by Jacob Bastacky, M.D., Adjunct Research Scientist, Children's Hospital Oakland Research Institute
Editor's Note: For more than a decade, we have known that the excessive mucus produced in cystic fibrosis lungs, pancreas and elsewhere is caused by an alteration in the fluid layer of these organs. This fluid, and how it is both distributed and disturbed, is the focus of a study by Dr. Jacob Bastacky of the University of California, Berkeley. His research involves the use of a scanning electron microscope, combined with x-ray microanalysis, to determine the ion and mucin concentrations in airway surface liquid. Dr. Bastacky was awarded a one-year grant from Cystic Fibrosis Research, Inc. last fall and is still in the process of conducting his research.
People, cells and living things in general are composed mostly of water. The light
microscope allows us, as researchers, to look at hydrated specimens of lung. But for
higher magnification, for example to see the parts of cells, we use electron microscopes.
When we study these specimens with electron microscopes, we have to dry them out before
they are placed in a vacuum. Otherwise, the moisture would compromise the vacuum. However,
biological material is best studied when it is not dehydrated. Over a twenty-year period,
we have perfected a method of studying water in tissues.
Using electron microscopical tools that we developed, we are able to achieve greater accuracy in studying wet tissue, such as the cystic fibrosis lung. Where do we obtain our specimens? We have been collaborating with several cystic fibrosis researchers who provide us with animal tissue from experiments in which ion transport is studied, as well as from humans with cystic fibrosis undergoing lung transplantation. By cooling the sample lung and its water to low temperatures (that of liquid air: -196 C) and keeping it cold during observation in the microscope, we are able to more accurately measure the fluid layer of the specimens. The benefits from these conditions are that we maintain a good vacuum during our research, and we are able to take images of the surface of the lung with its water present for many hours.
When studying the liquid lining in the lungs, researchers have been unable to accurately measure this lining because the fluid layer in the lungs is so thin, it is relatively inaccessible, and it is subject to almost instant change by the methods designed to obtain specimens. However, our technique of low temperature scanning electron microscopy, combined with X-ray microanalysis of rapidly frozen specimens, has been successful in generating reliable data on the elemental composition of airway surface liquid.
In our study, we have been investigating a couple of characteristics of the cystic fibrosis tissue. We have been looking along the airway to determine whether the liquid lining is continuous, and we have also been measuring its thickness. Learning the characteristics of the liquid lining layer is important for clearing inhaled particles from the lung and for protecting the delicate cellular lining of the airways. Knowing its thickness in both health and disease helps us understand how this protective layer functions and suggests ways in which its dysfunction may be corrected. We have found that the lining layer continuously covers most of the alveolar wall, that it varies in thickness with pools and thin areas in the small airways, and that in the trachea (of a mouse), it appears continuous, though again, it varies in thickness. In part, this variation in normal layer thickness is due to folding of the underlying layer of cells lining the airway. With continued research, we expect to learn more about the local patterns of airway surface liquid thickness which, we hope, will lead to a greater understanding of how cystic fibrosis affects the lungs. The ultimate goal of this study is to compare concentrations of ions in both normal and cystic fibrosis airway surface liquid, and to determine if and how mucin concentration of airway surface liquid is altered in cystic fibrosis.
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