Alcohol dependence and withdrawal in the rat An effective means of induction and assessment

Alcohol Dependence and Withdrawal in the Rat An Effective Means of Induction and Assessment

W. D. RUWE, L. BAUCE, W. W. FLEMONS, W. L. VEALE, AND Q. J. PI~MAN

Numerous problems have been associated with previous attempts to develop a suitable method for the induction and assessment of alcohol dependence and withdrawal syndrome in the rat. Using our modification of a common inhalation method for the long-term administration of ethanol, these problems can be eliminated. Adult male rats (Long Evans and Brattleboro) were exposed to ethanol vapor concentrations of 7 to 35 mgiliter of air, which cause rapid development of tolerance and physical dependence. With this inhalation method, it is possible to obtain and easily maintain high levels of ethanol in the blood (150 to 400 mg/ dl). When exposure to ethanol is terminated, ethanol is eliminated from the system within 1 to 6 hr. This rapid elimination of ethanol is accompanied by a high susceptibility to withdrawal reactions. The severity of the withdrawal syndrome was assessed within 6 to 24 hr after cessation of the ethanol administration by exposing each rat individually to a 60 to 120-set period of bell ringing. Con- vulsive seizures were observed in nearly 90% of the animals tested, with a mortality rate of less than 20%.

Key Words: Alcohol dependence; Withdrawal syndrome; Ethanol inhalation; Audiogenic seizures; Tolerance

INTRODUCTION

In order to study and understand the primary neurobiological, neurochemical,

and neuropharmacological mechanisms underlying alcohol addiction and with-

drawal, one needs an appropriate animal model. The ultimate objective is to obtain

a meaningful substitute that will closely resemble its human counterpart, both

biologically and behaviorally (Majchrowicz, 1981).

Pohorecky (1981) has suggested a number of relevant criteria by which an animal

analogue of alcohol dependence can be evaluated. With such a model, it should

be possible to: 1) produce pharmacological levels of alcohol in the blood that can

be maintained over a long period of time; 2) obtain metabolical and functional tolerance and a consequent development of physical dependence; and 3) easily repeat the addiction cycle. In addition, it is imperative that the health of the animal

be maintained throughout the duration of the experiment. Therefore, appropriate nutritional controls must be incorporated to prevent the excessive loss of body

From the Department of Medical Physiology, Faculty of Medicine, The University of Calgary, Calgary,

Alberta, Canada.

Address reprint requests to: W.D. Ruwe, Department of Medical Physiology, Faculty of Medicine,

University of Calgary, Calgary, Alberta, Canada T2N 4Nl.

Received May 6, 1985; revised and accepted July 29, 1985.

225

Journal of Pharmacological Methods 15, 225-234 (1986)

0 1986 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Avenue, New York, NY 10017

226 W. D. Ruwe, et al.

weight or dehydration due to water deprivation. Finally, the optimal animal analogue

of alcohol dependence will not require excessive auxiliary manipulation, such as

the use of drugs.

An approach that represents one of the closest approximations to such an ana-

logue is the continuous, involuntary inhalation model first developed by Goldstein

and Pal (1971) and subsequently refined by both Ferko and Bobyock (1977) and by

Rogers and coworkers (1979). However, traditionally there have been a number of

problems associated with this model. Included among the most frequently en-

countered problems are: 1) weight loss of IO-20%; 2) respiratory and mucosal

irritation; 3) potential for ethanol overdose, resulting in coma or death; 4) necessity

of drugs such as pyrazole to maintain stable levels of ethanol in blood; and 5) the

necessity of a lengthy acclimatization period (Rogers et al., 1979; Majchrowicz, 1981; Pohorecky, 1981). Perhaps the two most critical problems with respect to

investigation of the alcohol withdrawal syndrome using this paradigm are: 1) the

evocation of seizures in only 30-50% of the animals withdrawn and 2) the very high

mortality rate observed in those animals that do manifest signs of the alcohol

withdrawal syndrome.

To ameliorate these methodological shortcomings and some of the major prob-

lems associated with previous inhalation techniques, a modification of existing

systems was designed to provide a more effective paradigm for the study of the

ethanol withdrawal syndrome in rodents.

METHODS

Animals

To determine the efficacy of this technique, we selected age-matched male,

hooded Long Evans (LE) and homozygous Brattleboro rats. The Brattleboro or di-

abetes insipidus (DI) rat also was chosen because of our current interest in the

neuropeptide, arginine vasopressin (AVP), a substance that is absent in the DI rat

(Valtin et al., 1965). In addition, the use of this rat would help to determine if an

animal with numerous physiological anomalies could tolerate such a system.

