Rat experiment

i:n order to amplify ,investigate and quantitate this unex- piected finding in relation to the saline induced disease f'urther and to determine whether the change in air space dliameter in saline 'control instillations' was an evanescent phenomenon, a second group of experiments was designed to give single and repeated smaller, less haemorrhagic, doses, o>f elastase in order to produce alterations in air space transect length and to compare these results with saline instilled animals and untreated controls using both bioche- imical and morphologic analysis.

2 . 3a

Experimental protocol

The experiment was designed:

1. to examine the effects of elastase at 72 hours and 120 hours, to establish whether there was a transient or pro­ longed change in the distribution of air space size, the latter to be measured by documenting the transect length across airspaces.

2. to test whether such changes were produced by single or multiple doses of elastase, and whether effects were cumula­ tive.

3. to establish whether any effects on air space size were as a result of administration of exogenous elastase or whether they represented response to instillation alone.

4. To establish whether the urinary desmosine altered and if so, in what ways during the course of 72 and 120 hour experimental periods.

Method

Four-month old male Wistar rats, identified by ear punch numbering, were anaesthetised with halothane and oxygen using a Boyle's apparatus and 70% oxygen bubbled through halogene, to reach an eventual concentration of about 50%, within the chamber, so that oxygenation damage should not occur. This procedure renders an animal unconscious, with a slowed respiratory rate and relaxed epiglottis. The animals were given either three consecutive daily doses of 6 units of purified (porcine) pancreatic elastase (PPE) or a single bolus of 18 units.

Saline controls (three rats) of similar volume were also administered to paired rats in each elastase instilled group (see below). Three rats that had not undergone instil­ lation, but had been anaesthetised and handled were also used as controls.

Urinary desmosine was collected over twenty four hour periods for the period of the experiment, the animals being housed in North Kent (Dartford) metabolic cages specially designed for the collection of urine. The animals were all freely fed on stock diet and water. After collection, 12 mis of urine were freeze dried. The lyophilised sample was redissolved in hydrochloric acid and hydrolysed before analysis against controls using the radioimmunoassay of desmosine described further in the next chapter (3a.l2d). Reagents and the standard plot (Figure A . 1.1) are included

in appendix 1.#. but the results are displayed in Table 2.1 and discussed in parallel to the results obtained from the morphological analysis.

The animals were arranged in groups.

Group 1. a consisted of six rats which received a single bolus of 18 units of PPE [0.09mg in 0.5ml NaCl stored at 4°C] on day 1 and were killed on the 4th day at 72 hours after exposure.

Group l.b consisted of three rats which received saline on day 1 and were killed on the fourth day after 72 hours.

Group 2.a (also of six rats) received a bolus of 18 units of PPE on day 1 and were killed on the 6th day, 120 hours after exposure.

Group 2.b three rats received a saline bolus on day 1 and were killed at 120 hours after exposure, on the 6th day.

Group 3.a. (six rats ) received the triple dose ( 3 x 6 units PPE [0.27 mg]) as a single 6 unit instillation on the first three consecutive days (1, 2 and 3) and were then killed on day 6 after a maximum of 12 0 hours exposure to elastase (first instillation) and a minimum of 72 hours

(third instillation), whilst

Group 3.b. three rats received triple saline instillations and were killed on the sixth day.

Group 4. Three rats who were exposed once to anaesthetic and handled but not instilled were killed at 120 hours. No triple dose anaesthetics were given to untreated animals.

The rats were held in the left hand and the 500/il instilled with the neck fully extended. The procedure was swift, the animals were allowed to recover in their cages and handling was kept to a minimum.

[No attempt (i.e. by concomitant use of a non-irritant dye) was made to determine the route that the instillate had taken within the lung, but a bolus of 500/xl ought to be able to reach the lung surface before the mucociliary escalator has time to clear it and this volume represents a signif­ icant volume in relation to the rat lung, whilst not enough to cause damage akin to inhalation during drowning]

All animals were killed by progressive diminution of oxygen in inspired air and CO 2 asphyxiation.

The animals were immediately dissected and the lungs were exposed by incising the ventral cervical skin from the level of the chin to the point of the xiphisternum to allow access to the larynx and the thoracic inlet and the trachea was dissected free. The lungs collapsed when the ventral cres­ cent of the diaphragm was removed.

2.3b

Method of preparation of lungs for morphological analysis Fixation

After the initial dissection the rat lungs were infused with formol saline via a 16 gauge polythene tube. This cannula was tied into the cervical trachea and the lungs which had collapsed as the chest wall was opened, reinflated using a constant pressure apparatus filled with 10% formol saline at a pressure of 25cm H2O for one hour ( Figure 2.1). The thoracic pluck was then removed and suspended, after ligation of the trachea between the carina and the cannula.

Figure 2.1 Constant head apparatus for filling lung

The anaesthetised rat (or mouse in chapters 3 and 4) lies supine. A soft polythene tube inserted in through a tra­ cheostomy carries the formalin fixative (or Karnovsky fixa- tive in cha^s 3 and 4) into the lungs. The sternum and anterior ribs have been removed to allow complete filling and the pressure of the fixation is maintained by the head of fluid in the chamber which is clamped above the animal, having been calibrated to 25cms water.

25cm H2O calibrated

Figure 2.1