Astrocytic Responses to Binge Alcohol Intake in the Mouse Hindbrain

Abstract

Ethanol is the most commonly used toxic chemical in human cultures. Ethanol predominantly damages the brain causing various neurological disorders. Astrocytes are important cellular targets of ethanol in the brain and are involved in alcoholic symptoms. Recent studies have revealed the diversity of astrocyte populations in the brain. However, it is unclear how the different astrocyte populations respond to an excess of ethanol. Here we examined the effect of binge ethanol levels on astrocytes in the mouse brainstem and cerebellum. Ethanol administration for four consecutive days increased the glial fibrillary acidic protein (GFAP)-immunoreactive signals in the spinal tract of the trigeminal nerve (stTN) and reticular nucleus (RN). Another astrocyte marker, aquaporin 4 (AQP4), was also increased in the stTN with a pattern similar to that of GFAP. However, in the RN, the immunoreactive signals of AQP4 were different from that of GFAP and were not changed by ethanol administration. In the cerebellum, GFAP-positive signals were found in all four astrocytic populations, and those in the Bergmann glia were selectively eliminated by ethanol administration. We next examined the effect of estradiol on the ethanol-induced changes in astrocytic immunoreactive signals. The administration of estradiol alone increased the AQP4-immunoreactivity in the stTN with a pattern similar to that of ethanol, whereas the co-administration of estradiol and ethanol suppressed the intensity of the AQP4-positive signals. Thus, binge levels of ethanol intake selectively affect astrocyte populations in the brainstem and cerebellum. Sex hormones can affect the ethanol-induced neurotoxicity via modulation of astrocyte reactivity.

INTRODUCTION

Ethanol is the most commonly used drug in human history. It has both beneficial and harmful effects on the human body, that is, ethanol provides feelings of euphoria and well-being, whereas it also exerts acute and chronic toxicity, increasing the risk of neuronal disorders. Inappropriate levels of ethanol intake cause alcohol use disorder, which is a leading cause of preventable death worldwide, especially in younger generations. Ethanol predominantly affects and damages the brain causing synaptic dysfunction and apoptotic neuronal death. Previous studies have clarified the molecular and cellular targets of ethanol in the mammalian brain.¹⁾ Astrocytes are important targets of ethanol-induced neuronal toxicity. Astrocytes form neurovascular units at the blood brain barrier (BBB) with the blood vessel endothelial cells, pericytes, and neuronal processes.²⁾ In vitro studies have indicated that ethanol-treatment activates intracellular calcium signaling to alter gene expression.³⁾ Our previous study showed that binge levels of ethanol administration induced the retraction of astrocytic processes and disrupted the interaction with the blood vessels in the cerebral cortex.⁴⁾ Although astrocytes are diverse throughout the brain regions, the effect of ethanol exposure on astrocytes in other regions of the brain remains obscure.

Astrocytes express nuclear estrogen receptor ERα and ERβ as well as membrane type estrogen receptor and respond to estrogen to their reactivity.⁵⁾ In the mammalian brain, 17β-estradiol is produced by neurons and astrocytes, which plays important roles for neuroprotection against brain injury.^6,7) The neuroprotective action of astrocytes is reported to be dependent on estrogen, that may explain the increased risks of neurological disorders in menopaused women.⁸⁾ On the other hand, the epidemiological evidence has been provided for an association between alcohol consumption and blood estrogen levels,⁹⁾ making it important to know the physiological interaction between ethanol and estrogen in neurotoxicity. Indeed, some reports suggested that the metabolism and toxicity of ethanol are affected by sex hormones.^10–12) The sensitivity to ethanol of the dopaminergic neurons in the ventral tegmental area is decreased by estrogen receptor antagonist, indicating that estradiol sensitizes neurons to ethanol.¹³⁾ However, it has not been examined how the response of astrocytes to ethanol is affected by estradiol.

