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Short-Term Ethanol
Administration Does
Not Change the Total
Pyramidal Neuron Number
of the Rat Hippocampus:
A Stereologic Study
Ramazan Kozan, MD
Mustafa Ayyildiz, PhD, Assoc Prof
Department of Physiology
Ondokuz Mayis University School of Medicine
Samsun, Turkey
Orhan Bas, PhD, Assist Prof
Department of Anatomy
Afyon Kocatepe University School of Medicine
Afyon, Turkey
Süleyman Kaplan, PhD, Prof
Department of Histology and Embryology
Erdal Agar, PhD, Prof
Department of Physiology
Ondokuz Mayis University School of Medicine
Samsun, Turkey
ABSTRACT
In the hippocampus, short-term exposure to ethanol (EtOH) has been shown to
inhibit some functions, and nitric oxide (NO) is an important modulator of physi-
ologic processes. In this study, investigators explored the effects of EtOH on the
total number of neurons in rat CA regions and the possible neuroprotective role of
NO. The role of NO in rats given EtOH was examined with the use of a nonselec-
tive inhibitor of NOS (N[G]-nitro-L-arginine methyl ester [L-NAME], D[G]-nitro-L-
arginine methyl ester [D-NAME]), a central selective inhibitor of NOS (7-NI), and
a donor of NO (L-arginine). Toward this end, rats were randomly divided into
6 groups: control (saline 3 g/kg intraperitoneal [ip]), ethanol (ethanol 3 g/kg ip),
L-NAME (ethanol 3 g/kg ip + L- NAME 60 mg/kg ip), L-arginine (ethanol 3 g/kg ip
+ L-arginine 1 g/kg ip), D-NAME (ethanol 3 g/kg ip + D-NAME 60 mg/kg ip),
231
Advances
in Therapy®
Volume 24 No. 2
March/April 2007
Address correspondence to
Dr. Erdal Agar
Department of Physiology
Faculty of Medicine
University of Ondokuz Mayis
55139 Samsun, Turkey
Email: eragar@omu.edu.tr
©2007 Health Communications Inc
Transmission and reproduction of this material in whole
or part without prior written approval are prohibited.
0936
and 7-NI (ethanol 3 g/kg ip + 7-NI 40 mg/kg ip). Blood ethanol concentrations were mea-
sured 90 min after EtOH administration. Means (value±standard deviation) of total pyramidal
neuronal numbers in the right hippocampus were estimated using the optical fractionator
counting method. Values were as follows: 446,558±6207, 483,517±20,311, 464,588±30,637,
479,688±10,780, 458,294±17,770, and 477,281±7641 in the control, ethanol, L-arginine,
7-NI, L-NAME, and D-NAME groups, respectively. No significant differences were observed
between groups (P>.05). The results of the present study imply that short-term administration
of EtOH does not affect total pyramidal neuronal number in the right hippocampus of the rat.
Furthermore, NO does not change the effects of short-term EtOH on total pyramidal neuronal
number in the right hippocampal CA regions of the rat. Additional studies are needed to
clarify the role of NO and NOS inhibition in the effects associated with EtOH given on
a short-term basis.
Keywords: hippocampus; ethanol; nitric oxide; neuron, stereology
INTRODUCTION
The effect of ethanol (EtOH) has been associated with significant damage to the
heart, liver, gastrointestinal tract, and brain.1-3 Human and animal research studies
have provided evidence that the central nervous system (CNS) is vulnerable to the
damaging effects of EtOH exposure, as indicated by neurochemical,4electrophysio-
logic,5and neuroanatomic studies.6One particular form of neuroanatomic damage
is neuronal loss. EtOH administration is known to cause substantial neuronal loss in
several regions of the brain, such as a decrease in retinal ganglion cells in primates
after prenatal exposure to EtOH.7In particular, the hippocampal formation is vul-
nerable to the effects of EtOH exposure that occurs during the early postnatal peri-
od in the rat.8
A free radical gas known as nitric oxide (NO) plays an important role in a diverse
range of physiologic processes.9,10 NO is synthesized from the precursor L-arginine
by nitric oxide synthase (NOS), which transforms L-arginine into NO and cit-
rulline.10 NO is produced throughout the CNS in response to N-methyl-D-aspartate
(NMDA) receptor activation and other stimuli; it is an unconventional neurotrans-
mitter that freely diffuses across biologic membranes.11 It is known that NOS exists
in 3 isoforms: inducible (iNOS), endothelial (eNOS), and neuronal (nNOS).12 In the
adult rat or human hippocampus, nNOS expression can be detected in pyramidal
cells and interneurons throughout the CA1–CA3 region and in the granular cell
layer of the dentate gyrus.13 A recent study indicates that NO may play an important
role in dependence on substances such as opioids, nicotine, and EtOH.10 Bonthius et
al14 reported that NO limits EtOH-induced neuronal death in the CNS. Pharma-
cologic manipulations that decrease NO production can worsen the neurotoxic
effects of EtOH both in vitro and in vivo.14 Evidence suggests that NO-dependent
pathways modulate the acute effects of EtOH in vivo.15 It was found that intrahip-
pocampal injection of NOS inhibitors enhanced the anxiolytic effects of EtOH in
rats. Moreover, intrahippocampal administration of NO donors reduced the anxi-
olytic effects of EtOH.15 These findings suggest that NO may play a protective role
against the toxic effects of EtOH.
