The overall goal of this procedure is to evaluate changes in anesthetic sensitivity after a pharmacological intervention. This is accomplished by first carefully checking the exposure chambers to ensure uniform gas flow and temperature. The second step is to habituate animals to the chambers.
Next, the pharmacological intervention of interest is performed. The final step is to assess changes in anesthetic sensitivity by evaluating the animal's writing reflex in response to anesthetic exposure. Ultimately, writing reflex assays are used to show differences in anesthetic sensitivity caused by long or short acting pharmacological interventions.
Though this system can provide insight into changes in anesthetic sensitivity in mice, it can also be used in a variety of species to study the effects of any inhaled therapeutic. The testing apparatus consists of 24 clear acrylic cylindrical chambers, 10 centimeters in length, and five centimeters in diameter. This size is appropriate for a typical 25 gram adult mouse.
Every chamber has a port at each end for gas inlet and outlet. The outlet end is sealed with rubber gaskets to make it easy to remove. For animal insertion, mount each chamber on a rack that sits inside a water bath.
Place the rack so that only the lower portion of the chambers will be submerged. A support on the back end of the chamber provides stability and keeps the chambers horizontal, ensuring even contact of each chamber with the bath. After connecting an oxygen tank to a vaporizer and flow meter with polyethylene tubing, split the tubing into 25 small diameter resistors of equal length.
This ensures that equal flow is delivered to each chamber when connected. Next, connect the vacuum lines that exit each chamber at the opposite end of the gas inlet. The vacuum lines combine at a manifold to connect to an in-house suction line.
A pop-off valve along the main vacuum line ensures atmospheric pressure conditions within each chamber. Once everything is connected, fill the bath with enough water to fully contact the bottom of each chamber. Turn on the pump to circulate the water and maintain a constant temperature of 37 degrees Celsius.
Before proceeding with testing, the chambers are examined for leaks and proper airflow. First, set the oxygen to a rate of five liters per minute. Submerge each chamber under water and look for bubbles or water entry, both of which are indicat of leaks.
Replace any leaking chambers before beginning the experiment. For each chamber, connect a 500 milliliter per minute flow meter in line before the chamber to make sure that flows are balanced across each line. Any chamber not receiving the expected flow should have its tubing checked for obstructions.
Lastly, calibrate the agent analyzer to ensure a reading of 0.0%isof fluorine when 100%oxygen is flowing one week prior to habituation. Transponders are implanted to track body temperature during testing. After achieving the proper depth of anesthesia, sterilize the dorsal neck area with Betadine.
Next, use a sterile needle to inject the temperature transponders subcutaneously between the shoulder blades. Monitor the injection site daily for infection and migration of the transponder. Four days before the first assessment, all test animals are habituated to the chambers to reduce the effects of stress due to a new environment.
To do this, place an animal into each chamber with 100%oxygen flowing for two hours. Repeat this exposure daily until testing. After four days of habitation testing day begins with exposure to the pharmacological intervention of interest.
This intervention may be a stereotaxic injection into a specific brain region, an intravenous injection, or an intraperitoneal injection For pharmacological interventions. With a long duration of action, a stepwise determination of anesthetic sensitivity is performed. Begin testing by placing each animal into individual chambers with 100%oxygen flowing once all animals are in place.
Set the isof fluorine concentration to 0.4%for 15 minutes. During the last two minutes of this period, assess each animal's writing reflex by gently rolling the chamber until the mouse is placed on its back. The writing reflex is considered to be intact if the mouse is able to restore all of its paws to the floor of the chamber.
Within two minutes, record the state of writing reflex for each mouse and scan each mouse for temperature data. Next, increase the isof fluorine concentration by approximately 0.05%for 15 minutes, and assess the writing reflex as demonstrated earlier. Continue to increase the anesthetic concentration until all animals have lost their writing reflex.
Optionally anesthetic emergence may be measured by repeating the stepwise writing reflex assessment as anesthetic concentration is titrated downwards. To end the experiment, turn off the isof fluorine and flush the entire system with 100%oxygen for 15 minutes for interventions with a shorter duration. The time to emergence test is performed.
After placing each animal into individual chambers with 100%oxygen flowing, set the isof fluorine concentration to 1.2%After several minutes of anesthetic exposure, gently roll each chamber until each mouse remains on its back. Confirming the loss of writing reflex, maintain the isof fluorine concentration at 1.2%for 30 minutes to one hour depending upon the expected duration of action of the acute intervention. After turning off the isof fluorine flow 100%oxygen and measure the time until each animal places all four paws on the floor.
The writing reflex is considered intact when the animal can place all four paws on the floor three consecutive times. Here, the writing reflex was tested one week after I botanic acid was injected into the ventral lateral preop nucleus. A lesion to this brain region was predicted to decrease anesthetic sensitivity.
However, no significant differences in stepwise anesthetic sensitivity was found for either induction or emergence. In this example, the time to emergence test was performed immediately following bilateral I botanic acid microinjection. When injected into the VLPO, there was a markedly prolonged hypnosis as compared to injections since the nearby medial septum and vehicle injected controls.
After watching this video, you should have a good understanding of how to assess anesthetic sensitivity using the stepwise induction test or the time to emergence test while ensuring that every animal is exposed to identical gas flows and temperatures.
