Introduction: The presence of an endotracheal tube (ETT) increases the risk for microaspiration of secretions around the ETT. Biomarkers of pepsin and salivary amylase may be used to identify microaspiration in intubated patients because of their naturally occurring presence in the stomach or oral cavity and non-occurrence in the respiratory tract. Microaspiration may be difficult to detect until pulmonary complications, such as lung injury or infection, occur. This study assessed the presence of pepsin and salivary amylase in oral and tracheal secretions of ventilated adults. Method(s): This is a secondary analysis of data collected from 11 critically ill, adult patients on mechanical ventilation (MV) enrolled in a study to identify cues for ETT suctioning. Two paired samples of oral and tracheal secretions were suctioned when clinically indicated. Tracheal secretions were suctioned with a closed system, and oral secretions were obtained with an oropharyngeal catheter. Specimens were analyzed for total pepsin, pepsin A, pepsin C, and salivary amylase according to established assays. Results: Of 11 subjects, the majority were men (n=8), on enteral feedings (n=9) via a feeding tube placed in the stomach (n=7), and intubated with a continuous subglottic suction ETT (n=8). Median values: age, 62 years; duration of MV, 5.5 days; ETT cuff pressure 24 cm H2O; head of bed, 30degrees]. Pepsin was in measured in both oral (30.5 ng/mL; n=8) and tracheal secretions (11.1 ng/mL; n=7); Similar findings were noted for pepsin A (oral 14.7 ng/mL, n=7; sputum 7.4 ng/mL, n=6) and pepsin C (oral 14.7, n=7; tracheal 7.4, n=6). Salivary amylase (mean micro]mol/min/mL) was present in all oral secretions (359.8) and in the sputum of 6 subjects (1.8). Discussion & Conclusions: The majority of intubated patients on MV had both pepsin and salivary amylase in their sputum, likely due to microaspiration of secretions.; This finding suggests greater efforts are needed to reduce patients' risk. Ongoing strategies to prevent gastric reflux are important such as head of bed elevation and monitoring residuals. Presence of salivary amylase within tracheal secretions may indicate a need for more frequent oropharyngeal suctioning as part of routine care of intubated patients. Analysis shows no variations of the presence of pepsin or salivary amylase in relation to feeding tube placement or type of ETT. Generalizability is limited by the small sample size.

This study investigated the development potential of equine embryos in vitro after biopsy and vitrification. Twenty embryos were obtained from Quarter Horse, Thoroughbred, and mix-breed light mares between three and ten years old. The twenty embryos were divided into a biopsy (n=10) and control group (n=10). The biopsy group underwent microaspiration biopsy using a micromanipulator to obtain a small tissue sample from the embryo. Both groups were then vitrified using a commercially available technique originally described by Carnevale (2006) at Colorado State \ University. All 20 embryos were cultured in DMEM/Hams F-12 medium under oil at 37°C in 5% CO2 in air (Hinrichs et al., 1990). Embryos were monitored for expansion and hatching. Embryo development was statistically different between the two groups (p<0.05). The biopsy procedure did result in a much lower development potential in the biopsy group as compared to the control group (20% vs. 80%). However, embryos in the biopsy group did show expansion and hatching therefore the combined procedure did not preclude development potential in vitro. Based on these findings, more research needs to be done to increase the success of the combined procedure and the ultimate viability of the embryos needs to be confirmed with the establishment of successful pregnancies.

This thesis examines the dynamics of the formation of tethers, which are tubes of lipids 20 - 200 nm in diameter. In particular, this work investigates how the loading rate affects the observed threshold force at which a tether forms from a vesicle membrane. Tether dynamics are important to a myriad of biological processes such as signaling when white blood cells adhere to the walls of healthy and diseased blood vessels, or in the transport of intracellular material between neighboring cells. To understand the dynamics of tether formation in such systems more fully, the studies presented in this thesis focus on the dependence of the force needed to create a tether on the rate of force change. To conduct these experiments, I combined, for the first time, a microfabricated magnetic force transducer, or a microscale device that generates well-controlled and localized magnetic fields, and microaspiration, a technique to apply known tension to a lipid membrane. Using the combined global and local mechanical control of the joint system, I discovered a strong correlation between the threshold force of tether formation and the applied force ramp. An energy model, based upon that used to describe membrane rupture, characterized the observed dependencies and provided a mechanism to examine physically relevant quantities within the system. The usefulness of this combined approach was further substantiated by determining the influence of membrane modulators, including cholesterol, tension, adhesion site concentration, and phosphatidylserine, on the dependence seen between force threshold and force rates. Additionally, application of the experimental technique developed in this thesis led to the calculation of the inter-layer drag coefficient between membrane leaflets and to the first measurements of the thermal expansivity in aspirated 1-stearoy1-2-oleoyl- sn -glycero-3-phosphatidylcholine vesicles. This new tool for dynamic studies of membrane mechanics may further be extended to study how tethers form off of flowing cells or how phase regimes, induced by the presence of cholesterol, influence membrane dynamics.

Traumatic Brain Injury (TBI) accounts for 1,000,000 hospital admissions in the European Union every year and is the leading cause of death in individuals under 45 years of age in both Europe and the United States. This thesis examines the consequences to both the brain and lung following TBI using the lateral fluid percussion injury (FPI) in an in-vivo murine model. In the murine FPI model, alongside cerebral inflammation (associated with neuronal damage and the infiltration of inflammatory cells), there is significant neutrophil accumulation within the pulmonary interstitium 6 and 24 hours after TBI. This was associated with pulmonary haemorrhage and increased vascular permeability. In an attempt to reduce pulmonary injury, 17-DMAG, an HSP90 inhibitor, was applied but proved to be nonprotective. Since patients with TBI show increased susceptibility to bacterial infection, microaspiration and ventilator-induced lung injury, a double-hit model was established whereby mice first received the head injury and then received a lung injury. This demonstrated worse lung injury following intra-tracheal administration of hydrochloric acid after TBI. Depleting neutrophils with an anti-LY-6G depleting antibody improved outcome in this model, indicating increased susceptibility to damage was neutrophil dependent. To test whether neutrophil accumulation within the pulmonary interstitium was specifically related to brain injury, lung tissue following other distant organ injury such as renal ischemia-reperfusion injury (IRI) and renal transplantation were assessed. Significant pulmonary interstitial neutrophil accumulation was seen following both models and was associated with significant pulmonary haemorrhage. Inducing HSP70 activity with an HSP90 inhibitor was shown to be protective by reducing the degree of pulmonary haemorrhage in these models. In an attempt to identify the mechanisms behind neutrophil accumulation in TBI, renal IRI and renal transplantation, ICAM-1 (CD54), a marker of the reverse transmigration of neutrophils was investigated. No differences in ICAM-1 expression were seen following TBI, indicating that another mechanism must be responsible. This mechanism is the focus of on going work within the laboratory. Hypoxia is believed to contribute towards the development of secondary brain injury however little is known regarding its direct contribution. Working alongside chemists at the University of Edinburgh, a number of novel fluorescent hypoxia probes were designed and tested, but none proved to be able to detect hypoxia in-vitro. In conclusion, this thesis has demonstrated that following mild TBI, the lungs are “primed” with a massive interstitial neutrophil influx and that a subsequent micro aspiration of acid induces exaggerated lung injury. The mechanism by which this occurs is the focus of on-going investigation. Pulmonary sequestration of neutrophils is also a predominant feature of other distant organ injuries.