The hindbrain is located in the lower back part of the brain and includes the cerebellum and most of the brainstem, such as the medulla and pons. It integrates the incoming sensory information and coordinates motor responses. As with other brain regions, the cerebellum and brainstem are also sensitive to ethanol and the cerebellum is particularly vulnerable to ethanol. Ethanol exposure increases the release of γ-amino-butyric acid (GABA) from the Purkinje cells, molecular layer interneurons, and granule cells.¹⁴⁾ Chronic alcoholism causes cerebellar atrophy.¹⁵⁾ Similarly, the brainstem is also affected by ethanol. Ethanol directly activates the trigeminal sensory pathways, which is mediated by the neuronal nuclei in the brainstem. Brasser et al. proposed that the modulation of the trigeminal neuronal pathways by ethanol intake-induced neuronal activation contributes to alcohol-seeking behavior.¹⁶⁾ Thus, the hindbrain is important for the onset and progression of alcohol-induced neurological disorders. However, it remains obscure how the cells in the hindbrain are affected by excess ethanol intake. In this study, we examined the effect of ethanol intake on the astrocytic structure and reactivity in the cerebellum and brainstem in a mouse acute alcoholic model.

MATERIALS AND METHODS

Animals

Male ICR mice (8–10 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). Mice were individually housed at the animal facility at Kobe Pharmaceutical University and allowed to acclimatize for at least one week before the start of the experiments. All procedures involving the animal experiments in this study were conducted following the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The protocols were approved by the Kobe Pharmaceutical University Committee for Animal Care and Use.

Ethanol was intraperitoneally administered at 2 g/kg body weight (B.W.)/d as 20% (v/v) saline solution for four consecutive days.^4,17) For the control mice, an equivalent amount of saline was intraperitoneally injected. The sex hormone 17β-estradiol (50 µg/kg B.W./d; Sigma-Aldrich Japan, Tokyo, Japan) was dissolved in Coconad RK (Kao Corp., Tokyo, Japan) and subcutaneously injected every day until analysis.¹⁸⁾ Analysis was performed for two days after the final administration of ethanol.

Immunohistochemistry

Mice were deeply anesthetized with isoflurane, and 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) was transcardially perfused. The brain was fixed in 4% PFA in PBS at 4 °C for 6 h and cryoprotected in 30% sucrose in PBS at 4 °C overnight. The 30-µm coronal sections of the brainstem and cerebellum were prepared with a cryostat (SLEE medical GmbH, Mainz, Germany).

Immunohistochemistry was performed as described in our previous manuscript, with slight modifications.¹⁹⁾ Briefly, tissue sections were fixed in 4% PFA in PBS at room temperature for 5 min, briefly washed with PBS, and incubated in 10 mM citrate buffer (pH 6.0) (for glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP4)) or 10 mM Tris buffer (pH 9.0) (for Calbindin-D28k) at 75 °C for 40 min for antigen retrieval. They were then cooled to room temperature, washed in PBS three times, blocked with 1.5% fetal bovine serum (FBS) in PBS, and incubated with rabbit antibodies against GFAP (20334, DAKO/Agilent, Santa Clara, CA, U.S.A.), AQP4 (16473-1-AP, Proteintech Group, Inc., Rosemont, IL, U.S.A.), or Calbindin-D28k (14479-1-AP, Proteintech) at 4 °C overnight. The sections were then washed with PBS and incubated with Cy3-conjugated anti-rabbit immunoglobulin G (IgG) (Jackson ImmunoResearch, West Grove, PA, U.S.A.) diluted in PBS containing 1.5% FBS together with 4′,6-diamidino-2-phenylindole (DAPI), at room temperature for 3 h. They were washed in PBS and mounted with Fluromount-G (SouthernBiotech, Birmingham, AL, U.S.A.).

All images were captured using an Axio Scope A1 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) and processed with the GNU Image Manipulation Program (GIMP), ver. 2.10.32., an open resource software program for manipulating images. Number of immunoreactive signal-positive cells per area size, cell body size, and average signal intensity were measured using the ImageJ software. For cell body size and signal intensity of cell bodies, we quantified the values for at least 10 cells per individual and calculated the average of three mice.

