Cystic Fibrosis: A Rheological Catastrophe CISMM: Center for Computer Integrated Systems for Microscopy and Manipulation Collaborators: Bill Davis 2, Ric Boucher 2, Garrett Matthews 2,3 Project Director: Rich Superfine 1 Investigators: David Hill 1, Jeremy Cribb 1, Russell Taylor 1 December 2003 Cystic Fibrosis (CF) is a disease most often characterized by the accumulation of mucus over the lung airway epithelium. In healthy lung tissue mucus is transported continuously by the cilia to the glottis where it is expelled from the airways thereby removing adsorbed pathogens. The pathogenesis of CF airway disease involves abnormalities in this mucociliary clearance system; an increase in the concentration of mucin molecules in the patient’s airway surface liquid (ASL) causes a disruption of the mucus transport mechanism. This causes an increase in the viscoelastic properties of the mucus layer which leads to the establishment of bacterial colonies, elicits an inflammatory response, and degrades the surrounding tissue. Development of treatments to restore the flow of mucus out of the respiratory tract requires knowledge of the rheological properties of mucus at various concentrations. Viscous Mucus Stops Bacterial Clearance Microbead Rheology We can measure viscoelastic parameters, such as storage and loss shear moduli (denoted by G’(ω) and G’’(ω) respectively) by tracking the Brownian motion of micron-sized particles. The position versus time data of the particle is used to calculate its mean squared displacement,  r 2 (  ). From the mean squared displacement, the two dimensional complex viscoelastic modulus, G * (  ) is calculated and resolved into storage (G”) and loss (G’) components: The complex viscosity of the material is then calculated from the complex viscoelastic modulus: What’s Next? Microrheology in living cell-cultures: We plan to perform rheology experiments in the mucus and PCL layers of living lung-airway epithelial cells with beating cilia. Driven particle microrheology: By applying forces to a paramagnetic bead in the 3D force microscope, we can cause a measurable response in a field of non-magnetic beads. This would allow us to collect data at many points simultaneously in a heterogeneous sample. Two particle microrheology: Through high-speed video tracking, we will be able to reduce the impact of the bead on its rheological environment. Conclusion: Mucus is Elastic A 30 sec 2D trace of bead’s Brownian displacement. Histogram characteristic of Brownian motion in an elastic fluid Storage and loss moduli of 8% and 1.5% mucus Storage viscosity of several beads in 8% mucus, demonstrating heterogeneity CF and Lubricating Layer Collapse Microrheology of complex fluids such as mucus provide accurate measurements of the local viscoelastic properties with small sample volumes. By collecting data in multiple areas of the fluid sample, the heterogeneity of the fluid can be quantified. Normal CF 7  m PCL Mucus and PCL The Cystic Fibrosis Phenotype: Confocal microscopy images illustrating the difference in the height of the ASL in normal lung epithelium and in that of cystic fibrosis tissue. Water (marked with GFP labeled dextran) is adsorbed from the PCL. This concentrates the mucus (marked with red microspheres) and causes the height of the PCL to fall from ~7  m to ~3  m. This brings the concentrated mucus into contact with the cilia. Ciliary beat is stopped, and removal of mucus (and the pathogens against which it is intended to protect the tissue) is prevented. (Cell 95, , 1998) The airway surface liquid (ASL) is comprised of mucus, the periciliary layer (PCL) where the cilia beat, and the glycocalyx coating the cell surface. The rheology of each region can give valuable insight into the CF phenotype. Within each ASL region of cultured cells we will use the 3DFM to track the Brownian motion of sub-micron sized beads (blue, red, and green circles in the schematic below). From this data the viscoelastic moduli will be determined (see below). We will compare these in situ results with rheological measurements of mucin solutions with varying mucin concentration, varying pH, and mono- and divalent salt concentrations. Microrheology in Cell Cultures model bead mucin molecule 1 μm 1 UNC Physics & Astronomy, 2 UNC Cystic Fibrosis Center, 3 Univ. of South Florida Conclusions: Mucus is a heterogeneous fluid that’s rheological properties indicate a high degree of elasticity, which is dependent on the concentration of mucin molecules in solution.