Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

In this video, we will demonstrate how to construct and assemble alcohol vapor chambers. We will also describe an alcohol inhalation paradigm that exposes maas during the equivalent of all three trimesters of human development. The chambers are highly customizable to desired blood alcohol levels or exposure paradigms.

Our chamber design can house up to six standard size mouse cages where mice have ad lipid access to food and water. This paradigm produces relatively high blood alcohol levels in both pregnant mice and pups without the use of rosol. We hear from the laboratory of Fernando Valenzuela at the University of New Mexico.

In this video, we'll demonstrate the assembly of polycarbonate ethanol vapor chambers and describe their use in an all three trimester mouse model of fetal alcohol exposure. Anyone with basic carpentry skills will be able to construct the chambers and the dimensions can be modified to fit individual needs. The chambers can be housed in the animal facility throughout the exposure and will minimize stress related to transport and handling of the animals.

Due to differences in developmental stages, ethanol administration to pregnant rodents has been used to model the human exposure during first and second trimesters, whereas ethanol administration to neonates is used to model third trimester exposure. These chambers are designed to house multiple mouse cages simultaneously and expose pregnant dams as well as neonates housed with their mom. Some of the advantages to using this alcohol exposure paradigm include the ability to mimic all three trimesters of development, the reduction in stress to pregnant dams and pups, as well as the flexibility in your control over the level of ethanol exposure.

On five individual 24 by 48 inch pieces of one quarter inch thick, clear polycarbonate sheets measure and mark the given dimensions for the top, bottom, front, back, sides, and the door. These dimensions will construct one chamber 32 inches wide, 22 inches deep and 14 inches high. A chamber of this size will adequately fit six standard mice cages measuring 11 and a half inches by seven and a half inches by five and a half inches high.

It is important to remember that this provides only a single chamber, and this process will need to be repeated to end up with two identical chambers, one chamber for alcohol exposure, and one chamber for air only controlled exposures. The polycarbonate sheets can be cut with a circular saw equipped with an 80 tooth blade or a jigsaw equipped with a plastic or plexiglass specific blade. Cut the polycarbonate sheets to the measured dimensions being careful as possible to ensure straight cuts.

This will reduce gaps during the assembly process once all the pieces are cut. Measure out an eight inch by 16 inch rectangle in the center of the front panel to create an opening for the door. Using a three eight inch drill bit, drill a hole in the inside of each corner.

The holes are used to insert the jig sub blade through the polycarbonate sheet and cut away the opening. If using a circular saw, a plunge cut can be performed. A few back cuts may be needed to clean up the edges near the corners.

Once all the edges are cut, the centerpiece can be removed and you're left with an opening in the front panel. To begin the assembly of the door mark the holes needed to attach the piano hinge to the door. Using a five 16th inch drill bit drill counter sink holes at the marks, these counter sink holes will accept the head of the screw and should only be drilled halfway through the polycarbonate sheet.

With a three 16th inch drill bit, complete the holes through the sheet using the counter sink holes as your guide before assembly. Remove approximately two inches of the protective covering of the polycarbonate sheets to allow for welding of the pieces. The assembly process can go quickly, so it may be helpful to lay out the bottom back front and sides before welding the pieces together.

To weld the polycarbonate, use the fast setting weld on 16 solvent cement. To begin assembly, add a generous bead of weld on to the bottom edge of the side piece. Immediately wipe off any drips of weld on with a paper towel.

Place the side onto the bottom piece with assistance. Assemble the rest of the walls to the bottom of the chamber. Remember that for the dimensions given the side pieces will fit between the front and back panel.

Once the walls are assembled, fill any gaps between the joints from the inside with the Weldon. To attach the top of the chamber, add a generous amount of Weldon to the top of each wall. Set the top panel on top of the walls and align the edges.

Use bar clamps to hold everything in place while the weld cures. Alternatively, heavy textbooks can be used during the curing process to ensure that the door will rest flush on the outside of the chamber. A one inch by 12 inch piece of polycarbonate needs to be welded to the front panel one inch below the open.

The hinge will attach to this spacer and allow for the door to seal the chamber. To construct the inlet tubing, cut two pieces of half inch polyethylene tubing 12 inches long. Also cut a one inch piece of half inch polyethylene tubing to deliver the vaporized alcohol or air drill.

A five 16th inch hole approximately one inch from the end of each of the 12 inch pieces of polyethylene tubing. Assemble the air delivery system using two three eights inch plugs and one three-eighths inch T connector. Make sure that the holes you drilled in the polyethylene tubing are furthest from the T connector.

Attach the piano hinge to the door, making sure the counter sink holes are facing up. Using four 40 machine screws and nuts, attach the piano hinge to the door. Use the door as a guide and mark the holes for the piano Hinge on the one inch spacer on the front panel with a three 16th inch drill bit.