Statistical Analyses

Results were expressed as mean ± standard deviation. Data were statistically analysed by Student’s t-test, two-way factorial ANOVA, and/or Tukey–Kramer post-hoc test, using the EZR software for Windows.²⁰⁾ When a significant interaction was observed in the two-way factorial ANOVA, the Tukey–Kramer post-hoc test was performed to compare the mean values among four groups.

RESULTS

Ethanol Intake-Induced Changes in Astrocytic Reactivity in the Hindbrain

Binge levels of ethanol were administered to the mice for four consecutive days as described in Materials and Methods. The brainstem sections were immunostained with anti-GFAP antibody to visualize the reactive astrocytes (Fig. 1). In the brainstem of the normal saline-administered mice, GFAP-positive astrocytes were mostly found in the spinal tract of the trigeminal nerve (stTN, Figs. 1A, a1). There were no GFAP-positive signals in the spinal trigeminal nucleus (SPV) and a few GFAP-positive signals in the reticular nucleus/reticular formation (RN, Fig. 1a2). The ethanol intake increased the reactivity of astrocytes in the stTN (Figs. 1B, b1, D). In addition, many GFAP-positive astrocytes were observed in the RN (Figs. 1b2, E). The pattern of GFAP-positive signals in these regions was different: those in the stTN were seen as thin straight lines and dots, whereas those in the RN had a highly branched structure, which corresponded to the fibrous and protoplasmic astrocytes, respectively (Figs. 1b1, b2). Thus, astrocyte reactivity in the stTN and numbers of reactive astrocytes in the RN are increased by ethanol intake. The SPV also contained some GFAP-positive astrocytes with a morphology similar to those in the RN after ethanol administration, but the number was considerably less than in the RN (Fig. 1B).

Fig. 1. Increased Reactive Astrocytes in the Brainstem after Repeated Ethanol-Treatment

The brainstem sections of the saline- (A) or ethanol- (B) administered mice were stained with anti- glial fibrillary acidic protein (GFAP) antibody. An adjacent section of panel A was stained without primary antibody as a negative control (C). The yellow square regions in panels A and B are enlarged in panels a1, a2, b1, and b2, respectively. D: Quantification of fluorescent signal intensity for GFAP in the stTN. The values are normalized to the average value of control saline-administered mice. E: Number of GFAP-positive cells in the RN. Each circle indicates an individual animal. Error bars indicate standard deviation. The statistical significance of average values among groups was calculated with Student’s t-test. * p < 0.05. RN, reticular nucleus; SPV, spinal trigeminal nucleus; stTN, spinal tract of the trigeminal nerve. Scale bars: 100 µm.

AQP4 is a well-known astrocyte marker.²¹⁾ It localizes at the perivascular endfeet of astrocytes at the BBB and functions to control the barrier function of the BBB.^22,23) To examine the change in the localization of AQP4 by binge levels of ethanol intake, we immunostained the brainstem sections with anti-AQP4 antibody. AQP4-positive signals were observed in the stTN, where the signals were liner and punctate, similar to the GFAP-positive signals in this region (Figs. 2A, a1). The SPV and RN regions also contained AQP4-positive signals, where these signals surrounded the capillaries (Fig. 2a2). Ethanol administration did not change the number of AQP4-positive signals in the stTN, whereas it increased AQP4-immunoreactivity (Figs. 2B, b1, C, D). The number of AQP4-positive capillaries in the SPV and RN were not evidently changed by ethanol administration (Figs. 2B, b2, E). Thus, the AQP4 localization response to ethanol differs between the tissue regions.

