.03).
This comparative analysis provides a unique longitudinal perspective on the post-injury inflammatory response in two clinically relevant models, and demonstrates that the addition of septic stimuli to solid organ injury increases both the hemorrhagic insult and inflammatory response.
The recent military conflicts in Iraq and Afghanistan have seen an unprecedented increase in survival of combat casualties despite increasing injury severity scores (ISS) and complex injury patterns [1, 2]. These improvements in combat casualty care can largely be attributed to progress in care on the battlefield, medical transport and resuscitation strategies . As with civilian trauma, these severely injured combat casualties that survive the initial traumatic insult are at risk for developing an exaggerated inflammatory response with immediate and long term consequences such as sepsis, wound complications, heterotopic ossification (HO), invasive fungal infections, venous thromboembolic disease, multiple organ dysfunction syndrome (MODS), and multiple organ failure (MOF) [2, 4–12].
Despite extensive research, the underlying mechanisms for this dysregulated inflammatory response remains elusive both at the systemic and local tissue level. Attempts to mitigate the morbidity of this immune response through known pathways including blocking individual cytokines, co-stimulatory molecules, endothelial products, and complement cascade have yielded disappointing clinical outcomes in the face of promising preclinical results. This is in part due to the significant complexity and redundancy of the immune response to severe injury, which to date has not been adequately modeled in the preclinical setting by falling short on the model animal’s genetic similarity, degree of injury, goal directed critical care post-injury, and longitudinal evaluation of outcomes [10, 13]. Advances in preclinical modeling are required in order to better characterize and comprehensively differentiate the inflammatory response after various injury patterns, as well as to provide a better understanding for future interventions and clinical studies.
The nonhuman primate (NHP) is a highly valuable model which has significantly advanced our understanding of the physiological and immunological injury response patterns to acute trauma . As part of a preclinical immunomodulation study, we have developed two preclinical NHP traumatic injury models including a closed abdomen, laparoscopic liver trauma and hemorrhage model (THM) with a fixed resuscitation protocol , as well as a closed abdomen polytrauma hemorrhage model (PHM) involving combined laparoscopic liver and bowel injury, uncontrolled hemorrhage as well as an open full-thickness cutaneous flank wound with subsequent goal directed resuscitation . As previously reported, while the THM demonstrated a measurable systemic inflammatory response, only the few nonsurvivors developed end organ injury associated with limited resuscitation. Evolution of the PHM increased the mortality of the model, however, comparative analysis is required to better understand the complex interactions between polytraumatic injury, hemorrhagic shock, septic stimuli, and the inflammatory response. In developing this model the authors hypothesis was that a shift in the innate and adaptive immune responses would occur with the addition of septic stimuli and an associated increase in the systemic inflammatory response in the PHM despite the same liver injury in the THM consistent with a dysregulated response.
Pre-injury preparation THM and PHM
The animals were transported to the operative suite on the morning of surgery and underwent sedation with telazol (Zoetis Inc., Kalamazoo, MI), peripheral IV placement, as well as endotracheal intubation with isoflurane mask induction and ventilation. Adequate anesthesia was administered and maintained with isoflurane via Datex Ohmeda S/5 ADU Carestation. ECG, pulse oximeter, end tidal carbon dioxide, rectal temperature probe, and urinary foley catheterization were established for intra-operative monitoring. FiO2 was set at 0.21 for the pre-hospital phase, and ventilation was adjusted to maintain physiologic pCO2 values (Apollo /Drǟger Medical, Telford, PA). At this time the animal’s abdomen and bilateral groins were shaved, prepped with a chlorhexidine/alcohol-based solution, and sterilely draped. A right femoral cut down was performed and a tunneled right central venous line (PORT-A-CATH, Smiths-Medical, Dublin, OH) and arterial catheter (Cordis, Johnson & Johnson, Miami Lakes, FL) were placed into the femoral vein and artery, respectively. The arterial catheter was connected to a hemodynamic monitoring system (Philips IntelliVue MP70, Philips Electronics North America Corporation, Andover, MA) for continuous monitoring of arterial pressures and intra-operative lab draws. Attention was then turned to prepare the laparoscopic abdominal injuries. Access to the peritoneal cavity was performed via Hassan technique and a 12 mm trocar (ENDOPATH XCEL, Ethicon, Somerville, NJ) was left in place. Pneumoperitoneum was established with CO2 to an intra-abdominal pressure of 10–12 mmHg. A 10 mm 30 degree laparoscope was inserted through the port and peritoneal cavity was examined. A 5 mm trocar was placed in the left hypogastric region, and a second 5 mm trocar was placed in the right hypogastric region.
