This work was supported, in part, by Research grants SC CTSI NIH/NCRR/ NCATS -- UL1TR000130 (D.L.D.), AA022448 (D.L.D.), and the USC School of Pharmacy.

All procedures described here have been approved by the Institutional Animal Care and Use Committees of the University of Southern California Health Sciences Campus.
1. Experimental Setup and Assembly
<ol>
	<li>Acquire all of the following supplies and chemicals before the start of the study: mice, cages/metal cage tops, bedding, food, water, ethanol, pipets, sipper tops, shrink wrap, utility knife, zip ties, tape, Bunsen burner, scale, headlamp.</li>
	<li>Obtain C57BL/6J mice, either from a commercial source or an in-house colony, keeping in mind that mice can be group housed until time of testing. 
	&#8203;NOTE: The total number of mice procured depends on the complexity of the experimental design. Plan to accommodate roughly 12 - 15 mice per group, with pilot studies no smaller than 5 - 7 mice per group. In the representative results shown below we utilized a simple two-group set-up to assess the cause-effect relationship using a single dose (5 mg/kg) of the drug (MOX).</li>
	<li>Follow the steps below to assemble the bottles18.
	<ol>
		<li>Heat a utility knife using a Bunsen burner.</li>
		<li>Using this knife, cut roughly an inch off the top and bottom ends of a plastic 18 mL serological pipet.
		<ol>
			<li>Note that smaller volume pipets (i.e., 10 mL) can also be used to increase the precision of the measurement.</li>
		</ol>
		</li>
		<li>Warm the pipet under a heat gun.</li>
		<li>Insert the ball bearing sipper tube into the &#34;bottom&#34; end of the pipet (in other words, the opening nearest to the 18 mL dash-line).</li>
		<li>Seal the sipper tube in place with shrink wrap using a commercially available shrink wrap gun.</li>
		<li>Cap off the other opening with a silicone stopper.</li>
	</ol>
	</li>
</ol>
2. Animal Habituation
<ol>
	<li>Beginning at least 1 week prior to the start date of the experiment, transfer the mice to the room where the experimental procedures are to be carried out so that they may acclimate to the husbandry conditions (including the ambient temperature (21 &#177; 1 &#176;C) and 12-h reverse light/dark cycle, with lights off at 12 noon). Be sure to follow institutional guidelines and notify the appropriate channels before moving animals from one location to another.
	<ol>
		<li>If mice are being transferred from a standard light/dark cycle allow 2 weeks of additional habituation time.</li>
	</ol>
	</li>
	<li>Fill the newly made bottles to the brim with water. Make sure that the cap is closed securely and devoid of any air bubbles or leaks from the spout. If solution is leaking, re-secure the cap. Remove any air bubbles by simply tapping on the bottle so that the air can escape the tube.</li>
	<li>Upon arrival, single house each mouse in standard polycarbonate/polysulfone cages with bedding and a metal grid cage top; remove the cage lid as it will no longer be used.
	<ol>
		<li>Provide access to food and water bottle(s) ad libitum.</li>
		<li>Secure each bottle to the cage top by wrapping a plastic zip tie around each bottle to hold it in place. Trim any excess plastic from the zip tie to ensure that it does not protrude into the cage. 
		&#8203;NOTE: For the drinking in the dark (DID) procedure, only a single bottle of water is required. Habituation to the two bottle choice (TBC) paradigm, however, requires that the cage set-up include two bottles of water. If the metal grid hopper provided is designed to hold only a single bottle of solution, gently bend apart its bars to create space for an additional plate to accommodate the second bottle for TBC.</li>
	</ol>
	</li>
	<li>Set up at least 3 mouse free control cages. This will allow for the monitoring of any fluid loss caused by evaporation or spillage from the bottles, which is simply a natural occurrence that happens as cages are placed on and off the cage rack (see step 2.6.3, 3.6.1, and 4.6.1 for equations).</li>
