Oxygenator Is the Main Responsible for Leukocyte Activation in Experimental Model of Extracorporeal Circulation: A Cautionary Tale

Alessio Rungatscher, Maddalena Tessari, Chiara Stranieri, Erika Solani, Daniele Linardi, Elisabetta Milani, Alessio Montresor, Flavia Merigo, Beatrice Salvetti, Tiziano Menon, Giuseppe Faggian, Alessio Rungatscher, Maddalena Tessari, Chiara Stranieri, Erika Solani, Daniele Linardi, Elisabetta Milani, Alessio Montresor, Flavia Merigo, Beatrice Salvetti, Tiziano Menon, Giuseppe Faggian

Abstract

In order to assess mechanisms underlying inflammatory activation during extracorporeal circulation (ECC), several small animal models of ECC have been proposed recently. The majority of them are based on home-made, nonstandardized, and hardly reproducible oxygenators. The present study has generated fundamental information on the role of oxygenator of ECC in activating inflammatory signaling pathways on leukocytes, leading to systemic inflammatory response, and organ dysfunction. The present results suggest that experimental animal models of ECC used in translational research on inflammatory response should be based on standardized, reproducible oxygenators with clinical characteristics.

Figures

Figure 1
Figure 1
P38 and NF-κB activation in leukocytes in response to ECC was significantly higher in presence of oxygenator. Peripheral blood samples were collected at baseline, before and after ECC. Leukocytes were fixed and permeabilized before intracellular staining using Alexa Fluor 568-conjugated antibodies that recognize (a) Thr180/Tyr182 phosphorylated p38 or (b) Ser529 phosphorylated NF-κB or with isotype-matched antibodies as a control. After lysis of red blood cells, fluorescence of granulocytes or mononuclear cells was quantified by flow-cytometry (after gating of cells by size and granularity). Mean fluorescence levels were calculated after subtracting values from isotype-control antibodies. Mean values are shown with standard deviations. ∗P < 0.05 between the two groups at the same time point.
Figure 2
Figure 2
Representative flow-cytometry analysis at baseline (a), after ECC without oxygenator (b) and after ECC with oxygenator (c). A significant depletion of mononuclear cells and granulocytes after ECC + oxygenator is evident.
Figure 3
Figure 3
Systemic inflammatory response assessed by TNF-α (a), neutrophil elastase (NE) (b), and IL-6 (c) levels in plasma. ∗P < 0.01 versus the other group at the same time point.
Figure 4
Figure 4
Lung injury. Representative histological images (magnification 40x) of right lung fixed in formaldehyde, sectioned, and stained with haematoxylin and eosin (a). The acute lung injury score was calculated (b). ∗P < 0.01.

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