17beta-estradiol attenuates hippocampal neuronal loss and cognitive dysfunction induced by chronic restraint stress in ovariectomized rats.

Several lines of evidence suggest that hormonal changes after menopause may play an important role in the incidence of cognitive dysfunction, and also in the development of Alzheimer's disease. In this study, we investigated the effect of estrogen on cognitive function in rats under different stress environment. Female rats were divided into four groups: two groups were ovariectomized (OVX) and two were sham-operated. One group each of OVX and sham rats was kept in a normal environment, and the other groups were assigned to a daily restraint stress (6 h/day) for 21 days from 2 months after the operation. Following the stress period, subjects were tested for performance in novel object recognition test and then used for morphological and neurochemical analyses. The OVX plus stress (OVX/stress) group showed a significant impairment of recognition of novel objects, compared with the other groups. The OVX/stress group also showed a marked decrease in the number of pyramidal cells of the CA3 region and levels of brain-derived neurotrophic factor mRNA in the hippocampus. We further examined the effect of estrogen against cognitive dysfunction and hippocampal changes of OVX/stress rats. Vehicle or 17beta-estradiol (E2) at 20 microg/day was s.c. administered to OVX/stress rats from 2 days before the stress period to the end of behavioral analysis through an implantable osmotic pump. Chronic E2 treatment decreased stress response and improved the cognitive and morphological impairments relative to vehicle group. These data have important implications for cognition enhancing effect of estrogen treatment in postmenopausal women.

Ginkgo biloba extract EGb 761 attenuates hippocampal neuronal loss and cognitive dysfunction resulting from chronic restraint stress in ovariectomized rats.

We have recently found that a combination of ovariectomy (OVX) and chronic restraint stress causes cognitive dysfunction and reduces hippocampal CA3 neurons in female rats and that estrogen replacement suppresses the OVX/stress-induced behavioral and morphological changes. In this study, we examined the effect of Ginkgo biloba extract (EGb 761), a popular herbal supplement, on the cognitive dysfunction and neuromorphological change in OVX/stress-subjected rats. Female Fisher 344 rats were randomly divided into three groups: vehicle-treated OVX, EGb 761 (50 mg/kg) -treated OVX and vehicle-treated sham-operated control groups. Two months after ovariectomy, all animals received restraint stress for 21 days (6 h/day), and were then subjected to a novel object recognition test followed by morphological examination by Nissl staining. EGb 761 was orally administered once daily until the behavioral analysis was done. Treatment with EGb 761 improved memory impairment and neuronal loss of hippocampus in the OVX/stress-subjected group in the same ways as 17beta-estradiol. On the other hand, EGb 761 did not affect the loss of bone mineral density and increase in body weight after OVX, although 17beta-estradiol attenuated them. These results have important implications for neuroprotective and cognition enhancing effects of EGb 761 in postmenopausal women and suggest that the effects are mediated by a different mechanism from estrogen.

Thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns in single pancreatic acinar cells of mouse.

Modulation of the agonist-specific cytosolic Ca2+ oscillatory pattern by thimerosal has been investigated in single pancreatic acinar cells using patch-clamp perforated whole-cell recording to measure the calcium-dependent chloride current (I(C1)(Ca2+)). 1 microM thimerosal, which fails to evoke Ca2+ oscillation alone, clearly changed the pattern of Ca2+ oscillation from pulsatile spikes (evoked by low concentrations of activators) to sinusoidal or transient oscillations. The mimetic action of thimerosal was independent of extracellular Ca2+, was blocked by extracellular application of dithiothreitol or 10 mM caffeine, as well as by internal perfusion with heparin; but was unaffected by ruthenium red. We conclude that thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns mediated by sensitizing the InsP3-induced Ca2+ release.

Molecular analysis of abnormal satellite I DNA from a BUF/Mna rat thymoma.

BUF/Mna rats develop spontaneous thymomas in an autosomal dominant manner. We constructed recombinant plasmid library of 90 and 185 base-paired (bp) satellite I DNA fragments isolated from BUF/Mna rat thymoma DNA. Four unusual clones containing 93, 95, 95, and 173 bp inserts were isolated by colony hybridization with Wistar rat satellite I DNA. Nucleotide sequence analysis of the inserts of the 4 clones revealed abnormal sequence organization and unusual subunit structure of the rat satellite I DNA. Sequence comparisons between normal and abnormal satellite I DNA suggested that the unusual subunit structure could be generated by the change of the Hinf I recognition sequence to an Eco RI cleavage site, in addition to random deletions, insertions and base substitutions. The heptanucleotide sequence TGGGAAC, which is strictly conserved in normal subunits, was completely lost in all these clones. Southern blot hybridization revealed the amplification of abnormal satellite I DNA in BUF/Mna rat thymomas.

