Project protocol — Contents
Workflow, sampling, and experimental treatment
Reagents, supplies, and solutions
Procedure: ethanol dependence induction
Procedure: blood ethanol concentration determination
Procedure: ethanol withdrawal severity testing
Strains were tested on equivalent dose of alcohol. A strain of mice grouped within a cage was randomized into two groups. For details of the number of mice and EtOH dose given see Table 2 in Metten P, Crabbe JC, 2005.
Group 1: control group
Group 2: EtOH treated group
Each mouse in the control group was treated with saline injection and the same dose of Pyrazole HCL, as well an identical inhalation chamber filled with ambient air.
Each mouse in the EtOH treated group was treated according the workflow below and Table 2 in Metten P, Crabbe JC, 2005.
Day Step EtOH Treatment Pyrazole HCl 68.1 mg/kg i.p. 0 Induction 1.5 g/kg EtOH, 20% v/v i.p. No Yes 0 Maintenance 3-12% EtOH vapor chamber No Yes 1 Maintenance 3-12% EtOH vapor chamber Yes Yes 2 Maintenance 3-12% EtOH vapor chamber Yes Yes 3 Withdrawal testing None No* No 3 Withdrawal None Yes No
* Blood for BEC was obtained after determination of the severity of alcohol withdrawal.
- Inhalation chamber is described in detail in Crabbe JC et. al., 1994.
- Briefly the alcohol inhalation chambers were made of 0.5 inch Plexiglas measuring 71 cm x 66.5 cm x 114 cm in size, and each containing a vapor volume of 538 L. Within the chambers were three stainless steel 0.25 inch hardware cloth cages measuring 48 cm x 35 cm x 18 cm, suspended by a flat metal guide rail, and secured to each upper edges of the chamber. Each wire-mesh cage was then further divided into six compartments, in turn each was able to accommodate 5-6 mice; therefore, about 100 mice could be treated simultaneously/chamber. A separate door enabled access to each individual cage. A stainless steel drop pan 10 cm beneath the cages contained absorbent lining pads that were replaced daily.
- Ethanol piston pump (FMI LAB Pump Model G-6) provided continuous supply of vaporized alcohol.
- Vaporization was accomplished using liquid ethanol pumped through silastic flexible tubing, and passed through the chamber via 4.5 cm long 18 gauge needles with perforated tubing attached, for wicking ethanol through 2.5 cm x 85 cm filter paper.
- Efficient mixing of air and ethanol was accomplished using three small fans.
- Air was supplied, after passing through a desiccator (Drierite desiccant), onto control chamber at 55 L/ min (flow regulator, Matheson Model 70). Given the volume of the chamber, the air/ethanol was replaced about every 10 min.
- Vapor and air-flow measurements were measured using Cole-Parmer flow meters.
- Exhaust from air/vapor was eliminated from the chambers via 3.2 cm diameter hose into a built-in exhaust system.
- Chamber ethanol vapor (500 µL) concentration was measured about every 30 min using a gas-contained syringe (Hamilton model 1001) and a Hewlett-Packard Model 5890 gas chromatograph.
- Processed blood was centrifuged at 12,000 rpm using Beckman Microfuge 12.
- Processed blood (2 µL) was analyzed for alcohol content using an auto-sampler gas chromatography (Hewlett-Packard Model 7673A).
- Ethanol (EtOH), Pharmoco Inc, Brookfield CT, USA; EtOH solution was prepared at 20% v/v in saline.
- Pyrazole hydrochloride: Sigma Chemical Co., St. Louis, MO.
- Saline (physiological saline): 0.9%, Baxter Healthcare Corp, Deerfield IL, USA.
- Solutions and supplies for processing blood: 5% ZnSO4, 0.3N Ba(OH)2, distilled water, and ice bucket.
- Isopropyl alcohol: 10% for wipe downs between tests.
Acclimation to test conditions
Mice were acclimated to housing facility 1-3 wks.