Fig. 2. Ethanol-Treatment Increased Aquaporin 4 (AQP4)-Positive Signals in the Astrocytes in the Trigeminal Nerve Tract

The brainstem sections of the saline- (A) or ethanol- (B) administered mice were stained with anti-AQP4 antibody. The yellow square regions in panels A and B were enlarged in panels a1, a2, b1, and b2, respectively. The yellow arrows indicate examples of AQP4-positive blood capillaries in the reticular nucleus. C: Number of AQP4-positive signals in the stTN. D: Average signal intensity for AQP4 in the stTN. The values are normalized to the average value of saline-administered mice. E: Number of AQP4-positive capillaries in the RN. Each circle indicates an individual animal. Error bars indicate standard deviation. The statistical significance of average values among groups was calculated with Student’s t-test. * p < 0.05, ** p < 0.01. n.s., not significant; RN, reticular nucleus; SPV, spinal trigeminal nucleus; stTN, spinal tract of the trigeminal nerve. Scale bar: 100 µm.

In the cerebellum, four different astrocyte populations have been identified: Bergmann glia, protoplasmic astrocytes, fibrous astrocytes, and Fañanas cells.²⁴⁾ Immunostaining with anti-GFAP antibody stained these four populations in the cerebellum of the saline-administered control (Fig. 3A). Ethanol intake decreased the number of GFAP-positive signals of Bergmann glia in the molecular layer (Fig. 3B). The change in the number of Fañanas cells, which are a specialized subtype of Bergmann glia, was not conclusive in this study because they generally are a population with fewer numbers compared with the original Bergmann glia. The GFAP-positive signals on the protoplasmic astrocytes (yellow arrowheads in the Purkinje cell and granular layers in Fig. 3A) and fibrous astrocytes (white arrowheads in the white matter in Fig. 3A) were not apparently changed by ethanol administration (Fig. 3B). AQP4 was detected in the protoplasmic and fibrous astrocytes as well as some capillary-like structures in the molecular layer (blue arrowheads in Fig. 3C). Bergmann glia were negative for AQP4 (Fig. 3C). Ethanol administration did not change the localization of AQP4 (Fig. 3D). These results suggest that the effect of ethanol on astrocytes is largely different among the brain regions.

Fig. 3. Disappearance of Glial Fibrillary Acidic Protein (GFAP)-Positive Signals in the Bergmann Glia of the Cerebellum by the Ethanol-Treatment

The cerebellum sections of the saline- (A, C) or ethanol- (B, D) administered mice were stained with anti-GFAP (A, B) and anti-aquaporin 4 (AQP4) (C, D) antibodies. Blue arrows, white arrow, yellow arrowheads, and white arrowheads indicate the Bergmann glia, Fañanas cells in the molecular layer, protoplasmic astrocytes in the Purkinje cell and granular layers, and fibrous astrocytes in the white matter, respectively. GL, granular layer; ML, molecular layer; PL, Purkinje cell layer; WM, white matter. Scale bar: 100 µm.

Effect of Sex Hormones on Ethanol-Induced Neurotoxicity

The metabolism and physiological effects of ethanol depend on gender and sex hormones.^25,26) Therefore, we administered estradiol together with ethanol in our model. Estradiol or the control vehicle was administered subcutaneously from the first day of ethanol administration to the day before analysis (Fig. 4A). The absolute and change in B.W. between the first day and the day of analysis were not significantly different between the treatments (data not shown). The tissue weights of the brain, heart, and liver were not affected by the treatment with ethanol and estradiol either (Figs. 4B–D). The immunostaining of the sections of the brainstem with anti-AQP4 antibody revealed no significant change in the number of AQP4-positive signals in the stTN by the ethanol administration (Fig. 5I). In contrast, ethanol administration increased the signal intensity of AQP4-positive puncta and fragments by ethanol in the stTN with the vehicle-injection, similarly to the results in Fig. 2 (Figs. 5A, B, J). The administration of estradiol together with control saline had similarly increased the AQP4 signal intensity in the stTN (Figs. 5C, J). Co-administration of ethanol and estradiol decreased the intensity of AQP4-positive signals in the stTN, compared with those of the ethanol- or estradiol-single administered mice (Figs. 5D, J). The capillary-like signals in the SPV and RN were not apparently changed by ethanol administration (Figs. 5E, F), as seen in Fig. 2. The estradiol administration and ethanol/estradiol co-administration did not affect these signals either (Figs. 5G, H). These results indicate that the astrocytes in the trigeminal nerve tract increased their reactivity in response to both ethanol and estradiol, whereas the combined stimulation with ethanol and estradiol partially suppressed the response. Thus, each population of astrocytes shows different response patterns to ethanol in the presence of estradiol. Interestingly, the estradiol administration increased AQP4-positive signals in cells other than the capillary-surrounding cells (Fig. 5G), which was attenuated by the co-administration with ethanol (Fig. 5H).