Axon-sparing lesion of the preoptic area enhances receptivity and diminishes proceptivity among components of female rat sexual behavior.

Stereotaxic infusion of ibotenic acid deleted neurons in the medial preoptic area (POA) in the ovariectomized female rats. A well-circumscribed lesion was infiltrated by astrocytes; local axons of passage were spared. Following estrogen priming and progesterone supplement, the females with the lesion had higher lordosis quotients than the vehicle-infused controls, when males successfully mounted them. On the other hand, the treatment did not induce solicitation in females with the lesion nor reduced their rejection of male partners. Meanwhile, gradual and persistent suppression of the lordosis reflex followed electrical stimulation through electrodes placed in the POA lesion. Except that the females with the POA lesion needed less estrogen to obtain comparable prestimulation quotients with the controls, the lesioned and control animals responded similarly to the stimulation. Because an adjunct neural transection dorsal to the POA lesion abolished the stimulus-bound suppression of lordosis, the effect was due to the activation of axons of passage that presumably descend from the septum. It is concluded that the POA is the major target for estrogen in eliciting proceptive behavior; local POA neurons as well as septal efferents appear to inhibit the lordosis reflex, the principal receptive component in female rat sexual behavior.

Serum matrix metalloproteinase-3 and fibrin degradation product levels correlate with clinical disease activity in rheumatoid arthritis.

OBJECTIVE: Plasmin and matrix metalloproteinase-3 (MMP-3) have been linked to articular destruction in rheumatoid arthritis (RA). We compared circulating levels of plasmin-mediated fibrin degradation product (FDP D-dimer) and MMP-3 with traditional parameters of disease activity in RA to determine their clinical utility. MATERIALS AND METHODS: Serum levels of MMP-3 and D-dimer were determined by enzyme-linked immunoassays in 60 patients with RA. Twenty healthy females and 21 patients with systemic lupus erythematosus (SLE) served as controls. RESULTS: MMP-3 (436.8 +/- 474.2 ng/ml) and D-dimer levels (351.2 +/- 296.3 ng/ml) were markedly elevated in the sera from RA patients as compared with healthy controls (43.9 +/- 15.2 ng/ml and 63.0 +/- 64.1 ng/ml, p < 0.0001, respectively). Both levels strongly correlated with each other (r = 0.627, p < 0.0001) and were closely associated with various clinical parameters for the disease activity of RA, including the erythrocyte sedimentation rate (ESR) and the Lansbury's activity index (p < 0.0001). MMP-3 levels were more highly correlated with articular parameters such as the swollen and painful joint counts (r = 0.454, p = 0.0002), whereas D-dimer levels correlated well with C-reactive protein (CRP) levels (r = 0.581, p < 0.0001). In SLE patients, MMP-3 (239.1 +/- 199.6 ng/ml, p < 0.0001) and D-dimer levels (86.9 +/- 85.2 ng/ml, p = 0.0278) were also higher than in healthy controls. Both levels correlated with each other (r = 0.612, p = 0.0025), and were associated with ESR and CRP levels, as was observed in RA patients, but not with most of the other clinical indicators for SLE. CONCLUSIONS: Serum levels of MMP-3 and D-dimer are clinically useful indicators for disease activity in RA. Our results further support the hypothesis that MMP-3 and plasmin may interact in the inflammatory synovial tissues, and thus augment the articular destruction seen in RA. In SLE patients, however, MMP-3 producing cells could be different from in RA patients, and further studies will be required to clarify the pathogenetic mechanism underlying the raised serum levels of MMP-3 and/or D-dimer.

Anti-agalactosyl IgG antibodies and isotype profiles of rheumatoid factors in Sjögren's syndrome and rheumatoid arthritis.