I. Ethanol dependence induction
a. Strains with similar sensitivity to alcohol were grouped in a single inhalation chamber and exposed to the same dose of EtOH.
b. To facilitate the induction of ethanol dependence, the mice were initially injected with EtOH to achieve a BEC of about 1.50 mg EtOH/mL blood.
c. Blood levels of alcohol were stabilized by inhibiting its metabolism with injections of 68.10 mg pyrazole HCL/kg bw i.p. (days 0, 1, and 2).
d. Physical dependence to EtOH was accomplished by exposing the mice to EtOH vapor and through constant inhalation chamber for 72-h.
e. Due to strain variations in eliminating EtOH, the vapor concentration was adjusted according to strain-specific need to achieve equivalent levels of BECs (refer to Table 2 in Metten P & Crabbe JC, 2005) in all strains, which ranged from 3%-12% for the entire 72-hr maintenance.
f. Control mice were initially injected with saline, and with pyrazole daily, while contained in comparably setup chambers, but exposed only to air.
g. The apparatus is cleared and wiped with 10% isopropyl alcohol after each test.
II. Blood ethanol concentration determination
a. After 24-, 48-, and 72-hr exposures to EtOH vapor, the mice were removed in small groups and gently hand-restrained for blood collections and/or injections the same time every morning.
b. Before receiving their daily dose of pyrazole, the mice were nicked at the tail with a sharp scissor, and blood was collected using a 20 µL capillary tube.
c. Actual blood ethanol concentration was determined using a gas chromatographic assay (Terdal ES & Crabbe JC, 1994).
d. Control mice were equally manipulated without any samples collected.
III. Ethanol withdrawal severity testing
a. Shortly after removal from the chamber, before the last sample of blood was drawn, all mice were initially scored for handling-induce convulsion (HIC, Handling-Induced Convulsion Scale) weighed, and returned in their home cage.
b. For the next consecutive 10 hrs and at 24 hr and 25 hr, the mice were again tested and scored for HIC.
c. Scoring was based on 1-5 scale, modified from the original HIC Scale (Crabbe JC et. al., 1991), with "0" in the absence of convulsion, even with tail rotation, to "5" with tonic-clonic convulsions, with just tail lift, and no rotation (refer to Table 1 below and Table 1 in Metten P, Crabbe JC, 2005).
Table 1. Handling-Induced Convulsion Scale
Score Handling-Induced Convulsion Elicited behavior Handling details 5 tonic-clonic convulsion tonic-clonic convulsion tail-lift only 4 tonic convulsion tonic convulsion tail-lift only 3 tonic-clonic convulsion tonic-clonic convulsion tail-lift w/ gentle 180° spin 2 tonic convulsion tonic convulsion tail-lift w/ gentle 180° spin 1 none observed facial grimace tail-lift w/ gentle 180° spin
Data collected by investigator
Handling-induced convulsion scores were obtained for three consecutive measurements at 10-hr, 24-hr, and 25-hr.
After initial injections and after 24-h, 48-h, and 72-h exposures in EtOH-vaporized chamber blood EtOH concentration were measured.
- General index of handling induced convulsion severity was computed using the area under the entire 25-hr curve.
- Using the maximum HIC value as a reference plus two adjacent scores were used to obtain the highest running average or peak_EtOH value. In case of a tie, the earliest occurrence was chosen, and in case the scores occur at Hours 24 or 25, the average of Hours 24 and 25 was given as the peak.
- The hour of the first maximum score used to calculate peak was defined as the latency to peak hour (peak_latency).
- Withdrawal severity expressed as area under the 25 hour HIC curve, corrected by control values is considered the most important measurement in this study (withdrawal_EtOHadj), as noted by the submitting investigator.
Crabbe JC, Merrill CD, Belknap JK. Effects of convulsants on handling-induced convulsions in mice selected for ethanol withdrawal severity. Brain Res. 1991 May 31;550(1):1-6.
Crabbe JC, Metten P, Yu CH, Schlumbohm JP, Cameron AJ, Wahlsten D. Genotypic Differences in Ethanol Sensitivity in Two Tests of Motor Incoordination. J Appl Physiol. 2003 Oct;95(4):1338-51. Epub 2003 Apr 18.
Kamens HM, Phillips TJ, Holstein SE, Crabbe JC. Characterization of the parallel rod floor apparatus to test motor incoordination in mice. Genes Brain Behav. 2005 Jun;4(4):253-66.
Terdal ES, Crabbe JC. Indexing withdrawal in mice: matching genotypes for exposure in studies using ethanol vapor inhalation. Alcohol Clin Exp Res. 1994 Jun;18(3):542-7.