The overall goal of this paradigm is to assess the effectiveness of a potential anti-alcohol drug using rodent models of alcohol use disorder. Alcohol use disorder represents a major public health crisis. Current treatments are ineffective and ongoing pre-clinical research is needed.
Our protocol allows us to evaluate the safety and utility of a potential treatment as well as to take a look at the underlying neurobiology of the disorder itself. Begin the procedure by heating a utility knife on the Bunsen burner. Using this knife, cut roughly an inch off the top and bottom ends of a plastic 18 milliliters serological pipette.
Next warm the pipette under a heat gun. Insert the ball bearing sipper tube into the bottom end of the pipette. Then using a commercially available shrink wrap gun, seal the sipper tube in place with shrink wrap.
Cap off the other opening with a silicone stopper. For animal habituation, upon arrival, single house each mouse in the standard polycarbonate polysulfone cage with bedding, food and water and a metal grid cage top. Beginning on day four of the one week single housing acclimation, measure and record the mouse's daily body and food weights.
Measure the water intake using the etchings alongside the inverted bottle, to record the highest point of the meniscus. Once water intake has stabilized to plus or minus 10%variability from the mean of the last three days, begin ethanol access with either TBC or DID. To conduct TBS, prepare 10%ethanol solution in 1, 000 milliliters by adding 105.3 milliliters of 190 proof grain ethanol to 894.7 milliliters of water, then shake it thoroughly.
Given that ethanol evaporates quickly, replace the solution twice a week. On the first day of TBC, in each cage, empty one of the two water bottles and fill it to the brim with the freshly prepared ethanol solution. Given that ethanol and water are difficult to be distinguished visually, clearly label the bottles with their corresponding contents.
Add more solution to the water bottle as needed. Then place the bottle back in the cage, making sure that the cap is closed securely and devoid of any air bubbles or leaks from the spout. Remove any air bubbles by simply tapping on the bottle so that the air can escape the bottle.
Alternate the position of the bottle every other day as to correct for conditioned place preference related influences in drinking activity. Record ethanol intake level and analyze the 10%ethanol intake and preference ratio. As soon as ethanol intake has become stable, administer a single control intraperitoneal injection to each mouse during the daily measurement routine, to accustom the mouse to injection.
Once an ethanol baseline with low variability has reestablished, divide the mice into control and experimental groups using the ethanol intake values so that all groups have roughly similar average ethanol intake values. Begin daily drug dosing, either for an acute or multi day duration. To conduct DID, on each day of scheduled ethanol access, record the measurements for water volume, food intake and body weight and perform drug dosing.
Perform this during a preselected time during the light cycle that is chosen in accordance with the pharmacokinetics of the drug so that the compound is approaching the maximum brain concentration during the drinking period. Next, prepare 20%ethanol solution in 1, 000 milliliters by adding 210.5 milliliters of 190 proof grain ethanol to 789.5 milliliters of water. Then shake it thoroughly.
Fill the ethanol bottle before the start of the drinking session so that as soon as DID begins, the water bottle can simply be replaced with the alcohol bottle. During the entire DID session, use a red light headlamp as to not disturb the animal. At the start of the DID drinking session, which is scheduled to begin three hours into the dark cycle before the light cycle, record the volume of water for each mouse.
Then replace each water bottle with a bottle of 20%ethanol solution and record the starting ethanol volume. Read and record the final ethanol volume two hours later at the end of the drinking session on days one through three and four hours later on day four. Then analyze the 20%ethanol intake.
Subsequently, replace the ethanol bottle with water bottle and record water volume. This figure shows the assessment of the effects of an acute dose of moxidectin at five milligrams per kilogram on ethanol intake and preference. A single dose of moxidectin significantly reduced alcohol intake in excess of 45%compared with pre-moxidectin injections and preference in excess of 30%10%ethanol intake and preference both remain significantly lower than saline on the day immediately following MOX treatment.
Here, binge drinking was modeled using the DID procedure. Mice were administered the saline on days one through four with baseline 20%ethanol established on day four. On days one through three of the second weekly cycle, all mice received yet another daily saline injection.
The ethanol intake values from day three were then used to split the mice into two groups that subsequently received either one injection of moxidectin at five milligrams per kilogram or saline on day four. The following week, all mice received daily saline injections on days one through four and ethanol intake was measured yet again on day four. Acute administration of MOX was analyzed and found to significantly reduced alcohol intake in excess of 54%compared with pre-drug injections.
Keep in mind that future experiments using this protocol must be done to evaluate the mechanism, dosing and toxicity of a potential drug as well as to assess its effectiveness in relation to the current treatment options.
