Monitoring the healing of combat wounds using Raman spectroscopic mapping.

Soldiers wounded in modern warfare present with extensive and complicated acute wounds, confounded by an overwhelming inflammatory response. The pathophysiology of acute wounds is unknown and timing of wound closure remains subjective. Collagen gene expression profiles are presented for 24 patients. Impaired healing wounds showed a twofold decrease in the up-regulation of COL1A1 and COL3A1 genes in the beginning of the wound healing process, compared with normal healing wounds. By the final debridement, however, collagen gene expression profiles for normal and impaired healing wounds were similar for COL1A1 and COL3A1. In addition, Raman spectroscopic maps were collected of biopsy tissue sections, from the first and last debridements of 10 wounds collected from nine patients. Tissue components obtained for the debridement biopsies were compared to elucidate whether or not a wound healed normally. Raman spectroscopy showed a loss of collagen in five patients, indicated by a negative percent difference in the 1,665/1,445 cm(-1) band area ratios. Four healed patients showed an increased or unchanged collagen content. Here, we demonstrate the potential of Raman spectroscopic analysis of wound biopsies for classification of wounds as normal or impaired healing. Raman spectroscopy has the potential to noninvasively monitor collagen deposition in the wound bed, during surgical wound debridements, to help determine the optimal time for wound closure.

Combat Wound Initiative program.

The Combat Wound Initiative (CWI) program is a collaborative, multidisciplinary, and interservice public-private partnership that provides personalized, state-of-the-art, and complex wound care via targeted clinical and translational research. The CWI uses a bench-to-bedside approach to translational research, including the rapid development of a human extracorporeal shock wave therapy (ESWT) study in complex wounds after establishing the potential efficacy, biologic mechanisms, and safety of this treatment modality in a murine model. Additional clinical trials include the prospective use of clinical data, serum and wound biomarkers, and wound gene expression profiles to predict wound healing/failure and additional clinical patient outcomes following combat-related trauma. These clinical research data are analyzed using machine-based learning algorithms to develop predictive treatment models to guide clinical decision-making. Future CWI directions include additional clinical trials and study centers and the refinement and deployment of our genetically driven, personalized medicine initiative to provide patient-specific care across multiple medical disciplines, with an emphasis on combat casualty care.

Inflammatory biomarkers in combat wound healing.

BACKGROUND: Modern war ballistics and blast injuries inflict devastating extremity injuries, violating soft tissue, bone, and neurovascular structures. Despite advances in complex wound management, appropriate timing of war wound closure remains subjective. In addition, the pathophysiology of acute wound failure is poorly defined. METHODS: Patients with penetrating extremity wounds sustained during combat were prospectively studied and followed for 30 days after definitive wound closure. The primary outcome was wound healing. Wound dehiscence was defined as spontaneous partial or complete wound disruption after closure. Serum, wound effluent, and wound bed tissue biopsy were collected at each surgical wound debridement. Serum and wound effluent were analyzed with a multiplex array of 22 cytokines and chemokines, and wound tissue for corresponding gene transcript expression. RESULTS: Fifty-two penetrating extremity war wounds in 33 male patients were investigated. Nine (17%) wounds dehisced. Concomitant vascular injury, increased wound size, and higher injury severity score correlated with wound dehiscence. Both serum and wound effluent cytokine and chemokine protein profiles were statistically associated with healing outcome at various time points. Wound biopsy gene transcript expression demonstrated increased tissue inflammation associated with wound failure. Multiple protein and gene transcript biomarkers predictive of wound healing were identified. CONCLUSIONS: The cytokine and chemokine protein and gene transcript expression patterns demonstrate a condition of inflammatory dysregulation associated with war wound failure. A molecular biomarker panel may predict combat wound healing outcome and warrants prospective validation.

Perioperative blood transfusion in combat casualties: a pilot study.

