Long-term anaesthesia in rodents
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We have a scientist who is undertaking MRI work, with rats that can be anaesthetised for up to ten hours. The animals are induced with isoflurane, then given i/p urethane and i/m Vetergesic. Once fully anaesthetised their femoral b.v. are cannulated for monitoring blood gases, BP and for the administration of drugs and fluids. Their tracheas are exposed, and an endotracheal tube sutured in place.
During the surgery they are breathing naturally (O2 delivered via a nose cone, and rate noted. When the rat is moved to the MRI scanner, it is moved onto a ventilator to ensure a good quality blood gases are maintained. With lung capacity of a rat being about 7ml/kg, the machine is set to deliver 1.3- 1.5 ml of oxygen enriched (50%) medical air, at about 70 -80 breaths/minute, (mimic natural breathing rate for the individual). Peak Inspiratory Pressure (PIP) of approximately 6cm H20 and Peak Expiratory Pressure (PEEP) of 13 cm H2O is used.
However the rat becomes hypercapnic, and they have problems dropping the levels of blood CO2. The tube between the ventilator and the rat, carrying the gas, is approximately 4m, and needs to be as it cannot be in the same room as the MRI scanner. It is approx 2mm wide. The inspiratory and expiratory branches of the tubing separate about three cm above the endotracheal tube.
It seems likely to me that this is either related to a lack of suitable flow rate, pressure or too much dead space. The flow rate is partially determined by the pressure delivered by the machine, and the change in gas within the lungs must be related to the amount of pressure used to get gas in, and the amount of pressure used to get the gas out. But the long distance between the machine and the animal must be affecting the effectiveness of the pressure levels. It seems logical to me to increase the pressure levels of both PIP and PEEP, as well as the tidal volume delivered, but how high?
I used in the past Urethane or a combination of Urethane/Alpha-chloralose for physiological experiments (2-4h duration), surgery typically performed under Isoflurane anaesthesia with local anaesthesia or analgesia coverage. With BP monitoring in place we would transition with very slow bolus (~ 10 minutes) Urethane (25% solution in Hartmann’s) 1100 mg/kg (0.44 ml / kg / min) i.v. followed by Urethane (5% solution) 3.2 – 4.0mg/kg/min (0.064 – 0.080 ml/kg/min) i.v. It is not easy to maintain stable anaesthesia with Urethane and fine tuning and monitoring is required. With buprenorphine on board the above doses would require adjustment due to potentiation.
How did you maintain stable anaesthesia following a single IP bolus or urethane for 10h, or did you give repeat IP boluses or IV? Is it possible that, due to light anaesthesia, rats may fight the respirator? Have you tried just maintaining them with O2, Ringer’s infusion, homeothermia and no mechanical ventilation?
We didn’t use IP route because of urethane's effect on peritoneum (superficial organ damage and necrosis, leakage of plasma), SC route works fine (see Maggi & Meli, 1986 for a great review of this anaesthetic).
3 cm of additional tubing in the circuit will create a significant amount of rebreathing and could well be the cause of the hypercapnia you are seeing. I used exactly this approach and approximately the same tubing dimensions a long ago to produce experimental hypercapnia. Try getting the fresh gas/extract as close as possible to the trachea and see if that makes a difference.
I have not extensive experience with assisted ventilation, but from our fMRI studies it was very important to reassure a good gas exchange, keeping a good control of the body temperature (which I presume you have under control). Our setup allowed us for a shorter length of the tubing as 4 mm seems considerably long. I would agree that revising tidal volume and pressures would be useful; also undertaking regular gas blood analysis and manage it regularly with the depth of anaesthesia if possible (we only used isoflurane which allowed us a better management; we only run studies up to ~5hrs). There are also some compatible MRI ventilators that may be worth checking.
A couple of things to add - I would still measure the volume of the 2cm of tubing between ET tube end and the Y-connector. I am not clear why PEEP is being used; if you are getting progressive alveolar collapse, adding some regular “sighs” can fix that. Does the expiratory limb also have 4m of tubing leading back to the ventilator? I assume so, and if so this may be producing significant expiratory resistance - make it reasonably wide-bore (e.g. the 1cm “disposable” paediatric ventilator tubing you can buy in long rolls). Finally (and raise this tactfully!), could the endotracheal tube be in one bronchus?
Try to reduce the respiratory rate (RR) and increase your pressure. I went back to my old thesis with the nice old Harvard ventilator just with volume and RR, no pressures: 'A ventilation rate of approximately 45 breaths per minute and an end tidal volume of 4ml produced a capnography waveform, which indicated that minimal rebreathing was occurring, and a normal end-tidal CO2 concentration (approximately 35mm Hg)'.