V. A. Dolgachev1, S. V. Madathilparambil1, B. Thomas1, M. R. Hemmila1, K. Raghavendran1, D. Machado-Aranda1 1University Of Michigan,Acute Care Surgery,Ann Arbor, MI, USA
Introduction: Lung Contusion (LC) and Bacterial Pneumonia (PNA) are common in the critically ill trauma patients and in combination can be fatal. The emergence of multidrug resistant organisms has mitigated the efficacy of many antibiotics. Recent genomic studies have shown FER, a cytosolic non-receptor tyrosine kinase of unknown role in the lung, with increased gene expression among survivors from sepsis and pneumonia. Our hypothesis is that using electroporation-mediated (EP) delivery of the FER gene we can improve survival in a murine model of combined LC and PNA.
Methods: C57Bl/6 mice were subject to a lethal model of unilateral LC followed by intratracheal inoculation of 500 CFU of Klebsiella sp. at 6 hrs. In the treatment group, plasmid DNA encoding FER (pFER) was provided via hypopharyngeal instillation right after LC injury (time 0.2 hr). External electrodes were placed under each forelimb and 8 square wave electroporation pulses were delivered. Two control groups were used: one receiving Na+/K+-ATPase genes (shown to be benefitial in LC injury model) in similar LC+PNA injury conditions followed by EP (pPump); and a second with no gene delivery (LC + PNA/no EP). 7-day survival curves were recorded. In a parallel experiment, animals were euthanized at 24 hrs post-LC, lung mechanics were measured and bronchial alveolar lavage fluid (BAL), blood and lung tissue were collected. BAL cellular subpopulations were immunotypified by flow cytometry. BAL cytokines were assayed using ELISA kits for IL-1α, IL-6, IL-10, IL-33, MCP-1 and MIP-2. Quantitative bacterial counts were determined by serial dilution and plating of blood and lung homogenates.
Results: 7-day survival was markedly improved by pFER treatment compared to positive (pPump) and negative-controls (LC+PNA/no EP). Better lung compliance was found in pFER animals at 24 hr post-LC. At this same timepoint, pFER treatment group showed a robust inflammatory response with absolute increases in BAL monocytes, neutrophils and macrophages. In contrast, BAL of pPump treated animals was macrophage-predominant while negative-controls had a relative lower total of inflammatory cells, especially of macrophages, compared to the other groups. pFER treatment diminished levels of early response pro-inflammatory cytokines IL-1β, IL-6, MCP-1 and MIP-2, whereas increases were seen in modulation-cytokines IL-10, IL-33 and TNF-α. Lung bacterial counts were significantly lower in pFER (ANOVA p=0.023) with a trend towards better systemic containment.
Conclusion: EP-mediated delivery of the FER gene significantly improved mouse survival in a combination model of lung contusion and pneumonia. Better containment of bacteria within the lungs was possibly mediated by alterations in the immune cell and cytokine response.
