weighted more heavily than smaller ones. We stress that D1 and D2 do not provide conventional 3D stereological information about average airspace dimensions they simply emphasize the presence of a minority of enlarged airspaces. Nevertheless, as observed in Ref., these new indexes may prove useful as indicators of physiology MCE Company Safflower Yellow expected in early or mild emphysema but require rigorous validation. Herein, as a validation effort, we have applied these indexes post factum to a study of airspace enlargement in smoke-exposed mice and compared the results to conventional Lm measurements on the same histological images. Lung tissue samples from 20 female AKR/J mice were used in this study, with 10 exposed to mainstream cigarette smoke for 24 weeks and 10 age-matched controls, as described previously. At the end of smoke exposure, the mice were sacrificed by CO2 asphyxiation and exsanguinated. Next, the chest cavity was opened and the diaphragm incised. Lungs were then inflated to neutral buffered formalin for minutes, after which the trachea was tied off and the lungs excised and placed in a formalin bath for days. After fixation, lungs were trimmed and randomly oriented in preparation for sectioning. Lungs were embedded with paraffin and sectioned into 5 mm thick slices that were MDL28574 stained with hematoxylin and eosin. Slices were made in random directions, and eight random slices selected from all lobes of each mouse were placed on a slide. We note that this random method will result in some lobes being sampled multiple times, and the possibility exists that some lobes will avoid sampling altogether. Slices were then imaged at 2006magnification using a Nikon Optishot II microscope and Zeiss Axiocam digital camera; 12 images per mouse were acquired. Image locations were selected by using a random number generator to determine image coordinates. Major airways and vasculature were generally avoided in selecting fields to focus on peripheral parenchyma, as reported by others. When one of these was encountered, the microscope field was shifted in a randomly selected direction until the field included parenchymal tissue only. Digital images were 6066480 pixels and covered a field of approximately. Figure 1 shows representative H&E stained images from control and smoke-exposed mice, with color maps included to aid the eye in distinguishing airspaces. We note that gross examinations of morphometry of all healthy vs. smoke-exposed mice were insufficient for definitively deter