OBJECTIVE: Rheumatoid factors (RFs) in sera from rheumatoid arthritis (RA) patients bind better to agalactosyl IgG [gal(-) IgG] than to native IgG. Recently, a novel lectin-enzyme immunoassay (LEIA) which can detect all isotypes of the immunoglobulins was developed in Japan. Since RFs are also detectable in Sjögren's syndrome (SS), we determined anti-gal(-) IgG antibodies and RF isotypes in sera from primary or secondary SS and RA patients to elucidate the clinical significance of these antibody profiles. METHODS: A series of 128 patients with primary SS (35 pts.), RA (57 pts.), or secondary SS [n = 36 pts., the SS being associated with RA (RA-SS) in 12 pts., systemic lupus erythematosus (SLE-SS) in 17 pts., and mixed connective tissue disease (MCTD-SS) in 7 pts.] and 38 healthy females were examined. Anti-gal(-) IgG antibodies were measured with a LEIA kit (ED055) using human gal(-) IgG as antigen. IgG-, IgA- and IgM-RF were determined with an enzyme-linked immunosorbent assay kit using human IgG-Fc as antigen. RFs were also examined by a conventional assay (laser nephelometry; LN-RF). RESULTS: Serum anti-gal(-) IgG antibody titers were higher in RA than in primary SS, SLE-SS or MCTD-SS, but the incidence of the antibodies did not differ between RA and primary SS. In both RA and primary SS, the antibodies were positive in half of the LN-RF-negative patients, and were also detected in almost all of those patients who had at least one of the RF isotypes. LN-RF, IgG-RF, IgA-RF and IgM-RF were present more frequently and their titers were higher in RA than in primary SS, but IgA-RF levels were similar in both groups. In RA-SS, all of the antibody titers were notably higher than in RA or primary SS. In RA and primary SS, IgA-RF and IgM-RF were common RF isotypes, and anti-gal(-) IgG antibody levels correlated well with LN-RF, IgA-RF and IgM-RF levels. These antibody profiles did not relate to any of the clinical parameters in RA, but all the antibody titers correlated with anti-SS-A/Ro antibody levels in primary SS. CONCLUSIONS: The anti-gal(-) IgG antibodies are not specific for RA; they are also frequent in SS. Our LEIA for antibodies is a very sensitive method to detect all RF isotypes in both RA and SS. Most of the antibody profiles are common to both diseases, although antibody titers are higher in RA, especially in RA-SS.

Abnormal expression of apoptosis-related antigens, Fas and bcl-2, on circulating T-lymphocyte subsets in primary Sjögren's syndrome.

OBJECTIVE: To clarify the pathological role of the apoptosis-related molecules expressed on peripheral blood (PB) lymphocyte subsets in primary Sjögren's syndrome (SS). METHODS: The levels of apoptosis-regulating proteins, Fas and bcl-2, were determined in the PB lymphocyte subsets from 21 patients with SS and 14 healthy controls by 2-color flow cytometry. RESULTS: In the PB from SS patients, lymphocytopenia, especially CD4+ cell-lymphocytopenia, was prominent. As observed in previous studies, the percentages of CD4+ CD45RA+ cells were lower in the SS patients than in the controls, while activated (DR+) cells were increased in CD4+ cells from the patients. Fas+ cells were also increased in the patients' CD4+ cells and CD8+ cells, but not in their B cells or natural killer cells. Furthermore, we observed several positive correlations among the percentages of activated cells (DR+ cells or CD45RA-cells) and Fas+ cells recognized in the CD4+ and/or CD8+ cells from the patients. On the other hand, intra-cellular bcl-2 proteins measured as mean fluorescence intensity were significantly diminished in the CD4+ cells, CD8+ cells, CD19+ cells, CD45RO+ cells and Fas+ cells from 14 SS patients compared with 12 healthy controls. In addition, the numbers and/or percentages of CD4+ cells and Fas+ cells positively correlated with their expression of bcl-2 in SS patients. CONCLUSION: The abnormal balance between Fas and bcl-2 expression detected in the PB lymphocyte subsets from SS patients relates, at least partially, to the lymphocytopenia observed in the patients.

Interruption of the lordosis reflex of female rats by ventral midbrain stimulation.

