National Public Health Institute, Department of Mental Health and Alcohol Research, POB 33, 00251 Helsinki, Finland
* Author to whom correspondence should be addressed at: National Public Health Institute, Alcohol Research Center, POB 33, 00251 Helsinki, Finland. Fax: + 358 9 47448133; E-mail: kai.lindros{at}ktl.fi
(Received 1 September 2004; first review notified 15 September 2004; in revised form 19 September 2004; accepted 19 September 2004)
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ABSTRACT |
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INTRODUCTION |
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The purpose of the present study was to develop a method for efficient chronic ethanol administration to mice, in order to study specific pathogenetic factors in genetically modified rodent strains. We originally attempted to apply our oral liquid diet rat administration model, but we encountered technical problems. Lack of convenient equipment for down-scaled liquid diet administration made us consider alternative approaches. Previously, rats had been offered ethanol solution in agar gel blocks and in addition normal solid food (Landrigan et al., 1989; Gentry-Nielsen et al., 2001
). We reasoned that ethanol-containing liquid diet could be made semi-solid by addition of agar and that this gel could be offered without the need of specially designed liquid diet bottles.
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MATERIALS AND METHODS |
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Preparation and delivery of agar gel diet
We prepared a modified high-fat/low-carbohydrate liquid diet where the commercial LieberDeCarli (LD 101A; Purina Mills, Richmond, IN) provides 50% of the calories as previously described (Lindros and Järveläinen, 1998). Briefly, the fat content is increased from 35% (calories) to 44% by adding extra corn oil and the protein content maintained at 16% by adding casein (technical grade; Sigma, St Louis, MO). Vitamins and minerals are also added to equal the composition of the LieberDeCarli diet, but no carbohydrate, so that its content is reduced from 11 to 5.5%. Ethanol provides 34.5% of the calories. The control diet contains 40% carbohydrate (maltodextrose) to equicalorically replace ethanol. The exact composition of the LD101 diet, including its content of vitamins and minerals, can be found at the address http://www.dyets.com/710260.htm. Agar [Lab M Agar No 2, Amersham MC 6, 0.5% (w/w)] was added as described below.
Gel diet was prepared twice weekly as follows: agar powder was suspended and mixed in half of the final water volume and heated in a water bath with frequent agitation. The corn oil, the rest of the water and the LD101A dry ingredients were then blended together and added to the completely dissolved and slightly cooled agar. Finally the ethanol was added and the diet solution thoroughly mixed. The warm viscous diet was immediately poured into plastic boxes which were sealed and stored at +4°C. Loss of ethanol during the preparation and storage was found to be negligible.
A portion of the ethanol diet (2025 g) was inserted into Falcon tubes equipped with an 2 x 2 cm opening (Fig. 1). The Falcon tube was mounted in a tilted position inside the pellet grid of the cage using metal strings. The daily diet consumption was determined by weighing the Falcon tube. Control mice also received their diet in Falcon tubes. All mice also had access to a water bottle during the experiment.
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Biochemical assays and liver histopathology
Blood ethanol levels were determined by sampling from vena saphena (25 µl) from groups of mice at weekly intervals. After 6 weeks of treatment, mice were anaesthetized with sodium pentobarbital (60 mg/kg i.p.), blood samples collected by heart puncture and plasma separated and stored at 20°C. Pieces of liver were collected in buffered formalin, embedded in paraffin, cut in 6 µm sections and stained with hematoxylin/eosin. Steatosis was graded blindly from 04 as follows: 1 = <25% of cells containing fat, 2 = 2650%, 3 = 5175%, 4 = >75%.
The concentration of ethanol in blood and gel diet was determined by head-space gas chromatography (Hu et al., 1995). For assay of liver triglycerides as glycerol, 1 ml of liver methanolchloroform mixed homogenate was washed with sodium chloride, the resultant extract was dried and dissolved in 200 µl of tetraethylammoniumhydroxide (1:28 with 95% ethanol), incubated at 60°C for 30 min and mixed with 200 µl 50 mM HCl. Glycerol and serum alanine aminotransferase (ALT) activity were measured enzymatically by using commercial kits (Boehringer-Mannheim, Germany).
The data are expressed as means ± SD. Student's t-test was used to test statistical difference between groups. Pathological scores were compared using the MannWhitney test. A P-value < 0.05 was considered statistically significant.
The study had been approved by the Committee for Animal Experimentation of the National Public Health Institute in Helsinki, Finland.
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RESULTS |
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Blood ethanol levels and liver changes
Both groups of mice gained weight during the 6 week gel diet regimen, but the control mice more (6.7 ± 1.9 g) than the ethanol-treated mice (0.8 ± 1.8 g) (Fig. 2). The average daily diet intake by ethanol-treated mice during the 4 week period with the final concentration of ethanol in the diet (5.3%) was 16.3 ± 2.2 g. There were minor day-to-day fluctuations but the average weekly intake was quite stable. The controls were always given the equivalent amount of control diet. Occasionally some control animals did not eat all diet offered. The daily intake of ethanol diet corresponded to 2324 g absolute ethanol per kg body weight. The additional average intake of fluid from the water bottle was low both for ethanol-treated (1.6 ml) and control mice (1.4 ml).
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Histopathological evaluation of liver samples taken at termination revealed that after 6 weeks of ethanol gel consumption, the mice had developed marked mixed micro- and macrovesicular steatosis, reflected in an increase in the liver/body weight ratio and triglycerides (Table 2). Occasional mononuclear infiltration was also seen (results not shown). In addition, the serum activity of alanine aminotransferase was more than doubled.
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DISCUSSION |
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Although agar blocks have been used before as a vehicle for ethanol administration, the combination of liquid diet and ethanol is new. There are several advantages with this model. The method is simple and does not require any special equipment. The diet does not need to be prepared daily. Compared to ordinary liquid diet provided in bottles, there is no loss of diet due to bottle leakage or layering of diet during storage, which can be a problem with liquid diet. In the present study, control animals also received their daily portion in Falcon tubes, but the procedure could have been further simplified by simply offering the gel on Petri dishes. Furthermore, the agar gel contains enough water to satisfy the animal's daily fluid requirement, as evidenced from the minimal extra intake from the water bottle.
Evaporation of ethanol from the gel proved to be a greater problem than we had anticipated on the basis of published studies. Neither the study by Landrigan et al. (1989) nor the later study by Gentry-Nielsen et al. (2001)
appreciated the magnitude of this problem. Anticipating little evaporation, these studies reported values of daily intake of ethanol exceeding 40 g/kg body wt. These values are clearly too high, since the rat cannot metabolize more than 1418 g/kg body wt in 24 h (Lindros et al., 1983
). Thus, evaporation of ethanol from the agar blocks must have been massive, and consumption of ethanol from agar blocks cannot be calculated without correction for this.
In spite of pair-feeding, the control mice gained more weight in comparison with those given ethanol diet. This energy wastage syndrome has long since been recognized (Pirola and Lieber, 1976), but the phenomenon is still not fully understood (Suter, 2000
). The phenomenon may be accentuated in mice due to their fast metabolism. There also were much larger individual variations in blood ethanol levels during the experiment as compared to rats on an ordinary liquid diet. We suggest that this also can be related to the much faster rate of ethanol metabolism in the mice, so that occasionally most of the ethanol has disappeared before the animal eats more diet.
Finally, we suggest that although the present study was based on mice, it should be applicable for other rodents as well. For instance, by delivering the agar gel in bigger plastic tubes (200 ml) equipped with suitably sized eating holes the model should be useful in chronic studies with rats as well.
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REFERENCES |
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