	<li>Beginning on day 4 of the 1-week single housing acclimation, measure and record the daily body and food weights of each mouse, using a scale, (in grams) as well as the water intake, using the etchings alongside the inverted bottle to record the highest point of the meniscus (in mL).
	<ol>
		<li>While it is standard scientific practice to read the lowest point of the concave meniscus, because the bottles remain in an inverted position during the measurement, record the highest point of the meniscus.</li>
	</ol>
	</li>
	<li>Assess the parameters of 2.5 using the equations below:
	<ol>
		<li>Measure the Body Weight Change (g): weight of current day (g) - weight of previous day (g).</li>
		<li>Measure the Food Intake (g): weight of food on previous day (g) - weight of food on current day (g).</li>
		<li>Measure the Water Intake (mL): [volume of water on current day (mL) - volume of water on previous day (mL)] - average water loss from all control cages (mL).</li>
	</ol>
	</li>
	<li>Repeat step 2.5 consecutively, on days 5 - 7, to allow for the determination of a baseline for the three days immediately prior to the introduction of ethanol. If a consecutive recording cannot be collected, extend the acclimation period to allow for the evaluation of baseline measurements.</li>
	<li>Once water intake has stabilized to &#177; 10% variability from the mean of the last 3 days, begin ethanol access with either TBC (unlimited access) or DID (limited access). 
	NOTE: On rare occasion, one to two additional days may be needed for subjects to attain this stability; don&#39;t be alarmed if additional time is needed for the values to display &#177;10% variability from the mean of the last 3 days.</li>
</ol>
3. 24-Hour Two Bottle Choice (TBC)
Note: A schematic is prepared in Figure 1.
<ol>
	<li>Prepare a 10% (v/v) ethanol solution at a 500 mL volume by adding 52.65&#160;mL of 190 proof grain ethanol (~95% ethanol) to 447.35&#160;mL of H2O; be sure to shake thoroughly. Given that ethanol evaporates quickly, replace the solution in every 3-4 day&#39;s interval. 
	NOTE: Other concentrations of ethanol can be used as well, but the authors recommend a 10% concentration for this model.</li>
	<li>On the first day of TBC, (day 8 at the earliest) empty 1 of the 2 water bottles, in each cage, 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. Simply apply a piece of masking tape to the bottle and labeling it with a marker or by writing directly on the bottle.</li>
	<li>Add more solution to the water bottle, as needed.</li>
	<li>Place bottles back on the cage, making sure that all caps are closed securely and devoid of any air bubbles or leaks from the spout. If the solution is leaking, re-secure the cap. Remove any air bubbles by simply tapping on the bottle so that the air can escape the bottle.</li>
	<li>Alternate the position of the bottles every other day as to correct for conditioned place preference related influences in drinking activity (see Discussion for more).</li>
	<li>In addition to the daily measurements from 2.5 and 2.6, which have been ongoing, begin to read and record ethanol intake levels as well. Analyze the 10% ethanol intake and preference ratio using the following equations:
	<ol>
		<li>Measure the 10% Ethanol Intake (mL): [volume of ethanol on current day (mL) - volume of ethanol on previous day (mL))] - average ethanol loss from all control cages (mL).</li>
		<li>Measure the 10% Ethanol Intake (g/kg): [10% ethanol Intake (mL) x 0.07893 g/mL] / body weight (kg).</li>
		<li>Measure the Preference %: [10% Ethanol Intake (mL) / Water (mL)] x 100.</li>
	</ol>
	</li>
	<li>As soon as ethanol intake has become stable, administer a single control (saline) intraperitoneal (i.p.) injection (0.01 mL/g of body weight) to each mouse during the daily measurement routine. In this way, subjects become accustomed to the injection itself.
	<ol>
		<li>Stability is defined as&#160;&#177; 10% variability from the mean of the last 3 days (same as in section 2.8). 