Drill the holes in the front panel to attach the door with four 40 machine screws and nuts. Attach the door and piano hinge to the front panel. Tighten with a wrench.

Assemble the hold down. Toggle clamp as shown. Mark the holes of the toggle clamp on the front panel using the door as a guide.

Repeat this for two toggle clamps along the top and one on each side of the door with a three 16th inch drill bit. Drill the holes for the toggle clamps. Attach the toggle clamps with four 40 machine screws and nuts.

With the nuts on the inside of the chamber, tighten with a wrench to ensure an airtight chamber. Cut and attach three eighths inch wide rubber seal along the edge of the door. When the door is closed, the bulb will form an airtight seal between the door and the front panel of the chamber.

Mark the center of the top panel to create an inlet port for the chamber with a five eight inch boring drill bit. Drill the hole for the inlet port. Create an outlet port by drilling a half inch hole in the lower center of the back panel.

Insert a three eight inch through wall adapter into the exit port and attach from the inside of the chamber. At this stage, the protective covering can be removed. Install the air delivery system through the hole in the top panel from the outside.

Insert a three eight inch 90 degree elbow Drill a one half inch hole in the front of the door. Then insert a one half inch seal septa into the hole. This will serve as a port to measure the ethanol vapor.

At this point, the cutting and drilling is complete and the chambers can be cleaned. Assemble the rack and place the two chambers and air pub onto the rack. To help organize the airflow system, attach a scrap piece of polycarbonate to the lower shell and that can be used to help mount airflow regulators mark and drill three quarter inch drill hold for the three airflow regulators into the scrap piece of polycarbonate.

Attach the airflow regulators to the polycarbonate with the nuts provided. Attach a three eight inch through wall adapter to each port of the regulator for the ethanol vapor chamber. One airflow regulator will control the amount of air being bubbled through the liquid alcohol, and one will control the amount of air being mixed with the vaporized alcohol.

The last will control the amount of air for the second air only chamber. Assemble the alcohol flask as shown. Using three eights inch tubing.

Assemble the air delivery system as depicted in the diagram using T or Y connectors to split the lines. A single pump provides enough airflow for both air only control chambers and the alcohol chambers. Connect three eights inch tubing to the outlet ports.

Add 600 mils of one 90 proof ethanol to the flask and connect into the system. To test the level of ethanol vapor, use a syringe and a needle to withdraw five mils of air through the septin. Remove the needle and dilute the air sample one to 12 by pulling the syringe up to 60 mls.

With room air slowly push the air through the breathalyzer to measure the ethanol vapors. The levels of vaporized ethanol should be correlated to animal blood alcohol levels for each individual system. Once the chambers are located in the animal facility, remember to exhaust the chambers directly into the room's ventilation system.

Two to three month old female mice were group housed for at least one week, whereas two to five month old male mice were individually housed for two weeks. Female mice were paired with individually housed males for five days to allow for mating. Following mating, females were removed from the males individually housed in a new cage and placed within the vapor chambers.

Animals were exposed for ethanol for four hours per day. During the light cycle from G five to G 20. Females were periodically weighed to monitor pregnancy.

Animals were not exposed in the day of birth to avoid pup death. On postnatal day one, pups and moms replaced back into the chambers and exposed until postnatal. Day 12 pups were periodically weighed to monitor growth.

To start the vapor inhalation place. The individually housed pregnant dams into both the alcohol and air only. Chambers close and seal the doors.

Add the liquid ethanol flask connect to the system and turn on the pump. The airflow regulators control the amount of air that goes into the liquid ethanol and how much air is being mixed with the ethanol vapor. The ratio of ethanol vapor and air control the final ethanol vapor levels into the chambers.

The air only controls airflow is set to the combined airflow of both the ethanol and air. Following the ethanol vapor exposure, the food is changed to eliminate chow that may have absorbed vaporized ethanol. The chamber doors on both the alcohol and air only chambers are cracked to allow air exchange with the room.

In this video, we Demonstrated how to assemble ethanol vapor chambers and described a mouse alcohol vapor inhalation paradigm that exposes animals during all three equivalent human trimesters. The level of alcohol vapor can easily be adjusted to model low or high blood alcohol levels. One of the advantages of these chambers is how the chambers are large enough to house multiple cages at any given point.

This allows the animals to be housed within the chambers throughout the exposure. This minimizes any stress caused to the animal from handling or transportation. Furthermore, this paradigm can be customized to any trimester of interest and or level of exposure During the construction.

It is critical that all cuts are straight and square to ensure an airtight chamber when welding the pieces of polycarbonate. It's important to move quickly and efficiently because the welds dry rapidly. After watching this video, you should be able to construct your own vapor inhalation chambers.