The lordosis reflex, dorsiflexion of the vertebral column, is an estrogen-dependent, essential element of female sexual behavior in rodents. Unilateral electrical stimulation of the midbrain ventral tegmental area through a chronically implanted electrode in freely moving, estrogen-primed ovariectomized female rats caused a rapid and strong suppression of the lordosis reflex in response to either male mounts or manual cutaneous stimuli. The interruption occurred in a graded manner to increased stimulus intensity, with a threshold at 30 microA. The optimal frequency was at 75-125 Hz. After the termination of electrical stimulation, lordosis performance returned promptly to the pre-stimulation level. No aversive response accompanied the blockade of lordosis. Electrical stimulation specifically blocked lordosis, without disrupting the proceptive components of female sexual behavior. In 10 animals tested, concomitant injection of dopamine receptor blocker pimozide tended to offset the effects of electrical stimulation in 2 cases. Interruption of the lordosis reflex might be mediated by projections from the ventral tegmental area, which activate a descending pathway inhibitory to the lordosis reflex arc at or below the lower brain stem.

Melanotic neuroectodermal tumor of infancy in the mandible: report of a case.

A case of melanotic neuroectodermal tumor of infancy occurring in the mandible is described. The patient was a 1-month-old boy with a rapidly growing tumor of the mandible. Computed tomography showed 2 well-defined osteolytic lesions in the right mandible. Histopathologic diagnosis of a biopsy specimen was melanotic neuroectodermal tumor of infancy. The tumor was excised with removal of the surrounding bone, but 1(1/2) months later it recurred, and segmental mandibulectomy and reconstruction of the defect with a titanium miniplate was performed. Retrospectively, evidence of recurrence was noted on computed tomography taken on the tenth postoperative day. The recurrence was caused by incomplete removal of the tumor. Histopathologically, the tumor cells of the recurrent lesion were dispersed extensively in the bone marrow, and bone remodeling was active. The surgical procedure may have stimulated tumor cell proliferation and reactive bone formation. The patient was followed for 2 years with no evidence of recurrence or metastasis.

Changes in photically evoked blink reflex during sleep and wakefulness.

The effects of the state of arousal on photically evoked blink reflex were studied. Eyelid potential (ELP), the averaged electromyographic activity of the orbicularis oculi muscle, was used to define the onset latency and intensity of the reflex. ELP was maximum with the shortest latency when the subjects were awake and doing mental arithmetic. ELP amplitude gradually decreased and the latency lengthened with the advance in non-rapid eye movement (non-REM) sleep stages. In the REM stage, ELP was augmented and resembled that when awake. ELP is useful as an objective sign of the photically evoked blink reflex, which closely reflects cortical activities and brain stem function.

Thrombomodulin levels in the plasma and joint fluid from patients with rheumatoid arthritis.

The hemostatic mechanism is thought to contribute to the inflammatory process of rheumatoid arthritis (RA). Thrombomodulin (TM), an inhibitor of blood coagulation, is synthesized by various cells which are recognized in the inflammatory lesions of RA. To elucidate a possible relation of TM with the process of RA, therefore, we measured soluble forms of TM in the plasma and joint fluid (JF) from RA patients by a recently developed sandwich enzyme immunoassay using monoclonal antibodies. TM levels in the plasma and JF were not significantly elevated in RA patients, although TM levels in plasma were positively correlated with those in JF. The plasma TM levels were related to renal functions (serum creatinine levels), but the levels showed no connection with systemic inflammatory indices of RA such as erythrocyte sedimentation rates, serum C-reactive protein levels and Lansbury's activity index. In the JF, TM levels were not correlated with the numbers of neutrophils or monocytes/macrophages associated with articular inflammations. Our results indicate that TM levels in the plasma and JF do not reflect systemic and articular inflammations of RA, and suggest that TM molecules in JF are mainly recruited from circulating TM.

Comparison of antinuclear antibody and other immunohematological profiles among primary Sjögren's syndrome, secondary Sjögren's syndrome associated with rheumatoid arthritis or systemic lupus erythematosus, and corresponding systemic disease.

Sjögren's syndrome (SS) is currently classified into two groups (primary and secondary), because of differences in the disease in the two groups. We determined antinuclear antibody and other immunohematological parameters by using newer, more sensitive serologic methods on patients with primary SS, or secondary SS associated with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE), and patients with just the systemic disease free of SS. This study defined both distinctive and common features between primary SS and each systemic disease: High titers of fluorescent antinuclear antibodies (FANAs), anti-SS-A/SS-B antibodies, and rheumatoid factors (RFs), as well as leukocytopenia were considered the main features of primary SS. Elevated levels of RFs and C-reactive protein were prominent in RA patients. In contrast, high titers of FANAs and anti-single stranded or anti-double stranded DNA antibodies, positive anti-ribonucleoprotein or Sm antibodies, leukocytopenia, and hypocomplementemia were characteristic for SLE. Furthermore, patients with secondary SS plus RA or SLE were found to have mixed features of SS and the associated systemic disease. The results strongly suggest that patients with secondary SS have two separate diseases, SS and the associated systemic disease.

