Effects of low ethanol doses on heart rhythm in rabbits.

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Effects of Low Ethanol Doses on Heart Rhythm in Rabbits
K. Sh. Nadareishvili, I. I. Meskhishvili, D. D. Kakhiani,
G. L. Ormrtsadze, M. T. Khvedelidze, and E. T. Chitanava
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 9, pp. 306-310, September, 2004
Original article submitted August 18, 2003
Effects of low ethanol doses on the vagosympathetic mechanisms of heart rhythm regulation
were studied in rabbits. Analysis of heart rhythm variability showed that single intragastric
administration of 0.5 mg/kg ethanol caused tachycardia in animals with initial predominance
of vagal activity and bradycardia in animals with predominating sympathetic mechanisms.
This was associated with general activation of all regulatory effects on the heart rhythm and
a drastic increase in power spectrum for all frequency ranges, though with a certain deficiency
of vagal effects. However, after 24 h the vagal component of the spectrum drastically in-
creased in animals of both groups, while other parameters did not differ from the control.
Presumably, this rebound can be used as a physiological marker, ethanol tolerance measure,
formation of the abstinence syndrome and liability to alcoholism.
Key Words: ethanol; heart rhythm variability; rabbit; vagosympathetic balance
Institute of Radiobiology and Radiation Ecology, Academy of Sciences
of Georgia, Tbilisi. Address for correspondence: radiobio@caucasus.
net. Kakhiani D. D.
Effects of ethanol in low doses in various cardiovas-
cular diseases, including heart rhythm disorders, at-
tract much recent attention [2-6,9,10,12,15]. Experi-
mental and epidemiological reports [4,6,10] present
data unambiguously confirming the favorable effects
of moderate ethanol consumption at all levels of orga-
nization of biological systems, from molecular [7,11]
to population [14]. However, even very low ethanol
doses are harmful, for example, in some tumors or
during convalescence after hepatitis C, etc., [15]. Pos-
sible mechanisms of these effects remain a topic for
discussion, as it is not clear whether these effects are
caused by ethanol and its derivative acetaldehyde or
by their cooperation with other organic and mineral
components of alcoholic drinks [2,4,7,9]. The role of
individual and population emotional motivation and
social strain associated with personal life style and
with the genetic, ethnic, climatic, and other charac-
teristics determining the efficiency of “therapeutic”
alcohol doses and the formation of predisposition for
alcoholism [3,5,7,14] was never studied. Experimental
simulation of these conditions, including simulation in
humans, involves great difficulties. However, detec-
tion of primary physiological reactions to a single low
dose of alcohol can prompt approaches to experimen-
tal studies of not only above listed problems, but of
the mechanisms triggering the development of alcoho-
lism. The common pattern of activation of the neuro-
vegetative sphere and emotional motivation behavior
after a single low dose of alcohol is a measure of the
adaptive potentialities and liability of humans or ani-
mals to alcoholism [3,11]. The study of the main heart
rhythm parameters and their correlations with single
ethanol intake in low dose is interesting from the theo-
retical and practical viewpoints, because the parame-
ters of heart rhythm variability (HRV) can be more
informative than just the mean heart rate [3].
We previously studied the effect of single intra-
gastric low dose of ethanol on the rabbit HRV [8], but
the time course of changes in the structure of HRV
parameters in animals with initially different neuro-
vegetative status has not been studied. Now we inves-
tigated the effects of low ethanol doses on heart
rhythm of Chinchilla rabbits with initially different
vagosympathetic balance and studied changes in the
structure of HRV parameters reflecting the vagosym-
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272
pathetic balance and, presumably, neurovegetative sta-
tus in general [1].
MATERIALS AND METHODS
Experiments were carried out in autumn-winter on 14
male Chinchilla rabbits (2.5-3.0 kg). The animals were
born and bred in the local Breeding Center. At the age
of 4 months the rabbits were placed into separate cages
in which they were kept during the entire period of
observation under the same conditions with free access
to food and water. Before the experiments the neuro-
vegetative status of animals was evaluated [1] and
they were divided into vagotonics (VT) and sympatho-
tonics (ST). Spectral analysis of HRV included deter-
mination of the total power spectral density (msec2),
integral powers of spectrum densities for high fre-
quencies (msec2) at 1.7-0.4 Hz taken for the marker of
mainly vagal activity, for low frequencies (msec2) at
0.4-0.15 Hz taken for the marker of mainly sympa-
thetic activity, and for very low frequencies (msec2),
the genesis and physiological interpretation of which
remain disputable.
Analysis of time parameters included pulse rate,
mean R—R intervals, standard deviation of the mean
R—R interval, mode at 2 msec step of histogram plot-
ting, mode amplitude, range of R—R interval devia-
tions, and R. M. Baevskii’s index of strain adapted for
rabbits in standardized calculation.