BACKGROUND: In recent studies, blood transfusion has been shown to increase the rate of wound healing disturbances in orthopedic patients. Furthermore, our group has determined a correlation between delayed wound healing and elevations in inflammatory mediators in combat casualties. Therefore, we sought to determine the effect of blood transfusion on wound healing and inflammatory mediator release in combat casualties. METHODS: Prospective data were collected on 20 severely injured combat casualties sustaining extremity wounds. Patients were admitted to the National Naval Medical Center during a 13-month period from January 2007 to January 2008. Data variables included age, gender, Glasgow coma score (GCS), mechanism of injury, and transfusion history. Injury severity was assessed using the Injury Severity Score (ISS). Serum was collected initially and before each surgical wound debridement and analyzed using a panel of 21 cytokines and chemokines. The association between blood transfusion and wound healing, incidence of perioperative infection, intensive care unit (ICU) admission rate, and ICU and hospital length of stay was assessed. Differences were considered significant when p < 0.05. RESULTS: The study cohort had a mean age of 22 +/- 1, a mean ISS of 15.8 +/- 2.6, and a mean GCS 13.9 +/- 0.6; all were men and suffered penetrating injuries (90% improvised explosive device [IED] and 10% gunshot wound [GSW]). The cohort was divided into two groups. Patients receiving 4 units of blood initially (group 2, n = 9). There was no significant difference in age, ISS, GCS, or mortality between the two groups. However, group 2 patients had significant impairment in wound healing rate (54% vs. 9%, p < 0.05), higher ICU admission rate (78% vs. 9%, p < 0.01), perioperative infection rate (89% vs. 27%, p < 0.01), and a longer hospital length of stay (49.9 +/- 12.8 vs. 23.8 +/- 2.9, p < 0.05) compared with group 1 patients. In addition, there was a significant correlation between the initial mean serum cytokine/chemokine level of interleukin (IL)-10, IL-8, interferon inducible protein (IP)-10, IL-6, and IL-12p40 and the number of units of blood transfused (p < 0.05). CONCLUSION: Allogeneic blood transfusions in combat casualties were associated with impaired wound healing, increased perioperative infection rate, and resource utilization. In addition, the extent of blood transfusion was associated with significant differences in inflammatory chemokine and cytokine release.

Lymphocyte modulation with FTY720 improves hemorrhagic shock survival in swine.

The inflammatory response to severe traumatic injury results in significant morbidity and mortality. Lymphocytes have recently been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury. Experimental manipulation of lymphocytes following hemorrhagic shock may prevent secondary immunologic injury in surgical and trauma patients. The objective of this study is to evaluate the lymphocyte sequestration agent FTY720 as an immunomodulator following experimental hemorrhagic shock in a swine liver injury model. Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte sequestration agent, FTY720, (n = 9) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period after hemorrhage. Circulating total leukocyte and neutrophil counts were measured. Central lymphocytes were evaluated with mesenteric lymph node and spleen immunohistochemistry (IHC) staining for CD3. Lung tissue infiltrating neutrophils were analyzed with myeloperoxidase (MPO) IHC staining. Relevant immune-related gene expression from liver tissue was quantified using RT-PCR. The overall survival was 22.2% in the vehicle control and 66.7% in the FTY720 groups (p = 0.081), and reperfusion survival (period after hemorrhage) was 25% in the vehicle control and 75% in the FTY720 groups (p = 0.047). CD3(+) lymphocytes were significantly increased in mesenteric lymph nodes and spleen in the FTY720 group compared to vehicle control, indicating central lymphocyte sequestration. Lymphocyte disruption significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver immune-related gene expression in the FTY720 treated group. There were no observed infectious or wound healing complications. Lymphocyte sequestration with FTY720 improves survival in experimental hemorrhagic shock using a porcine liver injury model. These results support a novel and clinically relevant lymphocyte immunomodulation strategy to ameliorate secondary immune injury in hemorrhagic shock.

Lymphocyte depletion in experimental hemorrhagic shock in Swine.

BACKGROUND: Hemorrhagic shock results in systemic activation of the immune system and leads to ischemia-reperfusion injury. Lymphocytes have been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury, and immunomodulation of lymphocytes may prevent secondary immunologic injury in surgical and trauma patients. METHODS: Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte depletional agent, porcine polyclonal anti-thymocyte globulin (PATG) (n = 8) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period. Circulating lymphocytes were examined with FACS analysis for CD3/CD4/CD8, and central lymphocytes with mesenteric lymph node and spleen staining for CD3. Circulating and lung tissue16 infiltrating neutrophils were measured. Circulating CD3 lymphocytes in the blood and in central lymphoid organs (spleen/lymph node) were stained and evaluated using FACS analysis. Immune-related gene expression from liver tissue was quantified using RT-PCR. RESULTS: The overall survival was 22% (2/9) in the control and 75% (6/8) in the PATG groups, p = 0.09; during the reperfusion period (following hemorrhage) survival was 25% (2/8) in the control and 100% (6/6) in the PATG groups, p = 0.008. Mean blood loss and hemodynamic profiles were not significantly different between the experimental and control groups. Circulating CD3+CD4+ lymphocytes were significantly depleted in the PATG group compared to control. Lymphocyte depletion in the setting of hemorrhagic shock also significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver ischemia gene expression. CONCLUSIONS: Lymphocyte manipulation with a depletional (PATG) strategy improves reperfusion survival in experimental hemorrhagic shock using a porcine liver injury model. This proof of principle study paves the way for further development of immunomodulation approaches to ameliorate secondary immune injury following hemorrhagic shock.