Experimental Apparatus

A schematic representation of the experimental apparatus is depicted in Figure

1. Ethanol (95% ethanol) was delivered by means of a solvent metering pump (Accu- flo pump, Beckman Instruments Inc.) and a splitting device into each of eight 250-

ml vaporization chambers.

Each airtight vaporization chamber was maintained at 37°C by a water bath. A three-holed rubber stopper permitted access of the inflow tubing for ethanol (23-

gauge stainless-steel tubing) and ports for air inflow and outflow. Using a separate Optima air pump (Rolf C. Hagen Corporation) for each vaporization chamber, the ethanol vapor was delivered into each of the experimental chambers. Adjustments

of the speed of these air pumps (to provide flow rates of 2.5 to 4.0 Iitersimin) and/ or the metering pump altered the ethanol vapor concentration that was pumped into each experimental chamber. The animals were exposed to ethanol vapor con-

Induction and Assessment of Alcohol Withdrawal 227

OUTFLOW (Ethanol vapor)

ETHANOL (Via pump) -

SPLITTING DEVICE OUTFLOW

METERING PUMP

rine collection

EXPERIMENTAL CHAMBER

FIGURE 1. Schematic representation of the ethanol inhalation chamber for the long-term administration of ethanol. Modified after Rogers et al. (1979).

centrations of 7.0 to 35.0 mg/liter of air. The ethanol was routed into each of the eight vaporization chambers by the splitting device, which was directly connected to the metering pump by Tygon tubing. Each experimental chamber consisted of a

standard 48 x 26 x 16-cm Nalgene rodent holding cage with a Plexiglas top, cut to fit snugly. The chambers were angled slightly and a metal grid placed on the

floor of each chamber permitted urine and fecal material to fall through and flow

by gravity to collection trays for either disposal or subsequent analyses. A food tray

and water bottle were securely affixed to the experimental chamber, giving the animals free access to food and water. The inlet hole, 8 mm in diameter, was cut

at one end of the chamber just above the food tray, and an air sample portal was

cut in the opposite end. Daily measurements were obtained for each of the following parameters: 1) body

weight, 2) body temperature, 3) blood alcohol levels, 4) ethanol vapor concentra-

228 W. D. Ruwe, et at.

tions in the chamber, and 5) behavior. All measurements were made each day

between 07:OO and 09:OO hr. Each animal was weighed and its basal body temper-

ature was recorded on a YSI telethermometer connected to a YSI 401 temperature

probe inserted 6-8 cm beyond the anus.

Blood alcohol levels (BALs) were determined in 20-t.tl samples of blood taken

initially by amputating the tip of the tail and subsequently by simple removal of the

scab. This procedure did not appear to traumatize the animal.

This 20-~1 afiquot was mixed with 80 t.d of IO mM 2-propanol. After thorough

mixing, a 3+l sample of this solution was injected onto a gas chromatograph (Hew-

lett Packard). The gas chromatograph was calibrated by injecting a known (8 mM)

amount of ethanol and isopropanol.

Behavioral assessments of activity and alertness were made as these measure-

ments were being taken in order to detect the development of any untoward symp-

toms, including extreme lethargy and coma.

RESULTS

Blood Alcohol levels

Using the method described herein, it was possible to maintain BALs between 0

and 500 mg/dl. Figure 2 illustrates the relative stability of BALs that were achieved

during one experimental treatment.

During the initial 3 days, there is some variability because of the individual dif-

ferences in the ability of each rat to metabolize ethanol. However, during the

subsequent 15 days, mean BALs were maintained between 200 and 260 mgidl with

only slight fluctuations. With eight chambers, it was found that a total of 12 rats was an optimal number

for each experimental condition. This allowed considerable flexibility in maintaining

each animal at a specific level of blood alcohol. Thus, it was possible to establish

a gradient across the eight chambers of low (18 mg/liter) to high (40 mg/liter) ethanol

vapor concentrations in each chamber. During the initial stages of the experimentation, it was determined that if BALS

greater than 400 mgidl were obtained, the animals frequently became comatose and died. To eliminate this problem, individual animals could be moved from

chamber to chamber, depending on the BALs, to maintain a stable baseline between

200 and 265 mg/dl. The BAL patterns could be easily maintained and were quite similar over any

number of replications. It was important to make daily adjustments in ethanol vapor

concentrations within each chamber to avoid problems. If the concentrations were

increased irrespective of BALs, rats very rapidly increased BALs and became comatose. By careful observations on a daily basis, animals that showed any signs of

behavioral lethargy or whose BALs were inordinately high could be placed in a chamber with a lower ethanol vapor concentration, thus preventing morbidity.