Decay-accelerating factor (DAF, CD55)-negative T lymphocytes in the peripheral blood of Sjögren's syndrome patients.

OBJECTIVE: To clarify possible associations of decay-accelerating factor (DAF, CD55), expressed on circulating lymphocyte subsets and other hematologic cells, with corresponding cytopenias observed in primary Sjögren's syndrome (SS). METHODS: DAF expression on peripheral blood (PB) cells was determined in 21 patients with SS and 11 healthy controls by single or 2 color flow cytometry. RESULTS: In the PB from SS patients, anemia, monocytopenia, neutropenia, and lymphocytopenia were observed. Compared to the controls, the percentages of DAF-negative cells were higher in CD4+ and CD8+ T cell subsets from SS patients, but the expression of DAF was similar in the other PB cells, including CD19+ B cells, CD56+ NK cells, monocytes, granulocytes, and erythrocytes. The percentages of DAF-negative cells among the CD4+ and CD8+ cells were positively correlated in SS patients, but the numbers of cells in both subsets were decreased in those patients being treated with prednisolone. However, these proportional changes are thought to reflect a decrease in the numbers of DAF-positive CD4+ and CD8+ cells, because the absolute numbers of circulating DAF-positive CD4+ and CD8+ cells, but not DAF-negative cells, were significantly decreased in SS patients. In addition, DAF-negative cells were detectable in both CD45RA+ (naive) and CD45RO+ (memory) T cells from healthy individuals, and the expression of DAF was remarkably increased in both subsets after in-vitro activation with concanavalin-A. CONCLUSION: DAF-negative cells are proportionally increased among circulating CD4+ and CD8+ T cells in SS patients, although such changes are due to decreased numbers of DAF-positive cells within each subset. When considering previous observations, the DAF-negative CD4+ and CD8+ cells probably belong to activated T cell subsets in both SS patients and controls. However, the patterns of DAF expression seemed to be different between activated T cells recognized in the PB, and those induced by in vitro-stimulation.

[Basic studies on the photically evoked lid potential changes in rabbit, with special reference to orbicularis oculi reflex].

In order to elucidate the origins of the potential changes in the eyelid elicited by flash stimulation in rabbits, their physiological properties were studied under various conditions. In the present study, they were specially discussed from the standpoint of orbicularis oculi reflex. The photically evoked lid potential changes were obtained with the summation technique by the signal processor (7 T 08, SAN-EI, Tokyo). In addition, the average summated responses of photically evoked lid MV responses, electroretinogram (ERG) and the electroencephalogram (EEG) were recorded simultaneously under dark-adapted conditions and studied polygraphically. The results obtained were as follows: The average photically evoked lid potential changes in rabbits were shown to be composed of early rapid and slow components and late slow component. The early rapid and slow components corresponded to dominant vibrations of the evoked lid MV responses and the a and b waves with oscillatory potential of the ERG. The early rapid components, which corresponded to dominant vibrations of the evoked lid MV responses, consist of positive, negative and positive components with peak latencies of 24.8 +/- 2.7, 29.4 +/- 3.7 and 33.1 +/- 2.5 msec, respectively. In the average lid potential changes led off bipolarly from medial and middle parts of the superior lid, the electromyographic components appeared corresponding to the evoked lid MV responses, although the electroretinographic components and late slow component were observed to decreased considerably in amplitude or disappeared almost completely, as compared with those obtained by monopolar leads. In the average lid potential changes obtained by flash stimulation to a single eye, the electroretinographic and electromyographic components disappeared almost completely in the occluded side.(ABSTRACT TRUNCATED AT 250 WORDS)

[Basic studies on the potential changes in the eyelid elicited by flash stimulation, especially related to the adaptation of the eye].