Injury phase THM
Under laparoscopic visualization, animals in the hemorrhage model underwent a non-anatomic 60% left lobe liver resection creating uncontrolled hemorrhage which was designated time zero (t = 0 min). The pneumoperitoneum was then evacuated, all laparoscopic ports were removed, and the port sites were temporarily closed with 2–0 nylon sutures to achieve abdominal wall continuity.
Injury phase PHM
Under laparoscopic visualization, animals in the PHM underwent a 4 cm anti-mesenteric cecal injury to allow for abdominal sepsis. A 60% left lobe liver injury was then created (t = 0 min) and temporary abdominal closure was performed as described above. The animals in this model additionally underwent a right flank standardized circular (18.85cm2) full-thickness soft tissue injury down to the level of the muscle fascia.
Pre-hospital care THM and PHM
At t = 15 min, pre-hospital resuscitation was initiated with normal saline resuscitation (20 mL/kg) for both the hemorrhage alone and polytrauma groups . Anesthetic, cardiovascular, and pulmonary monitoring were continued throughout the 120 min pre-hospital phase.
Resuscitation phase/hospital care THM
Surgical resuscitation was initiated at the start of the hospital phase (t = 120 min) with a midline laparotomy. The hepatic injury was exposed and hemostasis was achieved by suture ligation, cautery, and Surgicel (Ethicon, Somerville, NJ). All intra-peritoneal blood and the resected liver segment were removed and the volumes were recorded as total hemorrhage volume, percent blood loss, and percent hepatectomy.  At t = 120 min the THM NHPs underwent continuous monitoring, oxygen therapy, blood-type matched and leukocyte- reduced blood transfusion at 10 mL/kg, active rewarming. The abdomen was closed and at t = 240 min the last blood draw was withdrawn and the arterial and venous catheters were removed, anesthesia was weaned, and the animals were extubated.
Resuscitation phase/hospital care PHM
The animals in the PHM also underwent surgical resuscitation at t = 120 min with similar hepatic repair and injury measurements as described in the THM. Once hemostasis was achieved the cecal perforation was repaired in two-layer closure. In addition, the peritoneal cavity was copiously irrigated and all fecal contamination was removed. The abdomen was closed at this time. At t = 120 min the PHM NHPs also underwent continuous monitoring, oxygen therapy, blood-type matched and leukocyte- reduced blood transfusion at 10 mL/kg, active rewarming. After the initial blood transfusion, NHPs underwent goal directed resuscitation to correct MAPs < 40 mmHg, base excess < − 4, and/or lactate > 4, and decreased urine output (< 0.5 mL/kg) with crystalloid resuscitation. NHPs with hemoglobin < 7 g/dL were resuscitated with blood transfusions. Once the animals were resuscitated, the flank soft tissue wound was then dressed (Hydrofera BLUE®, Hollister Wound Care, Libertyville, IL), and the arterial catheter was removed. The venous port was tunneled and remained in place with the port secured in subcutaneous space. The animals were then placed in a protective jacket (Lomir Biomedical Inc., Quebec, Canada), and they were extubated after weaning anesthesia.
Antibiotics THM and PHM
At the initiation of surgical resuscitation, animals in both groups received a dose of broad-spectrum antibiotics (enrofloxacin 5 mg/kg [Bayer HealthCare LLC, Shawnee Missino, KS]) and metronidazole 50 mg/kg [Henry Schein, Melville, NY].
Survival phase THM
The animals in the THM group were recovered in an “intensive care unit” setting with 24 h of continuous monitoring by trained research and veterinary staff. Animals were administered buprenorphine (0.1–0.3 mg/kg) every 4 h as needed for pain, peripheral intravenous normal saline (20 mL/kg) on post-injury day 1, 2, and 3, and a blood transfusion (10 mL/kg) for anemia (Hb < 9 g/dL). If clinically stable after 24 h, the animals were returned to their pre-operative housing. At least twice daily evaluations were performed in conjunction with veterinary staff, and euthanasia was performed based on adverse clinical states and complications, response to resuscitation, response to pain medications, and laboratory parameters to include indices of fulminant renal failure. If criteria were met, animals were euthanized with pentobarbital (Fatal-Plus; Vortech Pharmaceuticals, Dearborn, MI). All surviving animals were euthanized according to protocol on post-injury day 14 followed by a complete necropsy evaluation.