HRV parameters were compared using ANOVA
and MANOVA methods. The significance of differ-
ences was evaluated using Student’s, Fisher’s (F), and
Wilk’s (λ) tests. Such a wide spectrum of analyzed
HRV parameters was chosen for solving the concomi-
tant problem of determining the most informative or
minimum list of simple tests sufficient for reliable
evaluation of the vagosympathetic balance and facili-
tation of preliminary selection of animals. Other de-
tails of the experimental method and technology were
described previously [1].
Ethanol (0.5 mg/kg, 40% solution) was admini-
stered into the stomach through a nasogastral catheter
under local anesthesia with 2% dicaine (3-4 min be-
fore intubation). Control animals received the same
volume of normal saline (placebo). The study included
3 placebo-ethanol stages with 1-week intervals, quan-
titative analysis was made using weighed means.
RESULTS
All basal values of HRV parameters in VT and ST
rabbits, compared by Fisher’s test, except for power
spectral density at low and high frequencies (%) dif-
fered significantly from each other (Tables 1, 2). A si-
milar picture was observed in multiparametrical com-
parison of the time and frequency parameters, taken
separately and together, by Wilk’s λ test (λ=0.001-
0.005). These control values of individual parameters
of HRV and the entire picture of multiparametrical
differences between VT and ST are within the confi-
dence interval of previously published data [1] and
confirm the conclusion that the complex of HRV para-
meters can be used for grouping the rabbits by the
vagosympathetic balance, which with certain probabi-
lity reflects the general neurovegetative status. These
two groups of animals principally differ by changes in
their mean heart rates and R—R equivalent after low
doses of ethanol. VT developed relative tachycardia
after low doses of ethanol with an appreciable increase
in the pulse rate at the stage corresponding to maxi-
mum ethanol concentration in the blood (2-3 h), while
in ST the pulse rate decreased significantly at this
stage. Differences between these groups, were clearly
pronounced before ethanol administration, but then
leveled and became statistically negligible. After 24 h
the initial R—R values (and hence, heart rates) were
virtually restored in both groups.
It could be expected that after these drastic and
opposite changes in the heart rate and R-R the values
of other time parameters after ethanol intake would
change depending on the pulse rate changes in animals
of both groups. However, this did not happen: standard
deviation of the mean R—R interval and the range of
its deviations drastically increased in both VT and ST,
while the mode amplitude and strain index decreased
significantly. Significant differences between these
parameters in VT and ST rabbits persisted during all
periods of observation. Considering the possible chan-
ges in the vagosympathetic balance and its internal
structure, these changes in the time parameters cannot
be interpreted unambiguously. If the development of
tachycardia in VT is associated with ethanol suppres-
sion of the vagus tone [12], the development of brady-
cardia in ST can by no means be due to this factor.
We should like to emphasize that complex comparison
of the pattern of all time parameters by the λ test
shows that the significant differences between VT and
ST are retained during all stages of observation, though
they are less pronounced than in the control.
Spectral characteristics are changing in the same
direction: 1-3 h after administration of a low dose of
ethanol the total spectral power density and integral
values of power spectra in all frequency bands drasti-
cally increased in both groups, while specific contri-
bution of each of them is changed negligibly (Table
1). One-two hours after ethanol intake only the per-
centage of the integral spectral power density at high
frequencies decreased insignificantly in animals of
both groups compared to the control. Due to this, the
differences between all parameters in VT and ST were
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273
somewhat leveled. However, multiparametrical com-
parison of all spectral characteristics (λ test) still sho-
wed statistically significant differences, which was
explained by the gradient of changes in individual
parameters compared to the control. For example, at
different stages after ethanol administration the total
power of spectrum density in VT rabbits increased 3-
3.5 times vs. 7-8-fold in ST rabbits. For low frequen-
cies these values were 3.5-4 and 6-7-fold, for high
frequencies 2-2.5 and 3-4-fold, respectively. The per-
centage of integral power of spectrum density at high
frequencies increases in animals of both groups as
early as 3 h after the dose intake, while after 24 h this
increase was significant (p<0.05 vs. the control),
though there was no significant difference between VT
and ST.
Low ethanol doses caused activation of all regula-
tory effects on the heart rhythm, which was more pro-
nounced in ST. Activation of the vagus tone was some-
what delayed. Moreover, the percentage of integral
power of the spectrum density at high frequencies
taken as a marker of parasympathetic effects on the
heart rhythm changed specifically: a rebound was
observed. No difference in this parameter between VT
and ST was observed after 24 h, though rebound in the
former case surpassed the initial level by 57% and in
the latter by 33%. On the other hand, the absolute
power of spectrum density at high frequencies during
this period was in VT significantly higher than in con-
trols, while in ST complete recovery of this parameter
was observed.
