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Inhalation anesthesia for in vivo molecular imaging

Introduction

Inhalation anesthesia with isoflurane is quickly becoming the standard method of general anesthesia for rats and mice used in biomedical research. It holds many advantages over injectable agents: minimal animal handling, high throughput, large margin of safety, ease of anesthetic control, low cost of anesthetic agent, no controlled drugs, and quick recovery times. Moreover, low toxicity permits repetitive anesthesia, important for longitudinal in vivo imaging.

Gas Anesthesia Machine

Gas anesthesia requires the use of an anesthesia machine to dispense the gases that are necessary to induce sleep, immobilize and prevent pain to animals during procedures. In the anesthesia machine, the “carrier gas” oxygen flows under pressure (50 psi) through the vaporizer and picks up the anesthetic vapors. The O₂-anesthetic mix then flows through the breathing circuit and into the patient's lungs, by spontaneous ventilation (respiration). Exhaled and non-used gases are evacuated through a scavenging system.

Gas flow pathway:

O2 CYLINDER → REGULATOR FLOWMETER → VAPORIZER → PATIENT BREATHING CIRCUIT → SCAVENGING SYSTEM

Breathing Circuit

The patient breathing circuit is the pathway for anesthetic gas delivery to the patient. The goals of an anesthetic breathing circuit are to: 1) Deliver oxygen to the patient; 2) Deliver anesthetic to the patient; 3) Remove carbon dioxide that is produced by the patient.

In rodent anesthesia, non-rebreathing systems (e.g. Bain circuit, Mapleson-E circuit) are most commonly used. Non-rebreathing circuits use very high fresh gas flows that deliver the anesthetic gas and washes out the exhaled CO₂. Because there is no re-breathing, you do NOT need a CO₂ Absorber on your anesthesia machine. The basic design of a non-rebreathing circuit consists of two tubes. One delivers fresh gases (anesthetic and oxygen) from the anesthesia machine to the patient connection, then a second, usually larger tube, channels the exhaled (waste) gases to evacuation. In this type of circuit any exhaled/non-utilized gas is immediately directed toward the evacuation circuit. The waste gas tube is connected to either a disposable, scavenging activated charcoal canister or to an in-house evacuation system. The fresh gas tube can be located inside or outside the waste gas tube. The internal diameters and overall lengths of the fresh gas and waste gas tubes can vary greatly, and affects dead volume in ventilation calculations (see below).

Inhalation Anesthetics

Commonly used inhalation anesthetics include isoflurane and sevoflurane. Less commonly used are: methoxyflurane, halothane, desflurane, nitrous oxide. Clinical consideration of the selection of an inhalation agent is its route of metabolism. The major elimination route of inhalation anesthetics is via respiration. The percentage of metabolism by the liver and kidneys is an indicator of toxicity. Thus, a critical parameter to consider with frequent anesthesia, such as for immobilization purposes during in vivo imaging. The percentage of inhalation anesthetic as recovered metabolites in the liver and kidneys are as follows for each of the respective gases: methoxyflurane (50%), halothane (25-50%), sevoflurane (3%), isoflurane (0.17%), desflurane and nitrous oxide (no documented metabolism). Injectable anesthetics are largely metabolized by the liver and if used more than once daily, appear lethal to the subject. As isoflurane is minimally metabolized by the liver and kidneys and relatively affordable, it is the anesthetic of choice for rodent research.

Gas Scavenging Systems

The purpose of gas-scavenging systems is to eliminate waste anesthetic gases from the work area to minimize breathing by personnel. OSHA standards allow for 2ppm exposure level within the work environment. This is below the human odor detection limit for isoflurane. The human nose can't detect isoflurane until it reaches 50ppm. So if you can smell it, the exposure level is too high.

There are two primary methods of scavenging anesthetic gases: active and passive. Active scavenging methods used within research settings are capture systems using vacuum or, performing all anesthesia procedures within fume hoods. There is really only one commonly-used passive scavenging system and that’s charcoal filtration. This usually involves a pass-through charcoal canister filter in the exhaust gas line. Activated charcoal can absorb halogenated anesthetics at roughly 25% of its own weight by volume. Halogenated anesthetics include isoflurane, halothane, enflurane, desflurane, sevoflurane and methoxyflurane. Note that canisters do not absorb other anesthetic gases, such as nitrous oxide N₂O. The charcoal canisters should have their saturation levels monitored. Protocols should include weighing the canister prior to use, recording the initial weight on the canister and then reweighing the canister after each use. The canister should be replaced after a weight increase of 50 grams. (~twelve hours of anesthesia time at an O₂ flow rate of 2 liters per minute).

Since we are not intubating rodents routinely and we are opening and closing induction chambers routinely, there are a variety of pathways for the gas to travel besides through the filter which has a relatively high flow resistance. Take the following precautions into account:

Use a non-rebreathing circuit that delivers the gas from the machine to the patient, and then collects and contains the waste gas from the patient to an adequate disposal system.
Attempt to seal all leaks (particularly around the nosecone).
Use an oxygen flush assembly. Purge all waste gas from the induction chamber prior to unsealing the lid. If you are unable to "flush out" your induction chamber, you will have no choice but to work in a non-recirculating fume hood or a chemical fume hood with an activated carbon filter.
Use an induction chamber with a positive pressure seal. Or, use a vented induction chamber.
Use activated carbon filters that are reliable.

O₂ Flow Rate

It is important to determine the O₂ flow rate in order to prevent CO₂ rebreathing and consequent respiratory acidosis. The O₂ flow rate should be around 3 times the patient's minute ventilation. In practical terms, O₂ flow should be no less than 500 ml (0.5 liters) per minute and 1 liter for induction chambers. Non-rebreathing systems deliver high flow of dry cool gas to the patient, which causes significant body heat and humidity loss. In order to prevent hypothermia, it is recommended to place rodents on a heating surface or near a heat source. When using a heating blanket or heat lamp, ensure that the animals are not exposed to excessive heat. In case of long term anesthesia, a humidifier may be used to add moisture to the gases in the patient breathing circuit. Isoflurane anesthesia abolishes the corneal/blink reflex. In order to prevent corneal dehydration and blindness, it is recommended to apply an ophthalmic ointment such as Paralube® for ocular lubrication.

Minute ventilation is the volume of air that ventilates the lungs in one minute. It is calculated by multiplying the tidal volume (air moved in and out of the lungs with each breath) by the respiratory rate (200-300 breaths/minute in the awake mouse). For example, the tidal volume of a 25-g mouse is approximately .25 ml (1% of its body weight). The minute ventilation for this mouse would be approximately 50 ml (500cc per minute = O2 flow rate). During isoflurane anesthesia, the respiratory rate is markedly decreased.to about 25-50 breaths/min and the depth of each respiration is increased. Much of the gases that flow into the patient never reach the lung alveoli, i.e., where the actual exchange of O2 and CO2 occur. Much air occupies the space between the nose and the alveoli in the lungs: this is known as "anatomical dead space". Another significant portion of the fresh gases occupy the tubes and adapters between the anesthetic machine and the nose: this is called 'mechanical dead space'. Both the anatomical and the mechanical dead spaces are excluded from the gas exchange process and therefore must be added to the calculation of tidal volume and minute ventilation.