Lymphocyte depletion in experimental hemorrhagic shock in Swine

Abstract

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.

Figure 1

Experimental design. A) Liver injury was initiated at t = 0. Uncontrolled hemorrhage occurred until 1 hour, at which time the abdomen was packed and temporarily closed (pre-hospital phase). The animal was observed until 2 hours when hospital care was initiated. The liver was repaired and the abdomen definitively closed. The animal was then observed for a total of 72 hours. Blood transfusion was administered as indicated. Necropsy was performed when the animal expired or at 72 hours following euthanasia. The uncontrolled hemorrhage and pre-hospital phases were considered the hemorrhage period, while the hospital care phase was considered the reperfusion period. B) In the PATG experimental group, 4 doses of PATG (10 mg/kg) were administered starting 96 hours prior to liver injury.

Figure 2

Hemodynamic profiles during hemorrhage period for experimental and control groups. A) Heart rate. B) Mean arterial pressure. C) Cardiac output.

Figure 3

Kaplan-Meier survival curve for experimental groups versus control with log-rank test for statistical comparison. A) Overall survival. B) Reperfusion period survival. Dashed red line indicates end of hemorrhage and beginning of reperfusion period.

Figure 4

Peripheral lymphocyte counts during hemorrhage and reperfusion periods. One-way ANOVA with repeated measures design was used for statistical comparison. Error bars represent ± SEM.

Figure 5

CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocyte counts were quantified throughout hemorrhage and reperfusion A) Quantification of CD3⁺CD4⁺ and B) CD3⁺CD8⁺ lymphocytes throughout the experiment. One-way ANOVA with repeated measures design was used for statistical comparison. Error bars represent ± SEM.

Figure 6

Central lymphocyte counts at time of necropsy. A) Representative immunohistochemistry images ofanti- CD3 stains for normal, control, and PATG tissue. B) Quantification of CD3⁺ reactivity by automated cellular Imaging. * indicates p < 0.05 compared to normal, unmanipulated tissue. Data is depicted as mean ± SEM.

Figure 7

Peripheral neutrophil counts during hemorrhage and reperfusion periods. One-way ANOVA with repeated measures design was used for statistical comparison. Error bars represent ± SEM.

Figure 8

Lung tissue neutrophil counts at time of necropsy.A) Representative histology pictures of normal, control and PATG tissue. B) Quantification of MPO⁺ cell reactivity. * indicates p < 0.05 compared to normal, unmanipulated tissue. † indicates p < 0.05 compared to control group. Data is depicted as mean ± SEM.

Figure 9

Gene transcript expression in liver tissue of PATG relative to the control group. Median value of relative fold expression is depicted on a logarithmic scale. >2 fold difference from control was considered statistically significant and is indicated by *.