		&#8203;NOTE: It can take up to 1 week for ethanol levels to stabilize. This is especially true if mice are being re-used from a previous experiment and have had previous exposure to ethanol. The control is simply the solvent used to dissolve the drug.</li>
	</ol>
	</li>
	<li>Once an ethanol baseline with low variability is re-established, split the mice into dosing groups using the ethanol intake values so that all groups have roughly similar average ethanol intake values.
	<ol>
		<li>Designate one group as the control (continuing to receive saline) and the other as the experimental (i.p. injection of the investigated drug at 0.01 mL/g of body weight). Begin daily drug dosing, either for an acute or multi-day duration. Subsequently, the control can be re-introduced to test the post-drug effects (optional). 
		NOTE: Because drinking is monitored across a 24-h long period, the time of dosage administration is not dependent on the dark cycle.</li>
	</ol>
	</li>
</ol>
4. Drinking in the Dark (DID)
NOTE: A schematic is prepared in Figure 3.
<ol>
	<li>On each day of scheduled ethanol access (Days 1 - 4) record the measurements, for water volume, food intake, and body weight, and perform drug dosing. Do 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 at/or approaching the maximum brain concentration during the drinking period. 
	&#8203;NOTE: Remember days 1 - 3 are meant to simply acclimate the mice to drinking copious levels of ethanol in a short period of time. While mice do not reach BEC levels comparable to the human 0.08; these &#34;training&#34; days ensure that on day 4, during the slightly longer drinking session, they will in fact drink to that level.
	<ol>
		<li>Give all mice the control (saline) on days 1 - 3, and either the control or drug on day 4. This DID procedure occurs over the span of 3 weeks to include a pre-drug (week 1), drug (week 2), and post-drug (week 4) drinking session.</li>
		<li>NOTE: Note that during the drug-dosing week, day 3 ethanol intake levels are used to assign mice to either the control or drug group in a way where the ethanol intake levels of both groups have the least variability. This is unlike TBC, which assigns groups based on a 3 day average.</li>
	</ol>
	</li>
	<li>Prepare a 20% (v/v) ethanol solution (20E) at a 500 mL volume by adding 105.25 mL of 190 proof grain ethanol (~95% ethanol) to 394.75 mL of H2O; be sure to shake thoroughly.</li>
	<li>Fill the ethanol bottles before the start of the drinking session so that as soon as the DID begins the water bottles can simply be replaced with the alcohol bottles.</li>
	<li>During the entire DID session (steps 4.5 - 4.8), use a red-light headlamp as to not disturb the animals.</li>
	<li>At the start of the DID drinking session, scheduled to begin 3 hours into the dark cycle, record the volume of water for each mouse. Then, replace each water bottle with a bottle of the 20E solution and record the starting ethanol volume.</li>
	<li>Read and record the final ethanol volume 2 hours later, at the end of the drinking session on Days 1 - 3 and 4 hours later on Day 4. Analyze the 20% ethanol intake using the following equations.
	<ol>
		<li>Measure the 20% Ethanol Intake (mL): [volume of ethanol at end of drinking session (mL) - volume of ethanol at start of drinking session (mL)] - average ethanol loss from all control cages (mL).</li>
		<li>Measure the 20% Ethanol Intake (g/kg): [20% Ethanol Intake (mL) x 0.15786 g/mL] / body weight (kg).</li>
	</ol>
	</li>
	<li>On Day 4 only, immediately after recording ethanol volumes, and before re-introducing access to water, collect blood to assess the blood ethanol concentration of each mouse (optional). 
	&#8203;NOTE: Any non-terminal blood collection method may be used, such as retro-orbital sinus blood collection, or saphenous vein blood collection. For useful protocols see references for Parasuraman et al.21 and Yardley et al.20.
	<ol>
		<li>Perform analysis using various methods including LCMS, or commercially available machinery (see Table of Materials).</li>
	</ol>
	</li>
	<li>Replace all ethanol bottles with water bottles and record water volume.</li>
</ol>