Survival phase (PHM)
In the PHM group, the animals were also recovered in an ICU setting, but for at least 72 h with laboratory staff present for continuous monitoring and goal directed resuscitative interventions with crystalloid infusion to correct signs of poor perfusion (low urine output, elevated Cr or lactate) or blood transfusions to correct signs of anemia (Hb < 7 g/dL) and poor perfusion. Laboratory evaluation was performed every four hours as needed to evaluate the response to resuscitation while in the ICU setting. Pain management was performed with sustained release transdermal fentanyl patch (25mcg/hr. placed at t = 240 min) and buprenorphine (0.01–0.03 mg/Kg IM every 6 h as needed). The soft tissue wound was examined and dressed every 5 days. If assessed clinically stable, animals were advanced to a regular diet and returned to their pre-operative housing. Euthanasia with pentobarbital was administered at the veterinarian’s discretion or if the animals demonstrated signs of severe disability, severe infection, uncontrolled pain, or showed clinical/laboratory signs of deterioration and failed to respond to resuscitation. On post-injury day 14, surviving animals were euthanized and underwent a complete necropsy evaluation.
Laboratory and survival analysis (THM and PHM)
For the purposes of this study, laboratory analysis were contrasted between the two models’ comparable data points which included the blood collected at 2 weeks pre-injury as well as t = 0, 15, 60, 120, and 240 min, as well as on post-injury days 1, 3, 5, 7 and 14. Blood was analyzed for complete blood cell count, arterial blood gas, and basic metabolic profile. In addition, the serum profiles for a panel of 14 cytokines and chemokines (IL-6, IL-1ra, IL-10, G-CSF, MCP-1, IL-1β, IFN-γ, TNF, GM-CSF, IL-15, MIP-1α, IL-8, IL-2, TGFα) was determined using multianalyte bead based profiling (Luminex; NHP 14-plex; Millipore, Billerica, MA). Survival time was recorded according to time of intra-operative death (t = 0 to t = 240 min) or post-injury day.
Comparable variables from the two models including markers of injury, mortality, cytokine levels, and peripheral leukocyte counts were analyzed with IBMSPSS® Statistics (version 23.0, Chicago, IL, USA). Percent blood loss was calculated using the formula: [blood loss volume (ml)/total estimated blood volume (TEBV)] × 100. TEBV was calculated based on 5.4% of the animal’s weight according to previously published data on blood volume in Cynomolgus Macaques . Percent hepatectomy was calculated using the formula: [liver cut length (cm)/full liver length (cm)] × 100. An Injury Severity Score (ISS) was calculated for every animal in each group .
Markers of injury compared between the polytrauma and hemorrhage model (PHM) and the trauma and hemorrhage model (THM).
Pre-Hospital and Hospital Phase Markers of Injury
PHM (n = 21)
THM (n = 7)
Injury Severity Score
Left Hepatectomy (%)
60.6 ± 4.3
55.9 ± 3.76
Blood Loss (%)
68.1 ± 12.7
34.3 ± 2.33
24.6 ± 1.92
34.5 ± 6.5
90.0 ± 3.66
94.3 ± 0.38
7.1 ± 0.1
7.2 ± 0.1
Minimum Platelets (K/uL)
59 ± 25
205 ± 46
Experimental Injury Values
1.8 ± 0.36
3.08 ± 0.69
6.3 ± 0.71
4.88 ± 0.62
Maximum recorded cytokine values as well as fold change from baseline (2 weeks pre-injury) of systemic cytokines throughout the post-injury period (t = 0 to post-injury day 14) from the trauma and hemorrhage model (THM) and polytrauma and hemorrhage model (PHM)
Maximum Recorded Serum Level
Polytrauma and Hemorrhage Model
Trauma and Hemorrhage Model
7887 ± 2521
1076 ± 483
34,498 ± 5987
2511 ± 1229
13,411 ± 5598
617 ± 252
1652 ± 1912
201 ± 192
8419 ± 2297
4064 ± 1140
27 ± 71
0.5 ± 0.2
40 ± 126
4.7 ± 6.8
289 ± 558
2.6 ± 2.0
30 ± 41
1.4 ± 1.5
19 ± 18
13 ± 4.4
47 ± 52
12 ± 16
2056 ± 2190
1280 ± 737
19 ± 22
10.4 ± 5.5
50 ± 87
37 ± 15
Figure 3 demonstrates the cytokine levels throughout the post-injury period of those cytokines which were detectable and varied significantly from baseline. The PHM animals had a significantly larger cytokine elevation as compared to the THM typically around t = 240 min. However, significant differences occurred earlier between the two models at t = 60 min with IL-1ra and G-CSF, as well as at t = 120 min with IL-10. All cytokines began to down trend towards baseline by day-3 post injury.