In order to establish the most adequate method to lead the photically evoked lid potential changes, their physiological properties were studied from standpoint of dark or light adaptation, which is known to be due to the increase or decrease in the sensitiveness of the retina that is the receptor of the orbicularis oculi reflex. The photically evoked lid potential changes were obtained with the summation technique by the signal processor (7 T 0 8, SAN-EI, Tokyo) in normal subjects. In addition, the average summated responses of the photically evoked lid MV responses, the ERG, EOG and EEG were also recorded simultaneously and discussed polygraphically. The results obtained were as follows: The average evoked lid potential changes in healthy resting subjects with eyes closed were shown to be composed of early rapid component, initial slow component and late slow component, which corresponded to dominant rapid vibrations of the evoked lid MV responses, the a and b waves of ERG and the EOG, respectively. The electromyographic and electrooculographic components were observed to have peak latency of about 60 msec and 200 msec, respectively. The electromyographic and electrooculographic components were found to change corresponding to the level of cortical activity indicated by the EEG patterns. They were gradually suppressed in amplitude according to the decrease of cortical activity and disappeared in stage 2 of natural sleep, although the electroretinographic components remained. With the progress of dark adaptation after adapting from light to darkness, the electromyographic and electrooculographic components in addition to the electroretinographic component of the average lid potential changes were considerably increased in amplitude but hardly changed in peak latency.(ABSTRACT TRUNCATED AT 250 WORDS)

[Basic studies on the potential changes in the eyelid elicited by flash stimulation, especially the intensity of flash light stimulation].

The average photically evoked lid potential changes are known to consist of electromyographic components due to the orbicularis oculi reflex, electroretinographic and electrooculographic components, which corresponded to the average summating responses of the lid MV responses, electroretinogram (ERG), electrooculogram (EOG), respectively. In the present study the photically evoked lid potential changes were studied from the standpoint of the stimulus strength in healthy subjects and patients with an artificial eyeball. The photically evoked lid potential changes were obtained with the summation technique under various physiologic and pathologic conditions. In addition, the average summating responses of the lid MV responses, EOG, ERG and EEG were also recorded simultaneously and discussed polygraphically. The average evoked lid potential changes in healthy resting subject with eyes closed were shown to be composed of the electromyographic components dependent on the effector activity of the orbicularis oculi reflex, early and late slow components which corresponded to the ERG and the EOG respectively. The threshold stimulus necessary to elicit the electroretinographic, electromyographic and electrooculographic components was the intensity of flash stimulation, that is about 0.1, 0.3 and 4 joule in energy delivered to the stroboscope respectively. These components were observed to be increased in amplitude gradually according to the intensity strengthened. In the average lid potential changes obtained by random flash stimulation to a single eye, the electroretinographic components in the occluded side disappeared completely, but the other components were clearly observed and gradually increased in amplitude according to the intensity strengthened, although they were considerably decreased in amplitude than those obtained in the contralateral side of normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Combination of chronic stress and ovariectomy causes conditioned fear memory deficits and hippocampal cholinergic neuronal loss in mice.

We have recently found that the combination of ovariectomy (OVX) and chronic restraint stress (CS) causes hippocampal pyramidal cell loss and cognitive dysfunction in female rats and that estrogen replacement prevents the OVX/CS-induced morphological and behavioral changes. In this study, to clarify the mechanisms underlying the OVX/CS-mediated memory impairment further, we examined the roles of cholinergic systems in the OVX/CS-induced memory impairment in mice. Female Slc:ICR strain mice were randomly divided into two groups: OVX and sham-operated groups. Two weeks after the operation, the mice of each group were further assigned to CS (6 h/day) or non-stress group. Following the 3-week-stress period, all mice were subjected to contextual fear conditioning, and context- and tone-dependent memory tests were conducted 1 or 24 h after the conditioning. Overburden with 3 weeks of CS from 2 weeks after OVX impaired context- and tone-dependent freezing and the OVX/CS caused significant Nissl-stained neuron-like cell loss in the hippocampal CA3 region, although OVX and CS alone did not cause such behavioral and histological changes. Replacement of 17ß-estradiol for 5 weeks after OVX suppressed OVX/CS-induced memory impairment and hippocampal Nissl-positive cell loss. Furthermore, the OVX/CS mice exhibited a significant decrease in choline acetyltransferase in the hippocampus compared with other groups. The cholinesterase inhibitors donepezil and galantamine ameliorated OVX/CS-induced memory impairment. These data suggest that cholinergic dysfunction may be involved in the OVX/CS-induced conditioned fear memory impairment. Overall, our findings suggest that the OVX/CS mouse model is useful to study the mechanisms underlying estrogen loss-induced memory deficits.