Anesthesia of 10-day old mice. Ten-day-old pups can be anesthetised with 4.5% isoflurane in 4-5 mins, however, due to their small size, intubation under isoflurane anesthesia, delivered by a standard nose cone, is not feasible. We have previously used a ketamine/xylazine/atropine anesthetic regimen of 100/13/0.5 mg/kg, respectively, for thoracic surgery in 15- and 21-day-old pups and adults.4,7 In preliminary experiments that included oxygen supplementation we found this regimen resulted in an unacceptable mortality in 10-day-old pups. Given the inverse correlation between body weight and litter size of 10-day-old pups (R2=0.250, p<0.0001; Figure 1), we titrated the anesthetic regimen according to body weight groupings. Reducing the ketamine/xylazine/atropine dosage to 50/6/0.18 mg/kg, respectively, resulted in a sufficient depth of anesthesia to allow endotracheal intubation of spontaneously breathing pups weighing 5.5-8.10 g (Table 1) but this dose was not tolerated by lighter pups. Reducing the ketamine/xylazine/atropine dosage to 30/4/0.12 mg/kg enabled intubation of pups weighing 4.50-5.49 g, while further reduction of the ketamine dosage to 20 mg/kg enabled intubation of pups weighing 3.15-4.49 g (Table 1).
Table 1
Anesthesia regimen, number of intubation attempts and post-procedure survival of 10-day-old mouse pups
Anesthesia regimen: ketamine/ xylazine/ atropine (mg/kg); given in 10 mL/g body weight, ip | Body weight, g | Number of pups studied | Intubation attempts (A, 1-2; B, 3-4 or C, >4) and number of pups intubated, n (%) | Intubated pups proceeding to surgery, n (%) | Survival two days post-surgery, n (%) | Overall survival after intubation plus surgery, n (%) |
A | B | C |
20/4/0.12 | 3.15 - 4.49 | 22 | 8 (36) | 9 (41) | 5 (23) | 13 (59) | 12 (92) | 12 (55) |
30/4/0.12 | 4.50 - 5.49 | 20 | 13 (65) | 5 (25) | 2 (10) | 14 (70) | 12 (86) | 12 (60) |
50/6/0.18 | 5.50 - 7.30 | 20 | 13 (65) | 3 (15) | 4 (20) | 16 (80) | 14 (88) | 14 (70) |
p (Chi-square test) | | p<0.001 | | p=0.91 | |
R2 (Correlation coefficient, p value) | | | 0.995, p=0.04 | | 0.978, p=0.09 |
Intubation of 10-day old mice. Before intubation, the depth of anesthesia was assessed by the paw pinch reflex. For optimal intubation, the paw pinch reflex had to still be present but markedly reduced from that of a conscious animal. Intubation of 10-day old pups required specialised equipment (Figure 2A-C). After securing the anesthetised pup supine to an intubation platform (Figure 2B), the tongue was held with small forceps and a laryngoscope fashioned out of a piece of copper wire (Figure 2C) was used to expose the glottis and vocal cords, visualisation of the vocal cords being aided by trans-illumination with a flexible fibre-optic light (Figure 2D). A 24-gauge plastic intravenous (iv) cannula was used as an endotracheal tube. This consisted of a 19 mm length of plastic tubing (0.7 mm OD) attached to a 21mm plastic female luer lock adaptor (Figure 2B). The total volume of the cannula was 130 mL; the tidal volume of a mouse being ~8 mL/g.13,14 At the time of intubation, the tubing of the cannula was stiffened by insertion of a piece of copper wire via the luer lock adaptor (Figure 2B); the wire being removed immediately after intubation. The cannula, tilted so that the luer lock end was slightly lower (~10o) than the tip, was inserted as soon as the vocal cords separated.
The depth of anesthesia was assessed after intubation by the ability of the animal to breathe spontaneously. Successful tracheal intubation of spontaneously breathing pups was confirmed by briefly blocking the intubation catheter to check that this prevented chest movement. The endotracheal cannula was then connected to a ventilator delivering 100% oxygen. These procedures were performed rapidly (<15 sec) to minimise re-breathing.
Outcomes were best when intubation was achieved after only one or two attempts. Pups with a lower body weight were more difficult to intubate than heavier pups and required more attempts (p<0.001; Table 1). Survival post-intubation was inversely correlated with body weight (65, 70 and 80% for low-, mid- and high-weight groups, respectively, R2=0.995, p=0.04; Table 1).
Myocardial infarction surgery of 10-day old mice. For myocardial infarction surgery after intubation, anesthesia was induced with 4.5% isoflurane in 100% oxygen and tracheal intubation again confirmed by checking that the frequency of chest wall movement equalled that of the ventilator. Loss of spontaneous breathing followed by absence of a tail or paw pinch reflex indicated that a surgical plane of anesthesia had been reached (after ~4-5 min). Anesthesia was maintained with 2% isoflurane. The thorax was opened, and a myocardial infarction was induced by ligation of the left anterior descending coronary artery just distal to the left atrial appendage. After the ~10 min surgery, animals were allowed to recover by discontinuing the isoflurane. Spontaneous breathing resumed within a few minutes thereafter and then the pup was returned to the warmed pre-oxygenated chamber and monitored continuously during recovery until the righting reflex was regained, at which point the pup was extubated.
Of the pups that did not survive to follow-up at 48 h (Table 1), one from the low-weight group died 6 h after surgery, one pup from each of the mid- and high-weight groups died before being placed back with the dam, and one pup from each of the mid- and high-weight groups was cannibalized by the dam within 16 h of surgery, with small body parts or nothing remaining the next morning. Post-surgery cannibalism by the dam was reduced if pups were cleaned of blood and disinfectant, gently rubbed with home cage bedding, kept warm, were breathing regularly and were capable of spontaneous movement i.e. had fully recovered from anesthesia, before returning the dam to the cage.
Survival two days after myocardial infarction surgery was consistent between the different weight groups at 86-92% (p=0.91; Table 1). Infarcted myocardium, as assessed two days post-surgery by Alcian-blue perfusion of the heart, was evident by clear demarcation of stained, non-infarcted (blue) from ischaemic (unstained) tissue, distal to the ligation (Figure 2E).
Overall survival for the entire procedure (intubation plus surgery) was inversely correlated with pup body weight (55%, 60% and 70% for low-, mid- and high-weight groups, respectively, (R2=0.978, Table 1), although this correlation did not achieve statistical significance (p=0.09).