Severely injured polytrauma patients are at risk for shock, immune dysregulation, MODS, and MOF. The addition of septic insult in trauma patients and experimental animal models compounds the inflammatory response, and can increase both the morbidity and mortality of trauma [19–22]. Clinically effective strategies to mitigate the deleterious effects of this inflammatory response are principally centered on supportive care, and improvements in modeling this inflammatory response are required in order to better understand and target inflammatory mediators. In this study, we characterized and compared the physiologic injury and the systemic inflammatory response in two separate NHP trauma models, and demonstrated that the addition of septic stimuli to trauma and hemorrhage caused a distinct end organ injury pattern as well as an increased inflammatory response, hemorrhage, and subsequent morbidity and mortality despite improvements in resuscitation.
Both the THM and PHM animals developed evidence of renal injury and tissue hypoperfusion. The renal injury and mortality seen on post-injury day-1 of the THM, as previously described by our group, was more likely associated with inadequate resuscitation rather than systemic inflammation associated multiple organ dysfunction syndrome . In contrast, PHM NHPs demonstrated clinical observations and pathology results consistent with acute respiratory distress syndrome . It is postulated that the multiple inflammatory stimuli in these animals resulted in more significant distant end-organ damage, especially in the respiratory system, which ultimately lead to a trend towards higher mortality.
Interestingly, the NHPs in the PHM experienced significantly larger hemorrhage despite having a similar liver injury as the THM. This hemorrhage was associated with a relative thrombocytopenia, but not with other predisposing factors such as a significantly lower pH or temperature. Though we did not measure coagulation parameters in this study, we suspect that the thrombocytopenia and larger hemorrhage in the PHM was secondary to a sepsis induced coagulopathy [20, 22–31] with immune mediated endothelial activation and induction of coagulation cascade [23, 25, 26, 32, 33]. The coagulopathy of sepsis has been studied in detail, and specifically demonstrated in two prior NHP models [30, 34]. In a lethal intravenous E. coli NHP model, Taylor demonstrated that septic stimuli initiated not only an inflammatory response (elevated IL-6, TNF, and C5b-9), but also an early consumptive coagulopathy through endothelial activation (elevated Tissue Factor, Thrombomodulin, Endothelial Linked Adhesion Molecules) and induction of the coagulation cascade (depressed Fibrinogen) . Similar findings were also seen in a NHP peritonitis model in which the septic stimuli resulted in a significant inflammatory response and drop of both fibrinogen and platelets within 2 h of initial laparotomy . Recently, a traumatic shock rabbit model demonstrated that while hepatic injury and hemorrhage alone induce hypercoagulable findings on thromboelastography, the addition of bowel injury to the hepatic injury pattern induced a hypocoagulable state . In keeping with the reported literature, the increased hemorrhage, thrombocytopenia, and mortality associated with the septic insult in the PHM compared to THM reinforces the interconnected and interdependent immune, reticuloendothelial, and hemostatic systems, particularly in the setting of polytrauma.