Fig. 4. Co-administration of Ethanol and Estradiol Did Not Affect the Brain Weight

A: Experimental design. B–D: The brain (B), heart (C), and liver (D) weight relative to body weight (B.W.) of group A (administered with saline and vehicle), group B (administered ethanol and vehicle), group C (administered saline and estradiol), and group D (administered with ethanol and estradiol) were calculated. Error bars indicate the standard deviation. No statistical significance was noted among the brain weights of these treatment groups.

Fig. 5. Ethanol and Estradiol Affect the Astrocytic AQP4 Localization in the Spinal Tract of Trigeminal Nerve (stTN)

The brainstem sections of the saline-/vehicle- (A, E), ethanol-/vehicle- (B, F), saline-/estradiol- (C, G), and ethanol-/estradiol- (D, H) administered mice were immunostained with anti-AQP4 antibody. The yellow arrows indicate examples of AQP4-positive blood capillaries in the trigeminal nucleus (SPV). I: Number of AQP4-positive signals in the stTN. J: Average signal intensity for AQP4 in the stTN. The values are normalized to the average value of saline-/vehicle-administered mice. Each circle indicates an individual animal. Error bars indicate standard deviation. The statistical significance of average values among groups was calculated with two-way ANOVA followed by the Tukey–Kramer post-hoc test for multiple comparisons. * p < 0.05, ** p < 0.01. n.s., not significant. Scale bar: 20 µm.

Previous studies indicated that the ethanol intake damages the Purkinje cells to impair the cerebellar neuronal circuit.^27,28) As seen in Fig. 3, our binge ethanol administration model showed the disappearance of GFAP-positive Bergmann glial processes, suggesting that the cerebellum was damaged by the excess ethanol intake. Therefore, we checked the Purkinje cell morphology in our samples. Immunostaining with anti-Calbindin-D28k antibody showed no obvious morphological change in the Purkinje cells by ethanol, estradiol, and the combination of both (Fig. 6).

Fig. 6. Ethanol- and Estradiol-Treatment Did Not Affect Purkinje Cells in the Cerebellum

The cerebellum sections of the saline-/vehicle- (A), ethanol-/vehicle- (B), saline-/estradiol- (C), and ethanol-/estradiol- (D) administered mice were stained with anti-Calbindin-D28k antibody. An adjacent section of panel A was stained without primary antibody as a negative control (E). F: Average cell body size of Purkinje cells. G, H: Average signal intensity for Calbindin in Purkinje cell bodies (G) and in the ML (H). The values are normalized to the average value of control saline-/vehicle-administered mice. Statistical significance was not obtained with two-way ANOVA in these analyses. ML, molecular layer; PL, Purkinje cell layer; GL, granular layer; WM, white matter. Scale bar: 100 µm.