If we consider the neuroanatomical and functional
relationships between the central parasympathetic
structures in the organization of emotional motivation
and common adaptive behavior and some other as-
sumptions of the polyvagal theory [12], we must admit
that along with changes in the heart rate [3], vagus
activity rebound 24 h after a low ethanol dose can be
an additional and even more informative physiological
marker, a measure of ethanol tolerance, formation of
TABLE 1. Time Parameters of Rabbit HRV (VT as Numerator, ST as Denominator) before and after Intragastric Administration
of Ethanol (M±m)
Pulse rate/min 235.2±3.6 243.3±6.5 26+1.7±5.6* 281.0*±11.8 242.5±7.3
293.6±5.0 283.8±3.6 275.0±4.2 276.0±5.0 282.6±3.5
pF<<0.001 pF<<0.001 pF>0.12 pF>0.42 pF<0.001
R—R, msec 254.9±4.0 246.6±6.3 229.3±4.9* 213.5±9.0* 247.4±7.6
204.3±3.5 211.4±2.6 218.1±3.4 217.4±4.0 212.3±2.7
pF<<0.001 pF<<0.001 pF>0.86 pF>0.29 pF<<0.001
Standard deviation
of the mean R—R, msec 7.7±0.5 15.7±2.0* 14.5±2.1* 12.4±2.0 8.0±0.6
3.0±0.2 6.0±0.5** 6.5±0.6** 5.4±0.4** 3.5±0.3
pF<<0.001 pF<<0.001 pF<<0.001 pF<0.016 pF<<0.001
Mode amplitudes, % 14.6±0.8 8.7±1.0** 9.4±1.1** 13.3±2.0 14.1±1.3
29.5±1.6 18.1±1.5** 18.1±2.1** 21.2±1.4* 27.4±1.8
pF<<0.001 pF<0.001 pF<0.01 pF<0.016 pF<<0.001
Range of R—R interval
deviations, msec 37.1±0.4 69.2±6.1** 61.3±6.4* 55.0±6.1 39.2±3.0
15.2±0.2 28.2±2.3** 30.5±2.8** 25.6±1.6** 17.7±1.5
pF<<0.001 pF<<0.001 pF<0.002 pF<<0.001 pF<<0.001
Strain index 3.4±0.4 1.1±0.2** 1.5±0.4* 2.4±0.6 2.9±0.6
11.0±1.1 4.7±1.0* 5.3±1.4* 5.2±0.9* 10.5±1.2
pF<<0.001 pF<0.001 pF<<0.001 pF<0.05 pF<<0.001
MANOVA λ=0.0025 λ=0.03 λ=0.06 λ=0.04 λ=0.05
Note. Here and in Table 2: *p<0.01, **p<0.001 compared to the control.
Parameters
24321
Time after intragastric administration of ethanol, h
Control
K. Sh. Nadareishvili, I. I. Meskhishvili, et al.

274
the abstinence phenomenon and liability to alcoho-
lism. These problems deserve further investigation
with consideration for the species specificity of dose
dependence, significance of the initial vagosym-
pathetic balance from the viewpoint of evaluating
the contribution of total metabolic and circulatory
activation [13], on the one hand, and direct effect of
ethanol on the pacemaker and central and peripheral
nervous mechanisms of heart rhythm regulation, on
the other.
Selection of volunteers by psychophysiological
characteristics is a common scientifically-based re-
quirement to biomedical studies on humans. When the
same problems are solved in animal experiments, this
approach rarely used. It seems that the traditional cri-
teria of selection of objects of investigation (species,
genetic strain, age, weight, sex, etc.) are not always
sufficient for practical studies in experimental biology
and medicine. Selection by the HRV parameter is suf-
ficiently correct, noninvasive, and operative [1,8].
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TABLE 2. Rabbit HRV Frequency Parameters (VT: Numerator; ST: Denominator) before and after Intragastric Ethanol Intake
(M±m)
Spectrum density power, msec2
total 85.8±22.7 267.6±71.5 298.9±77.8 179.5±26.4 90.2±23.2
21.9±2.8 155.9±16.4** 167.0±30.1* 110.2±25.4* 17.3±1.5
pF<0.003 pF>0.6 pF=0.05 pF>0.25 pF<0.001
at very low frequencies 38.8±8.5 137.4±36.6 138.3±30.4* 74.7±14.7 32.7±10.5
6.6±1.3 2.9±12.5** 83.1±18.3* 42.6±9.5 5.8±1.0
pF<0.038 pF>0.47 pF<0.03 pF>0.16 pF<0.004
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2.7±0.2 7.3±0.9** 9.3±1.4** 7.8±0.8** 2.7±0.2
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38.0±3.0 42.2±4.5 38.8±4.5 33.6±6.3 34.8±4.2
pF>0.07 pF>0.31 pF>0.2 pF>0.14 pF>0.16
High frequencies, % 16.1±1.3 11.0±1.2 11.6±0.8 17.0±1.7 25.2±2.8
17.9±1.5 9.2±0.9** 11.3±1.0** 15.7±4.2 23.9±1.9
pF>0.48 pF>0.8 pF>0.1 pF>0.58 pF>0.7
MANOVA λ=0.05 λ=0.42 λ=0.06 λ=0.16 λ=0.08
Parameters
24321
Time after intragastric administration of ethanol, h
Control
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K. Sh. Nadareishvili, I. I. Meskhishvili, et al.