VENTAJAS DE LA LEY MINERA

in the fixative for 48 hours.

A constant pressure apparatus was developed in the work­ shops of the Department and built by Terry Bull, Keith James and me after initial trials of fixative and pressure.

I found that a pressure head of 25cms H2O resulted in even filling of the lungs, without distension and this was con­ firmed by filling lungs inside the thoracic cage in a few trial animals. I required that the lungs should look as though they were fully inflated but not overdistended. This figure of 25cms is in accord with other workers (Gil 1990). The apparatus delivered the fixative through a soft poly­ thene catheter into the tracheas of the experimental ani­ mals.

This technique was used for both the rats and the blotchy mice (see chapter 3) although the fixatives differed, the rats being inflated with formol saline and the mice with Karnovsky's modified fixative. [ In the case of the smaller animals, especially very young mice, a paediatric cardiac catheter was used of the most narrow calibre I could obtain.] The tubing was tied in under direct view , if necessary using a dissecting microscope.

Tying the tube in was essential to obviate the possibility of leakage and subsequent deflation. [In the mice, where inflation was always carried out in situ, the approach to the trachea was through a sub-laryngeal tracheostomy after partial thyroidectomy and after 3 0 minutes in situ inflation the whole thoracic pluck was dissected free, before this the contents of the chest were kept moist by saline irrigation, fixative was not used at this point in order that there should not be premature fixation of the pleura at the ex­ pense of the fixation of the lung from the intratracheal

route.]

In the case of the rats the lungs were supported in a beaker of saline before the transference at thirty minutes to formol saline, and further fixation with the fixed head apparatus was continued for thirty minutes, then the tube was removed, the trachea tied off and fixation continued for a further 24 hours.

Processing and section cutting

The lungs were processed for conventional light microscopy using paraffin embedding [ or for scanning electron micros­ copy in the mice, see chapter 3.b]. The lungs were separated from the heart and mediastinum.

Sections for computerised analysis using light microscopy were cut at 5/xm thickness. Sagittal blocks through the entire lung were stained using haematoxylin and eosin after routine processing.

The fixation constant was recorded as 0.75 using the tech­ nique described in chapter 1. of recording change in size of a block, using a micrometer, after fixation and subsequent processing. The use of sagittal sections was selected after inspection of slices of lung at several levels and orienta­ tions were assessed to ascertain that measurements were feasible and would be comparable in all animals used.

Measurement

Although a point counting method was considered and pilot studies were undertaken I had already assessed some of the blotchy mouse data in this way (see chapter 3) and subse­ quently had elected to use a linear measurement and image analysis procedure to obtain measurements across the air­ spaces rather than assessing the internal surface area. I

had also established that I did want to assess measurements other than the mean linear intercept or points counted.

2. 3c

Method of morphometric analysis

Morphometric analysis of the rat lungs was undertaken using a purpose built Cambridge image analysis system. This study in 1982-85 preceded the use of other now more common quanti­ fying systems and was not fully automated. In particular, the image was generated by back projection rather than the digitised television image now routinely used. The analysis produced histograms of measurements of slices of rat lung made at x 180 . Figure 2.2

The images of the slices of lung were back projected on to a translucent glass tablet. A dedicated Leitz microscope was fixed upside down and the image from the straight eye­ piece tube usually used for photography was reflected via a 45° mirror on to the tablet. The magnification factor was X 180 for the rat lungs[ and x 300 for the mice lungs]. These values were calculated using the digitised tablet and an etched calibration slide.

A clear acetate sheet on to which a series of roughly paral­ lel guide lines had been drawn was placed as an onlay over the image and the pen for the digitised tablet was used to measure the length of those lines which fell across air­

spaces, excluding bronchi. The cursor pen, using the onlay sheet as a guide, was used to record a line across and airspace, avoiding any ciliated space. The length of the

Figure 2 .2 Diagrammatic representation of method of measure­

ment. Stage I. The slide is placed on to the stage of the

inverted light microscope. The image passes up through a vertical camera tube instead of through the eye pieces and is reflected via a 45 ^ mirror on to the rear of a digitised clear glass screen at a magnification of 180 (rats) or 300 (mice). Stage II Measurement. A transparent onlay with parallel lines is taped over the image on the glass and the electronic cursor pen is used to record the distance across individual airspaces (transect length) . The observer records each length by saving it into an electronic file on the computer which sorts the transect lengths into 10/x catego­ ries .