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R. Kozan, et al
Effects of Ethanol on Rat Hippocampus
Investigators in this study used a nonselective inhibitor of NOS (N[G]-nitro-
L-arginine methyl ester [L-NAME], D[G]-nitro-L-arginine methyl ester [D-NAME]),
a central selective inhibitor of NOS (7-NI), and a donor of NO (L-arginine) to exam-
ine the effects of EtOH on total pyramidal neuron number in the right side of the
hippocampus and the role of NO in rats given EtOH. Few studies have used unbi-
ased stereology to assess alterations in total neuronal number in the CA region of
rats acutely exposed to EtOH.
MATERIALS AND METHODS
All experiments were conducted according to institutional guidelines. Animals
were treated in accordance with ethical principles (Principles of Laboratory Animal Care
[NIH publication no. 86-23, revised 1985]), and the institutional Ethics Committee
on the Use of Live Animals in Research approved the experimental protocol.
Appropriate measures were taken to minimize pain and discomfort. All rats were
housed 1 per cage with wood bedding and were given standard laboratory chow. Rats
were maintained on a 12 h:12 h light/dark cycle, and room temperature was main-
tained at 21±2ºC.
After 1 wk of acclimation, 36 male Wistar white rats weighing 195±25 g were ran-
domly divided for cell counting into 6 groups as follows: control (saline 3 g/kg
intraperitoneally [ip]), ethanol (ethanol 3 g/kg ip), L-NAME (ethanol 3 g/kg ip +
L-NAME 60 mg/kg ip), L-arginine (ethanol 3 g/kg ip + L-arginine 1 g/kg ip),
D-NAME (ethanol 3 g/kg ip + D-NAME 60 mg/kg ip), and 7-NI (ethanol 3 g/kg ip
+ 7-NI 40 mg/kg ip). Animals in study groups, other than controls, were given
EtOH as a single dose at 3 g/kg ip. Blood ethanol concentrations were determined
from tail blood samples drawn 90 min after EtOH was administered to separate
groups of rats (n=18).
Animals were deeply anesthetized with urethane (1.25 g/kg ip) and, 3 h after
EtOH exposure, were perfused via the left cardiac ventricle with 0.9% NaCl and 10%
formaldehyde that were preheated to 37ºC before use. Thereafter, the brain was
immediately removed from the cranium and was stored in the same fixative for at
least 2 wk.
Brains were coded in such a way that the experimenter was blind to the experi-
mental condition during cell counting. The right hemispheres of the brains, treated
through routine tissue processing, were embedded in Paraplast (Sigma-Aldrich,
Munich, Germany) and were sectioned at 40-µm thickness in the horizontal plane.
Slides were stored overnight in the oven (60ºC) and were stained with cresyl violet.