<ol>
	<li>Litten, R. Z., Falk, D. E., Ryan, M. L., Fertig, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+Development,+and+Adoption+of+Medications+to+Treat+Alcohol+Use+Disorder:+Goals+for+the+Phases+of+Medications+Development.">Discovery, Development, and Adoption of Medications to Treat Alcohol Use Disorder: Goals for the Phases of Medications Development.</a> Alcoholism: Clinical and Experimental Research. 40 (7), 1368-1379 (2016).</li><li>Grant, B. F., Dawson, D. A., Stinson, F. S., Chou, S. P., Dufour, M. C., Pickering, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+12-month+prevalence+and+trends+in+DSM-IV+alcohol+abuse+and+dependence:+United+States,+1991-1992+and+2001-2002.">The 12-month prevalence and trends in DSM-IV alcohol abuse and dependence: United States, 1991-1992 and 2001-2002.</a> Drug and Alcohol Dependence. 74 (3), 223-234 (2004).</li><li>Sacks, J. J., Gonzales, K. R., Bouchery, E. E., Tomedi, L. E., Brewer, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+and+State+Costs+of+Excessive+Alcohol+Consumption.">National and State Costs of Excessive Alcohol Consumption.</a> American Journal of Preventive Medicine. 49 (5), 73-79 (2015).</li><li>Litten, R. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medications+development+to+treat+alcohol+dependence:+a+vision+for+the+next+decade.">Medications development to treat alcohol dependence: a vision for the next decade.</a> Addiction Biology. 17 (3), 513-527 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Johnson+Medication+Treatment+of+Different+Types+of+Alcoholism.">Johnson Medication Treatment of Different Types of Alcoholism.</a> American Journal of Psychiatry. 167 (6), 630-639 (2010).</li><li>Litten, R. Z., Wilford, B. B., Falk, D. E., Ryan, M. L., Fertig, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+medications+for+the+treatment+of+alcohol+use+disorder:+An+evaluation+of+clinical+efficacy+and+safety.">Potential medications for the treatment of alcohol use disorder: An evaluation of clinical efficacy and safety.</a> Substance Abuse. 37 (2), 286-298 (2016).</li><li>Litten, R. Z., Ryan, M. L., Falk, D. E., Reilly, M., Fertig, J. B., Koob, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+Alcohol+Use+Disorder:+Understanding+Mechanisms+to+Advance+Personalized+Treatment.+Alcoholism:+Clinical+and+Experimental.">Heterogeneity of Alcohol Use Disorder: Understanding Mechanisms to Advance Personalized Treatment. Alcoholism: Clinical and Experimental.</a> Research. 39 (4), 579-584 (2015).</li><li>Schuckit, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-Wide+Search+for+Genes+That+Relate+to+a+Low+Level+of+Response+to+Alcohol.">A Genome-Wide Search for Genes That Relate to a Low Level of Response to Alcohol.</a> Alcoholism: Clinical and Experimental Research. 25 (3), 323-329 (2001).</li><li>Batki, S. L., Pennington, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Personalized+Medicine+in+the+Pharmacotherapy+of+Alcohol+Use+Disorder:+Targeting+Patient+Genes+and+Patient+Goals.">Toward Personalized Medicine in the Pharmacotherapy of Alcohol Use Disorder: Targeting Patient Genes and Patient Goals.</a> American Journal of Psychiatry. 171 (4), 391-394 (2014).</li><li>Koob, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+frameworks+and+mechanistic+aspects+of+alcohol+addiction:+alcohol+addiction+as+a+reward+deficit+disorder.">Theoretical frameworks and mechanistic aspects of alcohol addiction: alcohol addiction as a reward deficit disorder.</a> Current topics in behavioral neurosciences. 13, 3-30 (2013).</li><li>Yoneyama, N., Crabbe, J. C., Ford, M. M., Murillo, A., Finn, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+ethanol+consumption+in+22+inbred+mouse+strains.">Voluntary ethanol consumption in 22 inbred mouse strains.</a> Alcohol. 42 (3), 149-160 (2008).</li><li>Rhodes, J. S., Best, K., Belknap, J. K., Finn, D. A., Crabbe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+simple+model+of+ethanol+drinking+to+intoxication+in+C57BL/6J+mice.">Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice.</a> Physiology &amp; Behavior. 84 (1), 53-63 (2005).</li><li>Thiele, T. E., Navarro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Drinking+in+the+dark"+(DID)+procedures:+A+model+of+binge-like+ethanol+drinking+in+non-dependent+mice.">"Drinking in the dark" (DID) procedures: A model of binge-like ethanol drinking in non-dependent mice.</a> Alcohol. 48 (3), 235-241 (2014).</li><li>Crabbe, J. C., Spence, S. E., Brown, L. L., Metten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+preference+drinking+in+a+mouse+line+selectively+bred+for+high+drinking+in+the+dark.">Alcohol preference drinking in a mouse line selectively bred for high drinking in the dark.</a> Alcohol. 45 (5), 427-440 (2011).</li><li>Sprow, G. M., Thiele, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+binge-like+ethanol+drinking:+Evidence+from+rodent+models.">The neurobiology of binge-like ethanol drinking: Evidence from rodent models.</a> Physiology &amp; Behavior. 106 (3), 325-331 (2012).</li><li>Neasta, J., Hamida, B., Yowell, Q., Carnicella, S., Ron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+mammalian+target+of+rapamycin+complex+1+signaling+in+neuroadaptations+underlying+alcohol-related+disorders.">Role for mammalian target of rapamycin complex 1 signaling in neuroadaptations underlying alcohol-related disorders.</a> Proceedings of the National Academy of Sciences. 107 (46), 20093-20098 (2010).</li><li>Huynh, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+development+of+moxidectin+as+a+novel+therapeutic+for+alcohol+use+disorder.">Preclinical development of moxidectin as a novel therapeutic for alcohol use disorder.</a> Neuropharmacology. 113, 60-70 (2017).</li><li>Egli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+experimental+paradigms+and+animal+models+be+used+to+discover+clinically+effective+medications+for+alcoholism.">Can experimental paradigms and animal models be used to discover clinically effective medications for alcoholism.</a> Addiction Biology. 10 (4), 309-319 (2005).</li><li>Huynh, N., Arabian, N., Lieu, D., Asatryan, L., Davies, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+an+Orally+Dissolving+Strip+for+Pharmacological+and+Toxicological+Studies:+A+Simple+and+Humane+Alternative+to+Oral+Gavage+for+Animals.">Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals.</a> Journal of Visualized Experiments. (109), (2016).</li><li>Yardley, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ivermectin+reduces+alcohol+intake+and+preference+in+mice.">Ivermectin reduces alcohol intake and preference in mice.</a> Neuropharmacology. 63 (2), 190-201 (2012).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology and Pharmacotherapeutics. 1 (2), 87-93 (2010).</li></ol>

