Actividad ATPasa.

Anaesthesia was induced in an anaesthetic gas chamber, with 5% isoflurane (Baxter Healthcare Ltd, UK) administered in a 30:70% oxygen:nitrous oxide mixture. The depth of anaesthesia was assessed using the withdrawal reflex,

where the footpad of a hind limb was tightly squeezed to evoke withdrawal of the foot. When the rat showed no reflex the animal was considered deeply anaesthetised and it was therefore appropriate to proceed with surgery. The withdrawal reflex was assessed regularly throughout the surgical period to determine if the anaesthetic dose required adjustment.

Once deeply anaesthetised the rat was immediately transferred to a designated area within the laboratory and the fur overlying any planned incision sites was removed using electric clippers (Wella, Germany) and cleared using a handheld vacuum (Black & Decker, USA). The rat was then transferred to the operating table and artificially ventilated via one of two methods: oral intubation or surgical tracheotomy.

Surgical tracheotomy

In experiments where rats were not recovered following surgery a tracheotomy was performed. Upon transfer to the operating table the rat was positioned on its back and a facemask was used to deliver isoflurane at a reduced dose of 2-3% in the same gaseous mixture as previously stated. The neck area, which was previously shaved, was cleaned with 70% alcohol. An incision was made through the skin and fascia of the neck using blunt ended scissors and the underlying musculature was divided by blunt dissection to expose the trachea. The connective tissue surrounding the trachea was carefully separated using small round forceps and two ligatures of 2-0 thread (Davis & Geck, UK) were threaded under the trachea and loosely tied around the proximal and distal ends of the exposed trachea, approximately 1.5cm apart. A small incision was made between rings of tracheal cartilage using microscissors (World Precision Instruments, UK) and a ventilation tube (Linton Instruments, UK) was quickly inserted into the trachea and advanced down the trachea, towards the bronchi, approximately 2cm. Prior to insertion of the ventilation tube, a ruler was used to measure 2cm from the bottom of the tube and this distance was marked on the tube using a permanent marker pen. When inserting the tube into the trachea, care was taken to ensure that it was not advanced beyond this mark as this could lead to obstruction of one of the bronchi. The ventilation tube was then quickly connected to the ventilator (Ugo Basile, Linton Instruments, UK) where the stroke volume was set to ~3ml at a frequency of 45 strokes per minute to maintain anaesthesia. The two ligatures at the proximal and distal

ends of the trachea were then tied firmly around the trachea and ventilation tube to hold it in place and the neck was stitched using 4-0 silk suture (SoftsilkTM_{, Covidien, USA).}

Oral Intubation

In experiments where rats were recovered following surgery oral intubation was performed for artificial ventilation using a simplified rat intubation method (Jou et al., 2000). Following the initial induction of anaesthesia, animals were transferred to a cork board. A loop of 4-0 silk thread (SoftsilkTM, Covidien, USA) attached to the board was placed around the incisors of the animal and used to lift the animal into a raised position at an approximate angle of 45°. A fibre optic light (Schott, USA) was positioned over the neck of the animal and the tongue was pulled to one side. An intubation wedge made from a 2ml plastic syringe (BD PlastipakTM, Spain, Figure 2.1C) was inserted into the mouth of the rat with the bottom of the wedge touching the rat’s tongue and the roof touching its palate. The wedge was fixed firmly in the oropharyngeal cavity by natural pressure from the incisors. The wedge was held in a thumb and index finger grip and the tip of the wedge was elevated causing expansion of the oropharyngeal cavity and allowing visualisation of the vocal cords and trachea. A 16 gauge catheter, 45mm in length with a 70mm long wire stylet (MillPledge Veterinary, UK) was used as the endotracheal tube. The 5mm long sharp tip of the stylet was cut off to produce a blunt ended guide wire (Figure 2.1A). The wings of the 16 gauge catheter were also removed to enable the catheter to pass through the intubation wedge (Figure 2.1B). Under illumination of the fibre optic light the catheter attached to the guide wire was directed into the trachea. The guide wire was removed and the catheter was then further advanced 1-2cm into the trachea. The intubation wedge was then carefully removed with the aid of a cotton bud to hold the catheter in place. The catheter was then connected to a ventilator at a stroke volume of 3-4ml and a frequency of 55-60 strokes per minute. The dose of isoflurane delivered to the ventilator was set to between 2.5 and 3% to maintain anaesthesia. Condensation in the intubation tube and chest expansion and collapse synchronous with the ventilator provided confirmation that the intubation tube was correctly inserted into the trachea. The intubation tube was then secured in place by stitching it to the side of the mouth using 4-0 silk suture.

Figure 2.1. Rat intubation kit. Composed of a guide wire made from a blunt-ended 70mm long wire stylet (A), a 16 gauge catheter (B) and an intubation wedge prepared from a 2ml syringe (C).