Stereologic Procedures: Estimation of Total Neuron Number
in the CA Region
Total pyramidal neuron numbers in the CA region of the right hemisphere were
estimated using the optical fractionator counting method. In all, 15 to 20 sections
were used to make an unbiased estimation of total cell numbers in the CA region
through selection of every fifth section, according to a systematic random sampling
procedure.16 A sampling area of 294/22,500 µm2was used. Dissector height was
10 µm, and a 5-µm zone at the uppermost part of the section was excluded from the
analysis at every step as the upper guard zone. Thus, a thickness-sampling fraction
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Volume 24 No. 2, March/April 2007
of 10 µm/t was used, where trepresents mean section thickness. Stereologic analy-
ses of the CA region of the hippocampus were performed according to principles
described previously.17
At stereologic workstations that included a CCD (charge-coupled device) digital
camera (JVC, Tokyo, Japan), a personal computer with an image capture card
(FlashPoint 3D, Integral Technologies, Indianapolis, Ind, USA), a monitor (Hyundai,
Seoul, South Korea), a computer-controlled motorized specimen stage (Prior
Scientific, Rockland, Mass, USA), a microcator (Heidenhain, Traunreut, Germany),
and a light microscope (Leica DMR; Leica Microsystems, Inc., Wetzlar, Germany),
the total number of neurons in the CA region was estimated. A software program
(CAST-GRID\Computer Assisted Stereological Toolbox; Olympus, Copenhagen,
Denmark) was used to control, measure, and record stereologic data and to capture
digital images of all sections. This system reproduced microscopic images (obtained
through a 100×oil immersion objective, numeric aperture, 1.40) on the computer
monitor at a final magnification of 5140, which allowed accurate recognition and
counting of pyramidal cells in the CA region. Total neuron number in the sampled
sections was estimated using ‘’the unbiased counting frame’’ with 294 µm2in area.
A counting frame was placed onto sections in a systematic, uniform, randomized
manner,18 and appropriately sampled nuclei of neurons at the widest portion of the
counter were counted. Meander sampling of sectioned hippocampal profiles was
conducted over successive, systematically randomized steps. This ensured that all
locations within a sampled section were equally represented, and that all cell pro-
files had an equal probability of being sampled, regardless of shape, size, orienta-
tion, and location.
The total neuron number was estimated according to the formula given below:
N=ΣQ•1•1•1
ssf asf tsf
where ΣQ represents the total number of neurons counted in all optically sampled
fields of the hippocampus, ssf is the section-sampling fraction (1/5), asf is the area-
sampling fraction (294/22,500), and tsf is the thickness-sampling fraction (defined
by dissector height [10 µm] divided by estimated mean section thickness). The effi-
ciency of sampling and the convenient number of sampled cells were checked by
estimation of coefficient of error (CE). The coefficient of variation (CV) for each
group was also estimated to determine whether each group included an appropri-
ate number of animals.19
Comparisons between groups were carried out through 1-way analysis of vari-
ance with multiple comparisons performed according to Dunn’s post hoc method.
Statistical differences were significant at P<.05.
RESULTS
Blood ethanol concentrations were measured 90 min after EtOH administration
(in different groups, n=18). Values were: 0 mg/dL, 247.3 mg/dL, 252.8 mg/dL,
228.6 mg/dL, 247.4 mg/dL, and 231.5 mg/dL in the control, ethanol, L-arginine,
L-NAME, D-NAME, and 7-NI groups, respectively. The mean blood ethanol con-
centration value was 241.5 mg/dL in the groups to which EtOH was administered.
No significant difference was noted between groups given EtOH.
234
R. Kozan, et al
Effects of Ethanol on Rat Hippocampus
Means (value±standard deviation) of total pyramidal neuronal numbers in
the right hippocampus were estimated as follows after administration of a single
dose of 3 g/kg EtOH: 446,558±6207, 483,517±20,311, 464,588±30,637, 479,688±10,780,
458,294±17,770, and 477,281±7641 in the control, ethanol, L-arginine, 7-NI, L-NAME,
and D-NAME groups, respectively (Table). No significant differences were observed
between groups (P>.05) (Figure).
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Group ΣQ Step No. CE CV
Control, n=6 219 464 0.047 0.09
Ethanol, n=6 250 522 0.045 0.09
L-Arginine, n=6 234 491 0.046 0.14
7-NI, n=6 260 472 0.046 0.05
L-NAME, n=6 240 439 0.048 0.07
D-NAME, n=6 261 468 0.046 0.03
Mean Dissector Cell Number (ΣQ), Step Number, and CE and CV of Cell Estimation
Control
(n=6) Ethanol
(n=6) L-Arginine
(n=6) L-NAME
(n=6) D-NAME
(n=6) 7-NI
(n=6)
Neuron Numbers
600,000
550,000
500,000
450,000
400,000
350,000
300,000
The effects of acute ethanol administration (3 g/kg ip) on the total number
of neurons in the right hippocampus of the rat.