DLD and LA are inventors on a patent for the repurposing of ivermectin and related avermectins for the treatment of alcohol-use disorders. The authors have no other conflicts of interest and are entirely responsible for the scientific content of the paper.

Worldwide estimates indicate that as many as 76 million people meet the criteria to warrant a diagnosis for Alcohol Use Disorder (AUD). Unfortunately, pharmaceutical treatments currently available are largely ineffective and further development is necessary to offset the needs of this clinical population20. To this end, the following protocol aims to facilitate this endeavor by exemplifying two of the most basic rodent drinking paradigms: two-bottle-choice (TBC) and drinking in the dark (DID). Both models measure non-operant self-administration of ethanol, whereby mice ingest ethanol orally. In the TBC paradigm, ethanol (10% v/v) and water are both available continuously, resulting in comparably low blood ethanol levels that are a consequence of an episodic and gradual ethanol consumption pattern. This model is best for assessing lower levels of ethanol intake as a preference tastant and therefore, is said to be similar to human &#34;moderate&#34; drinking. Figure 2 above shows representative results for the TBC paradigm.
On the other hand, in the DID model ethanol (20% v/v) is only available for a limited amount of time. Unlike TBC, this model assesses the effects of a compound on behaviorally relevant concentrations of ethanol, taking advantage of the fact that C57BL/6J mice will consume large amounts of ethanol very quickly during the most active phase of their circadian cycle. These drinking sessions occur for 4 consecutive days, beginning at the 3rd h into the dark cycle, with a duration of 2 h on days 1 - 3 and 4 h on day 4. Using this procedure, mice typically consume enough ethanol to achieve BECs &#62;100 mg/dL and to exhibit behavioral evidence of intoxication. Our representative results for the DID paradigm are shown in Figure 4. Although we did not measure BECs for these mice, their drinking levels are similar to those reported in the literature that achieved BECs over 100 mg/dL13,14,15.
Both paradigms have limitations. During instances when oral gavages are the preferred method of administration for pharmacological testing of lead compounds, esophogeal trauma caused by the procedure may hinder ethanol self-administration in both paradigms. This is especially true for the DID model, which uses a higher concentration of ethanol (20%) and which may present instances where the pharmacokinetics of the drug require the drinking session to begin before the animals have had adequate time to recover from the procedure. This would be evident in a control group drinking at levels that correspond to BECs well below the expected 0.08. In our lab, we have experienced difficulty with force feedings both with the DID and TBC. We were able to work around these issues by formulating our compounds so that they could be delivered using an orally dissolving thin strip (ODS)19.
Moreover, in the DID model specifically, given the fact that the ethanol access period is so short in duration, drug dosing must be done during a preselected time during the light cycle that is chosen in accordance with the pharmacokinetics of the drug so that the compound is at/or approaching the maximum brain concentration during the drinking period. If a pharmacokinetic&#160;analysis cannot be performed, an alternative approach would be to conduct a time course assessment of the behavioral effects of the drug using TBC. Following this logic, by monitoring drinking activity on an hourly basis, and determining peak anti-alcohol effects, one could reasonably strategize when drugs should be administered19.
While the field of alcoholism has various animal models to investigate the different physiological and behavioral aspects of AUD8, the following protocol describes two commonly used paradigms, which allows for comparisons of results across laboratories. A second benefit is that these methods are straightforward, making their use convenient during acute and chronic studies similar to the examples provided above. Additionally, unlike other commonly used alcohol paradigms, such as operant conditioning and vapor chamber paradigms, the methodology we have described here can be carried out without the need for specialty equipment. Anecdotally, it is likely that nearly all components of the drinking apparatus can be found in a prototypical institutional vivarium. There are also various ways to alter the protocols described here so that they can best fit the goals of each experiment. For instance, our DID procedure is best for testing the acute effects of a drug on a single drinking session (during day Day 4). However, we have shown that multi-day drug dosing can be assessed by augmenting the drinking sessions to 4 consecutive days of 2 hour access, as opposed to the current 2 hours of access on days 1 - 3 and 4 hours on day 419. Mice can also be transferred from one paradigm to another, or re-tested for additional doses / different compounds, with a simple 1 - 2 week washout period in between.
It should be mentioned that any drug-induced change in drinking observed through these methods must be thoroughly investigated to determine whether the effects are selective for alcohol or if they might be the result of toxicity. For more information on controls the reader should refer to Yardley et al.20 No single paradigm can model all aspects of this condition. Instead, each paradigm typically examines a few of the key attributes associated with AUD. The TBC and DID models described here, have been linked to the binge/intoxication stage of the addiction cycle. For a more thorough understanding of the utility of the compound, multiple preclinical drinking models should be utilized.

