Surgical Methods & Physiological Control

The model was developed using male Sprague-Dawley rats weighing between 290g and 320g. Rats in this age range are young adults gaining weight primarily by adipose deposition. Due to the deep nature of the facial surgery, rats heavier than this range present increased surgical difficulty associated with the greater fat levels.

6.7.1 Anaesthesia & Monitoring

General anaesthesia was induced with a mixture of 3% halothane in a 40:60 O2/N2O gas

mixture, using a closed induction chamber. Once sedated, the animals were transferred to the NMR probe (Figure 6-13) where they remained for the duration of the preparation, surgery and NMR observation periods. Anaesthesia was maintained using 0.75 to 1.25 % halothane in the same oxygen-nitrous oxide mixture, delivered via a nose cone sited in the end of the probe. The halothane concentration was adjusted during the course of the experiment using heart rate, respiratory rate and temperature information as informants of the level of anaesthesia (discussed further at the end of this section). The rats breathed spontaneously throughout the experiment.

Heart rate was monitored continuously both on the bench during surgery and inside the magnet via subcutaneous ECG electrodes. These were sutured into place on the left side of the thorax and right axilla. Once inside the magnet, the movement of the electrodes with respect to each other during each respiratory movement, caused by the enlargement of the chest cavity during inhalation, caused a blip on the ECG trace which allowed the quick assessment of respiratory rate. Additional information about respiratory movements was also available as slow, deep breaths could be discriminated from rapid shallow respiration by the amplitude, frequency and spike length of the respiratory signal on the ECG monitor. During preparation and surgery, this information was obtained visually.

Core temperature was monitored using a thermocouple which was inserted into the anus and advanced six centimetres, to the level of the splanchnic colon. Temperature was displayed continuously on a digital meter and maintained at 37.0±0.2°C using radiant lamp heating whilst on the bench, and warm air current once inside the magnet bore.

When the probe was transferred inside the magnet bore, the thermocouple and ECG cables were connected in series with low-pass filters to avoid compromise of the RF shield.

6.7.2 Surgical Procedure

The rat was secured in the probe (shown in Figure 6-13), in the prone orientation, after attachment of the rectal thermocouple and ECG electrodes. Access to approximately 2mm of the MCA at the level of the olfactory tract was achieved by the established route of Tamura (Tamura et al., 1981) which is described in detail by Yanamoto et al (Yanamoto et al., 1998). Briefly, a 15mm diameter circular area of skin directly overlying the zygomatic bone and arch was removed and the facial muscles were dissected and retracted. A section of zygomatic bone of approximately 8mm was removed to reveal the lateral ligament of the temporo-mandibular joint, which was dissected apart. A small craniotomy, 2mm rostral and superior to the trigeminal groove was performed using a small dental drill, set at a moderately slow rotation speed (approx. 500rpm). Drilling was frequently paused and the cranium washed with isotonic saline solution at 37°C so as to avoid heating of the skull, and to remove small fragments of bone produced by drilling. The final, thin layer of bone was removed using fine forceps to reveal the MCA, olfactory tract and brain cortex covered with the overlying arachnoid mata and dura mata. The area thus exposed is illustrated in Figure 6-13.

It was noted during development of the surgical technique that heating of the brain due to constant (and non-cooled) drilling results in adherence of the dura mata to the cranium, with resulting damage when the final layer of bone was removed. Special care was therefore taken to avoid this; drilling was carried out slowly with frequent pauses and washing with iso-thermic saline.

The dura and arachnoid mata were pierced on either side of the MCA using a custom made cutting needle, so as to preserve a strip of dura approximately 400pm wide directly overlying the MCA (Figure 6-14). The rostral piercing was made slightly larger than the caudal to permit the insertion of the phosphor bronze hook, which was slid across and

arachnoid and dura mata, thereby occluding it, the tip of the hook locating in the caudal dura piercing - effectively locking the hook, MCA and dura.

The surgical procedure is essentially bloodless as major facial vessels such as superficial draining veins and arteries associated with the zygoma are tied and thermally coagulated prior to sectioning. Slight ‘weeping’ of blood from muscles and cut edges of skin quickly clot and therefore do not constitute a significant deviation from normal physiological volaemea.

Figure 6-14 A large craniotomy demonstrating the olfactory tract (A) and site of hooking (B) (post-mortem). The area exposed during routine surgery is shown by the larger dashed ellipse and the two durai holes by the smaller ellipses.

The time required from the induction of anaesthesia to the placing of the rat into the NMR scanner was approximately 2.5 hours. In general a slight reduction in core temperature resulted from transferring the rat from the bench to the magnet bore, and so a brief normalising period was necessary before quantitative NMR measurements were commenced. In practice this time was utilised productively with NMR setup procedures such as z-axis positioning, shimming, RF pulse calibration and the acquisition o f EPI phase correction datasets.