Body Temperature

Daily examination of one indicant of the overall health of the animals, body temperature, indicated that exposure to ethanol vapor in this inhalation system was


i

100

300

200

100

18 15 12 9 6 3 1 0 I 1 ,

DI

LE

RATS

RATS

DAYS PRIOR TO WITHDRAWAL

FIGURE 2. Blood alcohol levels (BALs) of Long Evans (LE) and Brattleboro (DI) rats on the day of withdrawal from ethanol (0) and on seven of the previous days of exposure to ethanol vapor. The BALs were determined by gas chromatography using blood sampled from the tail vein. The arrow indicates a 0.08% BAL, a level commonly associated with intoxication in humans.

not detrimental to the animals. Core temperature was maintained within the normal

range throughout the duration of the experiment. Figure 3 indicates that the animals

maintained their body temperature between 37 and 38°C for the entire period of ethanol exposure. This indicant was also helpful in assessing the deleterious effects

of exposure to high concentrations of alcohol. We have previously found that, if

core temperature dropped below 37”C, it was necessary to watch the animal carefully

to preclude the possible development of the severe hypothermia that frequently accompanies the onset of sequelae leading to coma.

Body Weight

Using this method, it was possible to prevent the excessive weight loss that has

been observed previously during exposure to high levels of ethanol. Since the Long Evans rats achieve a more pronounced gain in body weight than do the Brattleboro rats, the growth curves for the two rats are presented in Figure 4. Neither group

of animals showed the gradual increase in body weight that would occur over a 21-


-500

t-

GJ

$ -400

U z 3 -

&

g -300

-0 t

T

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L I L I I 1 I I 18 15 ‘? 9 6 3 1 0

LE RATS

DI RATS


FIGURE 3. Body weight, in grams (g), of Long Evans (LE) and Brattleboro (DI) rats on the day of withdrawal from ethanol (0) and on seven of the previous days of exposure to ethanol vapor.

day period. However, the body weights did not decrease significantly (t(62) = 1.54;

p > 0.10). Although some of the rats did show weight gain during this period, others

maintained very stable levels of weight, which kept the curves nearly flat.

Audiogenic Seizures

Audiogenic seizures were induced by exposing the unrestrained rat within 6-24

hr after removal of the ethanol to sound generated by a IO-cm electric bell for 60-

120 sec. The amount of sound generated by the bell was measured by a sound-

level meter (Type 2203, Bruel Kajer) and ranged from 101.5 dB at the origin of the source to 94.0 dB at a distance of 45 cm from the bell. The animals were placed individually in a 41 x 51 x 22-cm laboratory cage. The electric bell was suspended

from the top of the chamber on an open grid.

Alcohol Dependence and the Withdrawal Syndrome

Upon cessation of exposure to ethanol, the animals were observed closely for the appearance of specific withdrawal symptoms (Majchrowicz, 1981), both spon-

taneous and induced. A number of signs indicative of withdrawal occurred spon- taneously. These included: I) tail arching; 2) piloerection; 3) marked irritability and

tendency to fight; 4) broad-based gait; 5) intermittent tremor; 6) heightened startle

231

t

36.0

18 15 12 9 6 3 1 0 I I I I I I I 4


FIGURE 4. Body temperature, in “C, of Long Evans (LE) and Brattleboro (DI) rats on the day of withdrawal from ethanol (0) and seven of the previous days of exposure to ethanol vapor.

TABLE 1. Convulsion Frequency of LE and DI Rats

DURATION

OF ETHANOL

EXPOSURE NUMBER OF PERCENTAGE

RAT STRAIN (DAYS) CONVULSIONS CONVUSINC

LE (n = 18) 0 0 0

DI (II = 12) 0 0 0

LE (n = 12) 5 4 33

DI (n = 12) 5 1 8

LE (n = 6) 6 2 28

DI (n = 6) 6 2 28

LE (n = 6) 12 6 100 DI (n = 6) 12 4 66

LE (n = 7) 21 6 86

DI (n = 6) 21 6 100

Totals 5-6 9/36 25 12-21 22125 88


response; 7) alterations in normal activity; and 8) vocalizations. These symptoms

of the withdrawal syndrome were present in almost all animals examined.

When the animals were placed in the sound chamber and examined for the

response to audiogenic stimulation, nearly 90% (22/25) of the animals (both LE and

DI) examined displayed a very stereotyped behavior and rapid-onset seizures (Table

I). Following stimulus onset, the rats initially vocalized and then displayed very

rapid circling and running and decreased exploratory behavior and arched and

erect tails. These behaviors were followed by clonic-tonic seizures, which were of

a very rapid onset in these rats. These responses were observed in rats exposed to

ethanol for 12-21 days before withdrawal. In other animals exposed to ethanol for 5-6 days, only26% (g/36) developed a seizure in response to audiogenic stimulation.

In none of the 30 control animals tested was this type of response observed, and

none seized while exposed to the audiogenic stimulation.