For the past 25 plus years, significant effort has been put towards developing medications for the treatment of Alcohol Use Disorder (AUD)1.Although many advances have been made, AUD still remains a major public health problem, affecting over 18 million Americans, and costing over $220 billion annually2,3. Currently there are only three FDA-approved medications, disulfiram, naltrexone, and acamprosate, all of which have yielded inconsistent results in clinical trials and limited success even when combined with psychosocial intervention in the clinic settings4,5,6,7.
A primary reason for failures of current AUD therapy is linked to the heterogenous nature of AUD8. While both environmental and genetic factors contribute to the development of AUD, heritability accounts for an estimated 50 - 60% of the risk of onset9. Similar to the treatment of depression, it is widely accepted that patients suffering from AUD will need a variety of medications that are tailored to meet the needs of each patient10.
Clearly, there is an urgent need for more effective treatments that would be facilitated if the already arduous and time consuming process of drug discovery were streamlined3. To this end, the following protocol demonstrates the preclinical applicability of two rodent drinking models widely used to examine the neurobiological basis of AUD11. More specifically, the method introduced herein can assess the efficacy of candidate compounds at reducing alcohol consumption in both &#34;moderate&#34; and &#34;binge drinking&#34; scenarios utilizing the two-bottle choice (TBC) and drinking in the dark (DID) paradigms, respectively. Both paradigms examine non-operant ethanol self-administration, whereby mice ingest ethanol orally and at will, and therefore illustrate high face and construct validity as a model of human alcoholism11.
In TBC drinking, also known as free choice drinking, preference drinking, or social drinking, two bottles of solution are continuously available in the home cage. One bottle contains water, and the other contains a diluted solution of ethanol, whereby the concentration of ethanol can be varied (e.g., 5 - 30% v/v)11,12. The mice have constant access to both bottles, and therefore, can choose how much to drink from each bottle.
This model assesses the ethanol consumption of each mouse (g/kg), as well as the ethanol preference ratio (volume of ethanol consumed &#247; total volume liquid consumed). It is routinely used to compare drinking levels across different strains of mice, or after a specific genetic manipulation (e.g., gene knockout or knockdown) and results in blood ethanol concentrations (BECs) similar to what is found in humans when drinking in moderation13,14.
In the DID procedure, 3 h after the start of the dark cycle, the home cage bottle of water is exchanged with a bottle of 20% (v/v) ethanol solution for a limited access drinking session. The drinking sessions occur as a consecutive 4-day cycle, lasting 2 h on days 1 - 3 and 4 h on day 4. Days 1-3 serve as an alcohol-habituation period before the testing on day 4. Consequently, mice will reliably consume enough ethanol to attain BECs &#62;100 mg/dL and as a result, exhibit the behavioral effects of intoxication found in humans that are binge drinking13,14,15. Water access is available at all times other than the drinking session.
There are several variations of limited access drinking. For example, in the intermittent access model, mice receive two bottles (one containing water and the other containing 20% (v/v) ethanol) only on Monday, Wednesday, and Friday with a 24 h and 48 h withdrawal period on weekdays and weekends, respectively16. After several weeks of intermittent access, the mice will gradually and voluntarily escalate drinking levels, eventually attaining BECS similar to what is observed in the DID model. The DID, however, appears to be the most commonly used model to assess binge-like drinking behavior. Other models of intermittent drinking exist, but they rely on restricting access to food or vapor chamber induced increases in voluntary self-administration, which makes them less representative of voluntary human alcohol consumption16.