DISCUSSION

Effects of Ethanol on Diverse Types of Hindbrain Astrocytes

Ethanol exerts its neurotoxicity through multiple mechanisms.¹⁾ Among the various cell types potentially mediating ethanol-induced intoxication, astrocytes play important roles, with their high ALDH2 expression and association with the BBB.^29–31) On the other hand, recent single cell-level of transcriptome analyses revealed the molecular and functional diversity of astrocytes in the mammalian brain.^32–34) The results presented here indicate that the astrocytes in the stTN, where the sensory afferents from trigeminal ganglia are lined, responded to ethanol by increasing their reactivity and AQP4-immunoreactivity (Figs. 1, 2). This region contains fibrous astrocytes, like the white matter of the cerebrum, and our results also indicated this fibrous structure^35,36) (Figs. 1, 2). In contrast, the RN contained different astrocyte shapes, which are like protoplasmic astrocytes (Fig. 1a2). The protoplasmic astrocytes in the RN also increased their reactivity upon ethanol intake, whereas they are negative for AQP4 (Fig. 2). Thus, these two types of astrocytes have different characteristics, but both respond to ethanol by increasing their reactivity (Fig. 1). Fibrous astrocytes in the white matter contact with the nodes of Ranvier to regulate the action potential propagation through ATP release.^37,38) In contrast, protoplasmic astrocytes are the major astrocyte type found in the gray matter. They extend their processes to the synapses to modulate synaptic structure and activity. They also regulate the extracellular concentrations of ions, metabolites, and neurotransmitters.^39,40) Thus, excess ethanol intake changes the functions of these various astrocytic populations. The processes of protoplasmic astrocytes are reported to make contact with the blood capillaries.^2,41) Future study is needed to clarify whether the AQP4-positive cells surrounding the capillaries in the SPV and RN (Figs. 2a2, b2) are protoplasmic astrocytes which are activated upon ethanol intake or a different subset of astrocytes. Although AQP4 is known to be primarily expressed in astrocytes, electron microscopy data showed the existence of AQP4 protein in the blood vessel endothelial cells as well.⁴²⁾ The identity of AQP4-expressing cells around the capillaries in the SPV and RN will be important to understand the mechanisms of BBB maintenance against toxic chemicals.

AQP4 is a water-permeable channel molecule and implicated to play a role in edema formation.^43,44) On the other hand, AQP4 also contributes to water reabsorption and improves vasogenic edema.^45,46) Considering that the brain weight did not change by the ethanol-administration in our results (Fig. 4B), the increased AQP4 expression by ethanol did not induce apparent brain edema and tissue degeneration (our unpublished observation). Thus, AQP4 in astrocytes and/or endothelial cells after ethanol exposure may function to maintain BBB. Alternatively, it is possible that expression of other aquaporin channels is also regulated by ethanol, which attenuates the edema-causing activity of AQP4.

Microglia are also reported to be an important cell type in the regulation of ethanol intoxication.^47,48) However, our immunostaining analyses with anti-ionized calcium-binding adaptor molecule 1 (IBA1)/allograft inflammatory factor 1 (AIF1) antibody did not show any difference between control and ethanol-treated samples (our unpublished observation). The toxic effect of ethanol depends on the quantity and duration of intake; thus, our ethanol administration regimen exerts its effect without affecting the microglial activity.

The stTN contains axons of trigeminal nerve connecting craniofacial skin and muscles to transmit pain, temperature, and motor information. The gray matter of the brainstem, including the RN, is also important for the transmission of sensory and motor signals. In addition, the brainstem also coordinates motor behaviour and controls the sleep-wake cycle and vital functions.^49,50) Although the physiological functions and importance of astrocytes in the brainstem have not been intensely studied, they would play important roles for the neurological functions written in above. The binge levels of ethanol intake may result in the neurological defects related to the astrocytes. The functional consequences of the increased reactivity of astrocytes in the brainstem after ethanol and estradiol-treatment should be examined in future studies.

Effect of Ethanol on Cerebellum Astrocytes

Alcohol-dependent patients sometimes develop gait ataxia and lower limb postural tremor, due to the dysfunction of the cerebellum. Ethanol is known to interact with GABA-mimetic drugs, such as barbiturates, to potentiate GABA_A-receptor signaling.^51–53) Histologically, ethanol is reported to disrupt the molecular events at the mossy fiber–granule cell–Golgi cell synaptic sites and the granule cell parallel fibers–Purkinje cells (GPP) synaptic site, resulting in ethanol-induced cerebellar ataxia.⁵⁴⁾ However, the toxic effect of ethanol on the cerebellum astrocytes has not been clarified. With our regimen, ethanol selectively affected the Bergmann glia without affecting protoplasmic and fibrous astrocytes (Fig. 3). Our analysis did not conclude whether the Bergmann glia were damaged and eliminated from the Purkinje and molecular layers or they just lost their GFAP-immunoreactivity without structural changes. The physiological consequences of the loss of GFAP-immunoreactivity in Bergmann glia requires further investigation, as Bergmann glial processes cover the synapses on Purkinje cell dendrites and regulate the neuronal activity of Purkinje cells.⁵⁵⁾