No significant difference was noted between groups (P>.05): control (saline 3 g/kg ip), ethanol (3 g/kg ip),
L-NAME (ethanol 3 g/kg ip + L-NAME 60 mg/kg ip), L-arginine (ethanol 3 g/kg ip + L-arginine 1 g/kg ip),
D-NAME (ethanol 3 g/kg ip + D-NAME 60 mg/kg ip), and 7-NI (ethanol 3 g/kg ip +7-NI 40 mg/kg ip).
Data are expressed as mean±standard deviation (SD).
DISCUSSION
It has long been known that EtOH is easily administered in a noninvasive man-
ner, that it is rapidly absorbed, and that it produces neurodegeneration in specific
regions of the brain, including the hippocampus.20 Investigators in the present study
used a rat model and a nonselective inhibitor of NOS (L-NAME), D-NAME,
a central selective inhibitor of NOS (7-NI), and a donor of NO (L-arginine) to evalu-
ate the effects of a 3-g/kg single dose of EtOH on the total number of neurons seen
on the right side of the hippocampus, and to explore the role of NO.
In previous experimental studies, it was found that short-term administration of
EtOH results in many changes, such as a decrease in different types of cells in the hip-
pocampus.21,22 Nixon and Crews22 found that a 5-g/kg dose of EtOH decreased adult
neural progenitor cells by 40% at 5 h. They also reported that an acute dose of EtOH
decreased the number of BrdU+ cells in adult hippocampus 5 h after administra-
tion.22 In contrast to the results of Nixon and Crews22 and Cameron and McKay,21
investigators in the present study found that an acute dose of EtOH (3 g/kg) did not
significantly change the total number of neurons in the right hippocampus of the rat.
It should be noted that a higher dose (5 g/kg) of EtOH was used in earlier studies
than was used in the present study. It is also well known that the number of newly
created cells (ie, BrdU+ cells), when compared with the total cell number in the hip-
pocampus, is too low. Thus, the results suggest that even though EtOH decreased
the number of immunopositive cells in this region, this cell loss did not result in sig-
nificant cell death when compared with total outcomes. Berry and Matthews23
reported that a 2.25-g/kg dose of EtOH administered acutely selectively impaired
spatial memory, however, and that hippocampal formation is involved in the learn-
ing and memory process. Earlier investigators evaluated only the numbers of spe-
cific cell types such as BrdU+ or neural progenitor cells, whereas researchers in
the present study estimated the total number of cells. It is proposed here that EtOH
may change the numbers of cells in specific cell groups but may not cause a change
in the total number of cells.
It has been shown that alcohol inhibits NO production in vivo; thus, it may be
suggested that NO is of relevance in the pathogenesis of alcohol-induced brain dam-
age.24 Zhang et al25 reported that administration of an NO donor induces neurogen-
esis in the dentate gyrus of the hippocampus. Jang et al26 showed that acutely
alcohol-intoxicated rats produce a significantly reduced number of proliferating
cells and decreased NOS expression within the dentate gyrus of the hippocampal
formation. They also showed that alcohol injection inhibits the number of BrdU+
cells and the level of NOS expression in the dentate gyrus of the hippocampus in a
dose- and duration-dependent manner.26 In contrast, administration of a nonselec-
tive inhibitor of NOS (L-NAME), D-NAME, a central selective inhibitor of NOS
(7-NI), and a donor of NO (L-arginine) did not significantly change the total num-
ber of cells in the right hippocampus of the rat. Ikeda et al27 reported, however, that
NOS activity in various brain regions of mice, such as the frontal cortex, striatum,
hippocampus, and cerebellum, remained similar to that seen in controls after short-
(3 g/kg) and long-term (3.3 g/kg/d; 3.5 d) administration of EtOH.
236
R. Kozan, et al
Effects of Ethanol on Rat Hippocampus
CONCLUSION
Study results presented here show for the first time that short-term EtOH admin-
istration did not affect the total number of cells in the right hippocampus of the rat.
The same dose of EtOH may reduce the numbers of specific cell types such as NOS+
and BrdU+ cells, but not the total number of cells in the hippocampus. The inhibitor
of NOS and the donor of NO did not significantly change the numbers of cells pro-
duced. It is important to note that findings regarding the influence of NOS inhibi-
tion on the effects of EtOH are inconclusive. Additional studies are needed to clarify
the role of NO and NOS inhibition in the acute effects of EtOH.
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