Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Our objective was to evaluate if changes in on-pump cerebral blood flow, relative to the pre-bypass baseline, are associated with the risk for postoperative delirium (POD) following cardiac surgery. In 47 consecutive adult patients, right middle cerebral artery blood flow velocity (MCAV) was assessed using transcranial Doppler sonography. Individual values, measured during cardiopulmonary bypass (CPB), were normalized to the pre-bypass baseline value and termed MCAV_rel. An MCAV_rel > 100% was defined as cerebral hyperperfusion. Prevalence of POD was assessed using the Confusion Assessment Method for the Intensive Care Unit. Overall prevalence of POD was 27%. In the subgroup without POD, 32% of patients had experienced relative cerebral hyperperfusion during CPB, compared to 67% in the subgroup with POD (p < 0.05). The mean averaged MCAV_rel was 90 (±21) % in the no-POD group vs. 112 (±32) % in the POD group (p < 0.05), and patients developing delirium experienced cerebral hyperperfusion during CPB for about 39 (±35) min, compared to 6 (±11) min in the group without POD (p < 0.001). In a subcohort with pre-bypass baseline MCAV (MCAV_bas) below the median MCAV_bas of the whole cohort, prevalence of POD was 17% when MCAV_rel during CPB was kept below 100%, but increased to 53% when these patients actually experienced relative cerebral hyperperfusion. Our results suggest a critical role for cerebral hyperperfusion in the pathogenesis of POD following on-pump open-heart surgery, recommending a more individualized hemodynamic management, especially in the population at risk.

Background: Unresolved inflammation resulting in capillary leakage with endothelial barrier dysfunction is a major contributor to postoperative morbidity and mortality after coronary artery bypass graft (CABG). Angiopoietins (ANGs) are vascular growth factors, also mediating inflammation and disruption of the endothelium, thus inducing capillary leakage. We hypothesized that changes in the relative serum levels of ANG1 and ANG2 influence endothelial barrier function and perioperative morbidity after CABG. Methods: After approval and informed consent, serum samples (n = 28) were collected pre CABG surgery, 1, 6, and 24 h after aortic de-clamping. ANG1, ANG2, soluble ANG receptor TIE2 (sTIE2), and IL-6 serum concentrations were analyzed by ELISA. Human pulmonary microvascular endothelial cells (HPMECs) were incubated with patient serum and FITC-dextran permeability was assessed. Furthermore, ANG2 secretion of HPMECs was analyzed after incubation with IL-6-containing patient serum. Results: CABG induced an early and sustained increase of ANG2/ANG1 ratio (5-fold after 24 h compared to pre-surgery). These changes correlated with elevated serum lactate levels, fluid balance, as well as the duration of mechanical ventilation. Permeability of HPMECs significantly increased after incubation with post-surgery serum showing a marked shift of ANG2/ANG1 balance (18-fold) compared to serum with a less pronounced increase (6-fold). Furthermore, CABG resulted in increased IL-6 serum content. Pre-incubation with serum containing high levels of IL-6 amplified the ANG2 secretion by HPMECs; however, this was not influenced by blocking IL-6. Conclusions: CABG affects the balance between ANG1 and ANG2 towards a dominance of the barrier-disruptive ANG2. Our data suggest that this ANG2/ANG1 imbalance contributes to an increased postoperative endothelial permeability, likewise being reflected by the clinical course. The results strongly suggest a biological effect of altered angiopoietin balance during cardiac surgery on endothelial permeability.

Background. Cytoreductive surgery (CS) in late-stage ovarian cancer patients is often challenging due to extensive volume shifts, and high fluid intake may provoke postoperative complications. Expression of vasoactive mediators is altered in cancer patients, which may affect systemic vascular function. We sought to assess how serum levels of vasoactive markers and mediators change during CS in ovarian cancer. Methods. Following IRB approval and informed consent, pre- and postoperative serum samples were analyzed in 26 late-stage ovarian cancer patients using multiplex protein arrays and ELISA. Results. The proinflammatory cytokines and chemokines IL-6, IL-8, and CCL2 were significantly elevated after 24hrs compared to the baseline values, with IL-6 and IL-8 being most prominently increased. While ANGPT1 remained unchanged after surgery, its competitive antagonist ANGPT2 was significantly increased. In contrast, serum levels of the ANGPT receptor TIE2 were decreased to 0.6 of the baseline values. While VEGF-D, E-selectin, P-selectin, ICAM-1, and PECAM-1 remained unchanged, serum activity of both thrombomodulin and syndecan-1 was significantly increased following surgery. Conclusion. We identified a regulatory network of acute-phase reaction during CS in late-stage ovarian cancer. This suggests that IL-6 exerts positive regulation of other proinflammatory mediators and, by upregulating ANGPT2 and suppressing ANGPT1, induces a serum profile that promotes vascular leakage. This may contribute to the observed hemodynamic alterations during CS procedures.