56 I
21
pocampus and the importance of Ca for the functioning
diet except that sucrose was substituted isocalorically for and survival of hippocampal cells has been well estab-
ethanol. After 4–5 days, the mice which exhibited stage 2 lished. Calcium binding proteins have been implicated as
or 3 of intoxication signs according to Freund’s classifica- important regulators of neuronal degeneration in pathologi-
tion [10] were selected for morphological observation. The cal process [1]. Calbindin-D28k, which is a member of the
intoxication signs consisted of four stages, such as hyper- superfamily of calcium binding proteins is implicated in
reactivity and tremor stage 1; episode of rapid beating the regulation of intracellular calcium and as a marker of a
motion of the tail, slow, broad-based gait and rapid neuronal subpopulation [30]. In the adult mammalian
backward movements retropulsion; stereotype or repeti- brain, calbindin was thought to be present only in neurons,
tive movement stage 2; generalized tonic–clonic convul- which is believed to serve a neuroprotective role by its
sion stage 3; and death during convulsion stage 4. The calcium-buffering abilities [23].
approval of the Kagawa Medical University Animal Com- Astrocytes were originally thought to play only suppor-
mittee was obtained for this study. tive roles in the brain. Recently, there have been many
The animals were deeply anaesthetized with sodium reports concerning the functions of astrocytes including
pentobarbital 40 mg kg i.p.. To measure the blood neurotransmitter uptake [15], synthesis and secretion of
alcohol concentration BAC when the animals showed the neurotrophic factors [11]. Analysis targets of astrocytic
intoxication signs, blood was taken from the axilla-vessels reaction led the hypothesis that astrocytes are primary
of the mice and analyzed by the gas chromatograph targets of chemical injury and mediates the phenotypic
Shimadzu GC-8A, Tokyo, Japan. Four mice of the expression of chemical injury in the CNS [2]. Glial
control group and 6 mice of the experimental group were fibrillary acidic protein GFAP, which is the major protein
then perfused transcardially with a fixative of 10 for- of the intermediate filaments of astroglial cells, is the most
malin neutral buffer solution pH 7.4, Wako Co. Ltd., commonly used method to examine the distribution of
Osaka, Japan for preparation of paraffin embedded sec- astrocytes and the hypertrophy of astrocytes in response to
tions. Serial 7 mm-thick sections were cut coronally and neural degeneration or injury [21].
stained with cresyl-violet. The remaining animals of each Our previous report about the effects of short-term
group were perfused with 0.02 M phosphate buffered ethanol exposure on the suprachiasmatic nucleus SCN of
saline PBS, pH 7.4 followed by a fixative of 4 the hypothalamus of mice demonstrated that there was no
paraformaldehyde in PBS. The brains were removed and change on the calbindin D28k-IR with the increase on the
cryoprotected by immersion in 30 w v sucrose at 48C GFAP-IR [33]. The question arise that whether similar
until the brain sank. Serial 40 mm-thick frozen sections immunoreactivity changes are seen or not in the hippocam-
were cut coronally and processed for immunohistochemis- pus. Therefore, the aim of the present study was to
try. Free floating sections were immunostained for calbin- investigate the alteration calbindin D28k-IR neurons and
din D28k or GFAP using monoclonal antibody. To stain GFAP-IR astrocytes in the hippocampus of mice exhibiting
for either GFAP or calbindin D28k, the sections were intoxication signs in response to short-term ethanol treat-
incubated in the following solutions and between each ment. To understand which are particularly vulnerable or
steps the sections were rinse with PBS: 10 normal goat invulnerable to short-term ethanol exposure, the possible
serum in PBS with 0.2 Triton X-100 for 30 min; anti- interaction between short-term sensitivity to ethanol and
GFAP rabbit antibody 1:2000 dilution, DAKO, Glostrup, differences in the subfields of hippocampus were also
Denmark or anti-calbindin D28k mouse monoclonal anti- examined.
body 1:500 dilution, Sigma Chemical Company, St. Louis, MO overnight; biotinylated anti rabbit IgG or
biotinylated anti mouse IgG for 1 h; avidin–biotin complex
2. Materials and methods Vectastain, Elite ABC Kit, Vector Lab. Burlingame USA
for 1 h and 0.05 diaminobenzidine–0.03 hydrogen 2.1. Immunohistochemistry procedures
peroxidase for 5 min. Stained sections were mounted on gelatin-coated glass slides and air dried. They were then
Thirty male adult mice 7–8 weeks of BALB C strain dehydrated through graded 70, 80, 95, and 100 alcohol
from SLC Shizuoka, Japan, weighing 22–25 g, were to xylene and coverslipped using Entellan Merck, Ger-
housed in separate cages under controlled conditions with many. Control sections omitting the primary antibody
a constant temperature 23618C and kept in 12:12 light routinely developed to ensure that any observed staining
dark cycle. In this study the mice were divided into two was due to calbindin-D28k or GFAP. The morphology and
groups, ethanol-fed and pair-fed controls. The experimen- expression of calbindin or GFAP were studied with the aid
tal mice n516 received an unrestricted access liquid diet of a light microscope Nikon, YTHM, Japan.