<table border="0" cellpadding="0" cellspacing="0" style="width:1617px;" width="1616">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:483px;">1 L&#160; graduated cylinder</td>
			<td style="width:315px;">VWR</td>
			<td style="width:232px;">https://us.vwr.com/store/product/20935285/marisco-single-scale-cylinder-graduates-john-m-maris-co</td>
			<td style="width:588px;">To prepare ethanol solution.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1 L glass bottle</td>
			<td>Pyrex (Fisher Scientific)</td>
			<td>https://www.fishersci.com/shop/products/pyrex-reusable-media-storage-bottles-12/p-42752</td>
			<td>To prepare ethanol solution.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">100 mL graduated cylinder</td>
			<td>Fisher Scientific</td>
			<td>https://www.fishersci.com/shop/products/kimble-chase-kimax-class-a-to-contain-graduated-cylinders-8/p-4369311</td>
			<td>To prepare ethanol solution.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Analox</td>
			<td></td>
			<td></td>
			<td>One potential method of analyzing DID blood samples is by using the analox machine</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ball-bearing sipper tubes</td>
			<td>Ancare Corp.</td>
			<td>http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point</td>
			<td>Length: 2.5 inches, Diameter: 5/16 inches, Model: TD100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">C57BL/6J Mice</td>
			<td>Jackson lab</td>
			<td>https://www.jax.org/strain/000664</td>
			<td>May also come from internal breeding colony</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">disposable serological pipets</td>
			<td>VWR International (VWR)</td>
			<td>https://us.vwr.com/store/product/4760455/vwr-disposable-serological-pipets-polystyrene-sterile-plugged</td>
			<td>10 mL, 18 mL, or 25 mL&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ethanol, pure, 190 proof (95%), USP, KOPTEC</td>
			<td>Decon Labs (VWR)</td>
			<td>https://us.vwr.com/store/product/4542412/ethanol-pure-190-proof-95-usp-koptec</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">heat gun&#160;</td>
			<td>Master Appliances Corp.</td>
			<td colspan="2">http://www.masterappliance.com/master-heat-guns-kits/</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">heat shrink tubing</td>
			<td>---</td>
			<td>---</td>
			<td>Diameter: 3/8 inches</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">industrial knife/blade</td>
			<td>---</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">metal cage plate</td>
			<td>---</td>
			<td>---</td>
			<td>Should be available through the university/institutional vivarium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">mouse RO water</td>
			<td>---</td>
			<td>---</td>
			<td>Should be available through the university/institutional vivarium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">portable electronic scale</td>
			<td>Ohaus (VWR)</td>
			<td>https://us.vwr.com/store/product/4789377/portable-electronic-cs-series-scales-ohaus</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">red light headlamp</td>
			<td>nyteBright (Amazon)</td>
			<td>https://www.amazon.com/LED-Headlamp-Flashlight-Red-Light/dp/B00R0LMMF8/ref=sr_1_1?ie=UTF8&#38;qid=1499591137&#38;sr=8-1-spons&#38;keywords=red+lamp+headlamp&#38;psc=1</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">silicone stoppers</td>
			<td>Fisher</td>
			<td>---</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">thermometer</td>
			<td>Fisher Scientific</td>
			<td>https://www.fishersci.com/shop/products/fisher-scientific-hygro-thermometer-clock-large-display-2/p-4077232</td>
			<td>---</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">weigh boat</td>
			<td>VWR International (VWR)</td>
			<td>https://us.vwr.com/store/product/16773534/vwr-pour-boat-weighing-dishes</td>
			<td>The lid from a pipete tip box is an appropriate alternative</td>
		</tr>
	</tbody>
</table>

murine drinking models in the development of pharmacotherapies for alcoholism: drinking in the dark and two-bottle choice

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments are limited to three FDA-approved medications that are largely ineffective even when combined with psychosocial intervention, as is evident by the high relapse rate. As such, the search for more novel treatments represents an important public health goal. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds: two-bottle choice (TBC) and drinking in the dark (DID). The former allows mice to voluntary drink in moderation while the latter induces mice to voluntary consume a large amount of alcohol in a short period that mimics binge drinking. The simple and high throughput nature of both of these paradigms allow for rapid screening of pharmacological agents or for identifying strains of mice that exhibit certain voluntary drinking behavior.

