Modulating the Biologic Activity of Mesenteric Lymph after Traumatic Shock Decreases Systemic Inflammation and End Organ Injury

Abstract

Introduction: Trauma/hemorrhagic shock (T/HS) causes the release of pro-inflammatory mediators into the mesenteric lymph (ML), triggering a systemic inflammatory response and acute lung injury (ALI). Direct and pharmacologic vagal nerve stimulation prevents gut barrier failure and alters the biologic activity of ML after injury. We hypothesize that treatment with a pharmacologic vagal agonist after T/HS would attenuate the biologic activity of ML and prevent ALI.

Methods: ML was collected from male Sprague-Dawley rats after T/HS, trauma-sham shock (T/SS) or T/HS with administration of the pharmacologic vagal agonist CPSI-121. ML samples from each experimental group were injected into naïve mice to assess biologic activity. Blood samples were analyzed for changes in STAT3 phosphorylation (pSTAT3). Lung injury was characterized by histology, permeability and immune cell recruitment.

Results: T/HS lymph injected in naïve mice caused a systemic inflammatory response characterized by hypotension and increased circulating monocyte pSTAT3 activity. Injection of T/HS lymph also resulted in ALI, confirmed by histology, lung permeability and increased recruitment of pulmonary macrophages and neutrophils to lung parenchyma. CPSI-121 attenuated T/HS lymph-induced systemic inflammatory response and ALI with stable hemodynamics and similar monocyte pSTAT3 levels, lung histology, lung permeability and lung immune cell recruitment compared to animals injected with lymph from T/SS.

Conclusion: Treatment with CPSI-121 after T/HS attenuated the biologic activity of the ML and decreased ALI. Given the superior clinical feasibility of utilizing a pharmacologic approach to vagal nerve stimulation, CPSI-121 is a potential treatment strategy to limit end organ dysfunction after injury.

Fig 1. CPSI-121 prevents the systemic inflammatory response to T/HS.

Mean arterial pressure ranged from 52–55 mmHg throughout the final hour of T/HS lymph infusion, which was statistically lower than MAP of sham mice or mice subjected to T/SS (64–68 and 66–69 mmHg, respectively, p<0.05) (A). T/HS + CPSI-121 derived lymph maintained MAP throughout lymph infusion with MAP readings similar to sham and T/SS (66-73mmHg, p = 0.79 versus sham). Flow cytometry histogram comparing monocyte STAT3 phosphorylation (pSTAT3) before (blue) and after lymph infusion (red) (B). T/HS lymph resulted in a 2.3 ± 0.47 fold increase in pSTAT3 fluorescence, which was significantly higher than the pSTAT3 fold changes after sham or T/SS lymph infusion (1.0 ± 0.31 and 1.43 ± 0.12, respectively, p<0.05). T/HS + CPSI-121 lymph resulted in 1.44 ± 0.31 fold increase in pSTAT3 fluorescence, which was not statistically different from sham (p = 0.102) (C).

Fig 2. Acute Lung Injury Develops After T/HS Lymph Infusion and Is Attenuated by CPSI-121.

Acute lung injury (ALI) is present on lung histology after T/HS lymph infusion as demonstrated by alveolar hemorrhage (open arrow) and thickened hyaline membrane (closed arrow) (A). Histology from sham animals had normal histologic features, with thin alveolar walls free from cellular infiltrate. T/SS and T/HS + CPSI-121 infusion resulted in similar findings on histology with mild airway edema and cellular infiltration compared to sham, but attenuated compared to T/HS. Pulmonary injury score was significantly higher after T/HS lymph infusion (9.17 ± 0.6) compared to sham (2.83 ± 0.65, p = 0.0001), T/SS (3.83 ± 0.65, p = 0.0001) or T/HS + CPSI-121 (4.33 ± 0.6, p = 0.0022) lymph infusion (B). Lung permeability, another marker of ALI, was significantly elevated in animals subjected to T/HS derived lymph compared to animals injected with sham, T/SS or T/HS + CPSI-121 derived lymph. Average wet:dry ratio was 4.21 ± 0.271 in the T/HS group compared to 1.683 ± 0.531, 2.738 ± 0.533 and 1.362 ± 0.786 in the sham, T/SS and T/HS groups, respectively (C). Similarly, Evan’s Blue Dye absorbance was significance higher in the T/HS group (2.305 ± 0.69) compared to sham (0.449 ± 0.33), T/SS (1.051 ± 0.49) and T/HS + CPSI-121 (1.004 0078 0.136) (D).

Fig 3. Lung Macrophages Increase in Number Following T/HS Lymph Infusion.

T/HS lymph infusion resulted in increased amount of pulmonary macrophages present on flow cytometry (4.39% ± 0.75) compared to sham (1.41% ± 0.49, p = 0.026) and T/SS infusion (1.99% ± 0.51, p = 0.037) (A and B). T/HS + CPSI-121 lymph attenuated the pulmonary macrophage increase with quantity similar to T/SS (1.46% ± 0.27).

Fig 4. Immune Cell Infiltration of Lungs Following T/HS Lymph Infusion.

An increase in lung macrophages was present on immunohistochemistry (IHC) following T/HS lymph infusion as demonstrated by CD68 staining (A). T/HS lymph infusion resulted in an average of 19.1 CD68+ cells/high power field (HPF) compared to 4.8 (p = 0.0329) and 5.8 (p = 0.0418) cells/HPF in sham and T/SS, respectively (B). T/HS + CPSI-121 lymph had similar CD68+ cells/HPF to T/SS (4.9). Neutrophils were also present in increased numbers on IHC in lung samples followings T/HS lymph infusion (10.9 MPO+ cells/HPF) (C) compared to sham (1.7 MPO+ cells/HPF, p = 0.0117) (D). Neutrophil numbers were also increased in T/HS lymph infusion compared to T/HS + CPSI-121 (4.6 MPO+ cells/HPF, p = 0.0139).