Oriental Yeast Co. Ltd., Tokyo, Japan containing 6 v v ethanol 99.5 ethyl-alcohol, Wako Pure Chemical
2.2. Quantitative procedures Ind. Ltd., Osaka, Japan as the sole fluid source. The
pair-fed control mice n514 were fed an identical liquid For a morphometric study, the dorsal hippocampus was
I . Satriotomo et al. Brain Research 879 2000 55 –64
57
divided in 4 regions: the fields of the cornu Ammonis processes. The granule cell layer of the fascia dentate
CA1–CA3 and the superior limb of the dentate gyrus contained the smallest 10 mm neuronal cell bodies and
DG. The cornu Ammonis of CA1 until CA3 was divided was the most densely packed layer in the hippocampus. On
into sub-groups of stratum str. oriens, str. pyramidale, str. the other hand, the hilus of fascia dentate was composed of
radiatum and str. lacunosum moleculare. The dentate gyrus very large 25–35 mm and loosely packed polymorphic
was divided into molecular layers of fascia dentata or str. cells Fig. 1A. The intake of 6 v v ethanol in the mice
oriens, str. granulosum and hilus of fasciae dentata. Our exhibiting intoxication signs altered the neurons in the
observations were confined to the dorsal part of hippocam- hippocampus. Neuronal death or lysis was observed in
pus, as shown in Fig. 1. The number of labeled cells was fields of the cornu Ammonis CA1–CA3 and the dentate
measured quantitatively using image digital analysis in gyrus DG of the experimental group Fig. 1C and E
sections of the ten mice of both groups. The first of these compared with the control group Fig. 1B and D.
sections which
contained dorsal
hippocampus was
The calbindin-D28k positive neurons were moderately numbered as the first section, with all subsequent sections
stained in CA1, CA2 and dentate gyrus subfields of mice being numbered sequentially. For each animal, a number
hippocampus. CA1 subfields contained a higher proportion between 1 and 4 was selected randomly in order to
of calbindin D28k positive pyramidal cells and were more determine the first section. Every 4th section was picked
immunoreactive than the CA2 subfields, whereas pyrami- up and the number of calbindin-IR neurons and GFAP-IR
dal cells in the CA3 subfield remained unreactive Fig. astrocytes for example, the 2, 6, 10th sections etc. were
2A. The single neurons lying in the str. oriens, str. selected for calbindin-immunohistochemistry IH and the
radiatum and str. lacunosum-moleculare were intensely 4, 8, 12th sections were chosen for GFAP-IH were
labelled in the control groups Fig. 2C. These single counted, respectively. About 10 sections from each animal
neurons were characterized by the arborizated slender were used in this study. Photographs 3400 for calbindin
processes Fig. 2E. The short-term ethanol exposure led to and 31000 for GFAP that were taken from selected
significant differences when compared with the control sections with a light microscope Nikon eclips E600 were
group Fig. 2B. The number of calbindin-positive neurons captured by CCD color camera Inotech, Japan and
was decreased and less intensity of immunostaining was transformed into digits with the help of Viltz Universal
seen when compared with the control group Fig. 2D. software Vers. 1.5.1, Interwere Corp. in Power Macintosh
Moreover the single neurons that located in str. oriens, str. 4400 200 personal computer. Photographs of individual
radiatum and lacunosum-moleculare showed a lack of cells were used in order to avoid interference from
processes in the experimental group Fig. 2F. Table 1 overlapping images. The scanned images were placed
shows the estimate of the number mean6S.D. of
2
permanently on the computer screen then analyzed by calbindin-D28k IR neurons mm in various subregions in
National Institute of Health NIH image software. The the hippocampus of ethanol treated and control mice. The
area fraction of the total number of IR-cells proportion of statistical comparison between these groups revealed sig-
4
calbindin-IR area per square measuring fields of 1.35310 nificant differences in all hippocampal subfields, except str.
2
mm and GFAP-IR area per square measuring field of
pyramidale of CA1 subfield and str. granulosum of dentate
3 2
1.4310 mm , respectively were taken. The number of
area. IR-positive cells mm-square tissue from each region of the
In the control mice, GFAP-IR perikarya and processes hippocampal sections of the alcohol treated animals was
were observed throughout the hippocampal formation calculated and then compared with the respective control
including CA1–CA3 and DG cells of the hippocampus group Tables 1 and 2. The statistical analysis of data
Fig. 3A. Both pyramidal and granular cell layers were from each region were performed using Statview-J-4.5
clearly distinguished from adjacent layers, because only Abacus Concepts, CA with Student’s t-test P,0.05 was
fine GFAP-IR were seen in these layers Figs. 3C and G. taken as being significant.
The GFAP-IR astrocytes were observed to have small bodies with the short processes in the control group Fig.
3E. The short-term ethanol exposure resulted in distinct
3. Results alteration of GFAP-IR astrocytes in the dorsal hippocam-