Clinical studies and animal models evaluating longitudinal cytokine response to trauma
Study (reference #)
Mean Peak (pg/mL)
Mean Peak (pg/mL)
Mean Peak (pg/mL)
Kubiak 2011 
Animal (Insult): Swine (Trauma, IRI, Sepsis)
Survival: 48 h
Trauma (laparotomy) + IRI (SMA clamp × 30 min) + Sepsis (fecal clot) with passive drainage at 12 h. n = 5
Kinasewitz 2000 
Animal(Insult): NHP (Trauma, Sepsis)
Survival: 14 days
Trauma (laparotomy), Sepsis (E coli fibrin clot peritonitis). n = 5 (non-survivor)
~ 31,000 (death)
Trauma (laparotomy), Sepsis (E. coli fibrin clot peritonitis). n = 4 (signs of sepsis)
~ 12,000 (sepsis)
Trauma (laparotomy), Sepsis (E. coli fibrin clot peritonitis). n = 4 (well appearing)
~ 12,000 (well)
Martin 1997 
Clinical Study (Sepsis vs. Trauma and Survivors vs. Nonsurvivors) and (Sepsis vs. Trauma and Hemorrhage)
Septic Shock (mean APACHE II score = 27) non-survivors n = 14
9th blood draw
Septic Shock (mean APACHE II score = 27) survivors n = 11
3rd blood draw
Trauma (mean ISS = 27) non-survivors n = 7
8th blood draw
blood draw 13
Trauma (mean ISS = 27) survivors n = 53
blood draw 11
Trauma (mean ISS not reported) + Hemorrhagic Shock n = 8
2nd blood draw
8th blood draw
Jastrow 2009 
Clinical Study (Trauma +/− MOF)
Trauma (mean ISS 26) with MOF. n = 11
Trauma (mean ISS 25) with nonMOF. n = 37
PHM Animal: NHP (Trauma, Hemorrhage, and Sepsis)
Survival: 14 days
Trauma (laparoscopic liver/bowel injury, flank soft tissue injury) + intra-abdominal hemorrhage/ sepsis × 2 h. n = 20
THM Animal: NHP (Trauma and Hemorrhage) Survival: 14 days
Trauma (laparoscopic liver injury) + Hemorrhage (uncontrolled hemorrhage × 120 min)n = 5
Sheppard 2017 
Animal: NHP (Trauma and Hemorrhage)
Survival: 24 h
Trauma (Lapartomy and Femur Fracture) + Decompensated Hemorrhage (pressure controlled)
< 6 h
Trauma (Laparotomy and Femur Fracture) + 60 min Hemorrhage (Pressure Controlled)
< 6 h
Deitch 1996 
Animal: NHP (Trauma and Hemorrhage vs. Trauma)
Survival: 8 h
Trauma (neck dissection and partial removal of clavicle) + Hemorrhage (40 mmHg × 3 h or reached -5 mEq Base Excess). n = 5
~ 4 h
Trauma (neck dissection and partial removal of clavicle)
~ 8 h
Namas 2009 
Clinical Study (Trauma)
Human Trauma (mean ISS 24) + Hemorrhagic Shock n = 4 (non-survivors)
within 6 h
within 6 h
within 6 h
Baker 2012 
Animal: Swine (Trauma vs. Hemorrhage vs. Trauma and Hemorrhage)
Survival: 300 min
Trauma (femur fracture and lung contusion) + Hemorrhage(20-30 mmHg × 60 min). n = 6
Trauma (femur fracture and lung contusion). n = 5
Hemorrhage(20-30 mmHg × 60 min)n = 5
< 15 min
Namas 2009 
Animal (insult): Swine (Trauma and Hemorrhage vs. Hemorrhage)
Survival: 2 h after resuscitation
Trauma (anterolateral thoractomy and pericardial window) + Hemorrhage (MAP < 30 mmHg × 10 min, MAP < 20 mmHg for 10 s, or MAP>/= 30 × 90 min). n = 4 (non-survivors)
Hemorrhage (MAP < 30 mmHg × 10 min, MAP < 20 mmHg for 10 s, or MAP >/= 30 × 90 min). n = 3 (non-survivors)
Trauma (anterolateral thoractomy and pericardial window) + Hemorrhage (MAP < 30 mmHg × 10 min, MAP < 20 mmHg for 10 s, or MAP>/= 30 × 90 min). n = 7 (survivors)
Hemorrhage (MAP < 30 mmHg × 10 min, MAP < 20 mmHg for 10 s, or MAP >/= 30 × 90 min). n = 9 (survivors)
As has been previously reviewed , compared to solid organ injury and uncontrolled hemorrhage models such as the THM, the pressure controlled hemorrhage and trauma models listed in Table 3 result in a relatively limited inflammatory response [35, 38, 41, 43]. The addition of septic stimuli and soft tissue trauma in the PHM significantly increased the inflammatory response with a greater than 6100-fold elevation in IL-6 levels from baseline in the PHM, compared to the only 1100-fold increase in the THM. Preclinical studies favor sepsis as the major driver of the inflammatory response [36, 50], and clinical studies have shown IL-6 elevation has been tied to sepsis in trauma [21, 47, 51–57]. Table 3 also demonstrates that sepsis alone or the addition of sepsis to trauma results in a more pronounced inflammatory response than trauma alone, hemorrhage alone, or trauma with hemorrhage [20, 21, 30]. The highest IL-6 peaks (> 15,000 pg/mL) in Table 3 occurred in animals with persistent septic stimuli [20, 30] and non-surviving patients suffering from septic shock . As seen in our PHM, relatively lower cytokine levels seen within the first 24 h in NHPs who underwent intra-peritoneal sepsis × 2 h with subsequent peritoneal washout and repair of bowel injury were reflected in other studies in which the animals were able to clear their septic stimuli  and patients who were able to recover from septic shock . Though comparing cytokine levels between studies can be challenging given inconsistencies in analysis technique and variation in response to inflammatory stimuli across species , it is interesting that the IL-6 threshold (> 4110 pg/mL) predictive of MOF in trauma patients demonstrated by Jastrow et al., also distinguishes the IL-6 levels between the PHM and THM, as well as in the results of other studies seen in Table 3 contrasting trauma alone vs. sepsis ± trauma in both animal models and clinical studies. This may be suggestive that this IL-6 threshold distinguishes early peak inflammation in response to certain inflammatory stimuli that predicts subsequent immune dysregulation. Comparing the reported TNF and IL-10 levels to the literature, as seen in Table 3, demonstrates that the levels in the PHM cross the MOF predictive threshold of TNF and IL-10 described by Jastrow , however, the differential response per inflammatory stimuli in other studies is not as consistent as is seen with IL-6.