DISCUSSION

The development of an effective method for inducing and assessing the ethanol

withdrawal syndrome in the rat is an extremely important component of the overall

research schema designed to delineate the complex effects of alcohol on the central

nervous system.

Previous attempts to design a suitable paradigm by which the ethanol withdrawal

syndrome could be induced and assessed have been characterized by numerous

drawbacks and methodological problems. The system that has most effectively

induced the ethanol withdrawal syndrome is that designed by Goldstein and Pal

(1971). The approach they chose, the involuntary inhalation technique, was very

simple and cost effective. However, in order to stabilize the level of alcohol in the

blood, these investigators introduced an additional confounding variable, the drug,

pyrazole. In addition to promoting the desired effect of blood alcohol stabilization

through an action that inhibits alcohol dehydrogenase, this drug also has a number

of additional effects: first, pyrazole independently effects depression within the

central nervous system (Rydberg and Neri, 1972); second, it enhances alcohol de-

pendence (Littleton et al., 1974); and, third, pyrazole interacts with a number of

biological systems with resultant toxicological effects (LeBlanc and Kalant, 1973; Lieber and DeCarli, 1973; Goldstein, 1978). Moreover, the inhalation method as

originally proposed has been criticized because it: 1) provides no nutritional con-

trols, 2) does not involve oral exposure to ethanol, 3) introduces the possible de-

velopment of respiratory irritation, and 4) presents a situation in which the risk of

ethanol overdose is greatly heightened (Pohorecky, 1981).

Subsequent modifications by Ferko and Bobyock (1977), as well as by Rogers and colleagues (1979), have overcome some of these problems. Ferko and Bobyock

(1977) designed an inhalation procedure that eliminated the use of pyrazole, yet, as Rogers et al. (1979) pointed out, in the Ferko-Bobyock method, BALs were not stable (BALs increased from less than 1.0 mg/ml on day 5 to more than 3.0 mg/ml

on day IO), nor were these BALs maintained at a high level (greater than 100 mg/


dl) for more than 4 days. Additionally, the animals were subject to coma and, in

some cases, death. The system designed by Rogers et al. (1979) offered a further refinement of the

inhalation technique and reduced the number of problems associated with this

method. Implementing a number of modifications, Rogers et al. (1979) were able

to induce chronic levels of blood alcohol that could be maintained at relatively high

levels over an extended period of time without apparent detriment to the animal.

Furthermore, these investigators provided a means by which the animals exposed

to ethanol were able to maintain normal body weight throughout the period of

exposure (Majchrowicz, 1981). Consistent with reports on the viability of studying the alcohol withdrawal syn-

drome in rats, only 30-50% of the animals tested, using the Rogers modifications,

exhibited convulsions induced by auditory stimulation. Moreover, in those in which

running fits and clonic-tonic seizures did occur, 90-95% died within 1 hr following

the seizure. This observation, too, is consistent with previous findings in which

very high rates of mortality have been reported for animals undergoing seizures

induced by audiogenic means (Majchrowicz, 1981). With the experimental paradigm that we report now, these latter two problems

have been eliminated. While audiogenic seizures were induced in almost 90% of

the animals, a mortality rate of less than 20% was obtained in each of the replications.

It is possible that the lower mortality rate and the higher percentage of withdrawal seizures observed are due to the very careful maintenance of individual BALs within

a much narrower range than in previous investigations (e.g., Rogers et al., 1979).

Only by closely monitoring the levels of alcohol in the blood on a daily basis is it

possible to prevent the marked variations in BALs that otherwise occur in individual

rats. By maintaining very stable levels of alcohol in the blood (200-260 mg/dI) over a

long period of time (12-21 days), respiratory irritation was kept to a minimum and

there was very little evidence of rats becoming comatose and dying due to ethanol

overdose. Our modifications of previous inhalation paradigms to incorporate daily

measurement of BALs, measurement of body temperature to assess the physiolog-

ical impact of a given BAL, and the maintenance of a consistently high BAL indicate

that the inhalation method offers a relatively simple and inexpensive means by

which animals can be made tolerant to and dependent upon ethanol. We also have

been able to demonstrate its utility for other rat strains, including the physiologically

“fragile” Brattleboro rat with diabetes insipidus.

Utilizing this method, it may be possible to delineate more clearly the neuro-

chemical alterations that occur during the development and maintenance of tol-

erance to ethanol. This method may provide a means by which the underlying cause

of the ethanol withdrawal syndrome and its biological and biochemical substrates

may be identified.

This work was supported by MRC. WDR is an AHFMR Fellow, WWF an AHFMR Summer Student, and

QJP an MRC Scientist and an AHFMR Scholar.

We thank G. Olmstead for typing the manuscript.


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Alcohol dependence and withdrawal in the rat An effective means of induction and assessment