In the following representative investigations, social drinking was modeled using the two-bottle choice (TBC) paradigm. Briefly, mice had access to two bottles of solution, one of which contained water, and the other a 10% (v/v) ethanol solution. Subjects were subsequently split and evenly assigned to drug treatment groups, moxidectin (MOX) vs. saline control, so that each group would have averaged ethanol intake levels with the least amount of variation.
Initial baseline 10E intake during the 24-h period stabilized at 14.46 &#177; 1.85 g/kg (n = 8) before injections began, and subsequently re-stabilized at 14.14 g/kg post saline injections. To assess the effects of an acute dose (5 mg/kg) of MOX on ethanol intake and preference, drinking activity was assessed pre MOX, MOX, and post MOX. We found that a single dose of MOX significantly reduced alcohol intake in excess of 45%, compared with pre MOX injections [F (2, 22) = 26.33, p &#60;0.0001] (Figure 2A), and preference [F (2, 14) = 17.35, p &#60;0.0001] (Figure 2B) in excess of 30%. 10E intake and preference both remained significantly lower than saline on the day immediately following MOX treatment (by more than 25%, and 15% respectively) shown as post MOX injections in Figure 2).
In an unpublished pilot study, binge drinking was modeled using the drinking in the dark (DID) procedure whereby mice had daily limited access (2 h) to one bottle containing 20E beginning 3-h into the circadian dark phase, for 3 consecutive days, with a longer access period (4 h) on day 4 (Figure 4). Female mice (n = 12, 6 mice/group) were administered saline injections (i.p.) on days 1 - 4 with baseline 20E established on day 4 (Pre-Drug). On Days 1 - 3 of the 2nd weekly cycle, all mice received yet another daily saline injection. The ethanol intake values from day 3 were then used to split the mice into two groups (n = 6/ group) that subsequently received either one injection (i.p.) of MOX (5 mg/kg) or saline on Day 4 (Drug). The following week all mice received daily saline injections on days 1 - 4 and ethanol intake was measured yet again on Day 4 (post Drug). Acute administration of 5 mg/kg MOX was analyzed and found to significantly reduced alcohol intake in excess of 54%, compared with pre-Drug injections (t = 7.635, p &#60; 0.0001)
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57027/57027fig1.jpg" /> 
Figure 1. Two-Bottle Choice Schematic&#160;&#160;<a href="/files/ftp_upload/57027/57027fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57027/57027fig2.jpg" /> 
Figure 2. Two Bottle Choice (TBC). MOX (5 mg/kg) reduces 10% v/v ethanol (10E) intake (A) and preference (B) in female C57BL/6J mice using a 24-h access two-bottle choice paradigm. After attaining stable drinking levels for 3 consecutive days, MOX was administered. Bars represent average 10E intake levels from the day prior to the MOX injection (white; Pre MOX), the day of the MOX injection (black; MOX) and the day after the MOX injection (gray; Post MOX). Values represent the mean &#177; SEM for 12 mice. *P &#60;0.05, **P &#60;0.01, ***P &#60;0.001, and ****P &#60;0.0001 versus Pre MOX (leftmost horizontal line) or Post MOX (rightmost horizontal line), Tukey&#39;s multiple comparison post-hoc test. Modified from Huynh N. et al.19 <a href="/files/ftp_upload/57027/57027fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57027/57027fig3v2.jpg" /> 
Figure 3. Drinking in the Dark Schematic&#160;&#160;<a href="/files/ftp_upload/57027/57027fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57027/57027fig4.jpg" /> 
Figure 4. Drinking in the Dark (DID). MOX (5 mg/kg) reduces 20% v/v ethanol (20E) intake in female C57BL/6J mice using a drinking in the dark paradigm. Bars represent average 20E intake levels on the week prior to the MOX injection (Pre-Drug), the week of the MOX injection (Drug) and the week after the MOX injection (Post-Drug). Values represent the mean &#177; SEM for 12 mice (6/group) between saline and MOX groups. ****P &#60;0.0001 versus Pre-Drug (leftmost horizontal line) or Post-Drug (rightmost horizontal line), Tukey&#39;s multiple comparison post-hoc test. <a href="/files/ftp_upload/57027/57027fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice at JoVE.com

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is&#160;required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments ...

murine-drinking-models-development-pharmacotherapies-for-alcoholism

Universidad San Sebastian

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Medicine

Unit 1

Drug Abuse and Addiction

SCIENTISTS IN ACTION

7.8K Views.  University of Southern California. Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Video: Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...