The leukocyte data presented in this study reveals the expected post-injury changes associated with severe trauma including leukocytosis, neutrophilia, and relative lymphocytopenia in both the THM and PHM [58–67]. Interestingly, despite a more significant systemic cytokine response, the NHPs in the PHM had a lower WBC and neutrophil count at t = 240 min, and a higher neutrophil:lymphocyte ratio at t = 60 min. The neutrophil: lymphocyte ratio has emerged as an important prognostic factor in inflammatory conditions such as those found in critically ill patients and cancer patients [66, 68]. The PHM’s higher neutrophil:lymphocyte ratio in addition to the relative neutropenia and leukopenia compared to the THM are consistent with the reported literature that increased tissue trauma and the intra-abdominal sepsis induced stress-induced lymphocyte apoptosis  as well as greater neutrophil and lymphocyte sequestration [59, 66, 70–72] both at the site of sepsis and in distant organs such as the lungs  seen in prior studies. These early alterations in the balance of innate/adaptive immune responses in trauma imply long term consequences of immune dysregulation and increased mortality .
Physiologically relevant and highly reproducible preclinical animal models of polytraumatic injury are required to reproduce the complex and heterogeneous pathobiology of human polytraumatic injury and to develop novel therapeutic strategies. In an attempt to move from the bench to the bedside with the aim to test candidate therapeutic immunomodulatory agents that may dampen the early trauma-induced inflammatory/immune response, we have evaluated the inflammatory response in two preclinical NHP trauma models. Analysis of comparable variables between the two models demonstrated that the addition of septic stimuli in the PHM leads to a larger hemorrhage and a mixed inflammatory systemic cytokine response, as well as early alterations in innate/adaptive immune response compared to the THM. This study’s novel longitudinal comparison of systemic cytokines in the two separate models demonstrated that the addition of sepsis crosses predictive threshold of IL-6, IL-10, and TNF for immune dysregulation. Further, this analysis supports clinical studies demonstrating acute traumatic coagulopathy with polytrauma, as well as prompts further exploration of hemostatic complications of even transient septic stimuli in trauma patients. Future efforts in model development will focus on extended ventilator support phase for recovery, as well as expanded coagulation, systemic biomarkers, tissue immunohistochemistry, and flow cytometric studies to further characterize the effects of trauma and sepsis and potentially elucidate therapeutic targets to mitigate the inflammatory response of trauma.
Cary Honnold, DVM, Diplomate ACVP, ACVPM and Pathology Department at Naval Medical Research Center.
This effort was supported (in part) by the U.S. Navy Bureau of Medicine and Surgery under the Medical Development (PE 0604771 N) and Office of Naval Research work unit number BUMED 6.5 604771 N.C210.001.A0812 and DMRDP – Intra – ATTD BUMED GDF 602115HP.4130.001.A1105.
Availability of data and materials
Please contact author for data requests.
DV, MB, TD, EE and BB conceived of the study. DV, MB, BB, CL, EE, TD carried out the animal study protocols including the injury phase and post-injury care, as well as the post-injury analysis tissue and plasma. DV, BB, MB, and TD drafted the manuscript and CL and EE contributed to revisions of the manuscript. All authors read and approved the final manuscript.
The study protocol was reviewed and approved by the Walter Reed Army Institute of Research/Naval Medical Research Center (NMRC) Institutional Animal Care and Use Committee in compliance with all applicable Federal regulations governing the protection of animals in research.
The authors declare that they have no competing interests.
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