Thus, the effect of ethanol is largely different among cell types and brain regions. Our previous study indicated that the astrocytes in the cerebral cortex are suppressed and released from their interaction with blood vessel endothelial cells after ethanol exposure.⁴⁾ The exact mechanisms how ethanol exerts different effects on astrocytes and brain structure in different regions are not yet clear. The difference in the sensitivity toward ethanol among astrocyte subtypes may explain the diverse effects. In addition, it is also possible that there are region-specific target molecules for ethanol.¹⁾

Interaction between Ethanol Toxicity and Sex Hormone Signaling

Astrocytes are one of the major cellular targets of estrogen. Neuron- or astrocytes-derived 17β-estradiol increases reactivity of astrocytes and protect neurons from the injury-induced neuronal damages.^6,7) Estradiol has been presented to suppress AQP4 expression in cultured astrocytes with an ischemic condition.^56,57) Our results indicated that the single administration of estradiol increased the signal intensity of AQP4 in astrocytes in the stTN, whereas the combined administration of ethanol and estradiol decreased the AQP4 signal intensity (Fig. 5). Interestingly, AQP4 expression is increased by estradiol in mouse uterine.⁵⁸⁾ From these findings, it can be speculated that estradiol may upregulate AQP4 expression in static conditions but downregulate it in activated or stimulated conditions. The combinatory effect of ethanol and estradiol is further discussed in below.

Ethanol has a toxic effect on the reproductive system and sexual behaviour. Therefore, the interaction between the sex hormone signaling and ethanol toxicity is a research topic gathering attention from researchers. In humans, common and mild levels of alcohol intake increase the blood estradiol levels and decrease progesterone levels.⁵⁹⁾ Thus, the combined effect of ethanol with estradiol is important to understand the ethanol toxicity, especially in women. An in vitro study with intestinal Caco-2 cells indicated that the ethanol toxicity was enhanced by the co-administration with estradiol.¹⁰⁾ In contrast, another study demonstrated the neuroprotective effect of estradiol against ethanol toxicity in the developing rat cerebellum.⁶⁰⁾ This effect may be mediated by the enhanced expression of brain-derived neurotrophic factor.¹¹⁾ Both ethanol and estradiol decrease the secretion of transforming growth factor (TGF)-β1, but increase that of TGF-β3 and basic fibroblast growth factor, from the pituitary cells.⁶¹⁾ Thus, estradiol signaling has a coordinating effect on ethanol toxicity, although the effect is dose- and context-dependent. In our results, the co-administration with estradiol weakened the increased astrocytic reactivity by ethanol in the brainstem, although the administration of estradiol itself caused changes in astrocyte reactivity (Fig. 5). Thus, ethanol toxicity and estradiol signaling are mutually affected and the co-existence of ethanol and estradiol may exert novel physiological consequences. Future studies should be performed to examine the differences in ethanol-induced neurological dysfunction under different sex and menstrual/pregnant conditions.

Acknowledgments

We thank Dr. Kimie Nakagawa, Laboratory of Hygienic Sciences, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, for her kind suggestion on the immunostaining method. We also thank Dr. Kei Nakayama, Dr. Koji Teramoto, and Ms. Tomoko Okuno, Laboratory of Hygienic Sciences, Kobe Pharmaceutical University, for their helpful support for laboratory management and technical assistance.

Funding

This work was supported by JSPS KAKENHI (JP21K07306 to HH).

Conflict of Interest

The authors declare no conflict of interest.

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