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. Author manuscript; available in PMC: 2026 Jul 17.
Published in final edited form as: Eur J Pharmacol. 2026 Jul 1;1030:179104. doi: 10.1016/j.ejphar.2026.179104

Repeated Ethanol Exposure in Female Rats Impairs Cerebral Endothelial Function and Increases Cerebrovascular Stiffness

Emily J Baumoel a,*, Madison C Heitkamp a,b,*, Anduela Thaqi a, Jared Luchetta a, Gianna M DeMarco a, Anushree N Karkhanis a,b,c, Abbie C Chapman a,c
PMCID: PMC13373886  NIHMSID: NIHMS2193728  PMID: 42385974

Abstract

There are several lifestyle factors known to increase the risk for age-related cognitive decline and dementia, including chronic alcohol consumption and alcohol use disorder. Chronic alcohol use in adulthood causes decreased gray matter and atrophy in brain regions central to learning and memory, including the hippocampus. Further, people with alcohol use disorder have a high incidence of cerebral white matter hyperintensities, the hallmark of cerebral small vessel disease, suggesting a link between alcohol consumption and cerebrovascular disease. However, how ethanol exposure impacts the cerebral circulation remains unclear. Here, we investigated the effects of a single ethanol vapor exposure (12 hours), repeated ethanol exposures (12 hours/day for 10 days) or air exposure (controls; n=8/group) on cerebrovascular function and structure in adult female Wistar rats, as women are more sensitive to alcohol-related brain injury. Endothelial function and myogenic reactivity of isolated and pressurized posterior cerebral arteries (PCAs), the pial artery responsible for perfusing the hippocampus, were assessed ex-vivo using pressure myography. Both single and repeated ethanol exposure impaired PCA endothelial function, however, had a differential effect on myogenic tone. Repeated ethanol exposure caused oxidative stress, vascular inflammation and reduced myogenic tone of PCAs compared to a single exposure. Further, PCAs from rats repeatedly exposed to ethanol were stiffer than arteries from controls and a single ethanol exposure, which commonly occurs with aging. These findings demonstrate that ethanol exposure rapidly impairs endothelial function in the cerebrovasculature of females and suggest that repeated exposures may result in disrupted hemodynamics and accelerated vascular aging.

Keywords: Alcohol use disorder, cerebrovascular stiffness, cerebral endothelial dysfunction, ethanol exposure, vascular inflammation

Graphical Abstract

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1. Introduction

It is estimated that 1 in 10 individuals over the age of 65 have dementia and approximately 40% have at least one form of memory dysfunction (2022; Brito et al., 2023; Cahill, 2020). While age is the primary non-modifiable risk factor, aspects of lifestyle, such as alcohol consumption, are modifiable risk factors that accelerate age-related cognitive decline. Epidemiological studies support chronic alcohol consumption and alcohol use disorder as major risk factors for all types of dementias, particularly early-onset dementia (Rochoy et al., 2019; Schwarzinger et al., 2018; Zahr, 2024). Although a relationship between chronic alcohol use and development of Alzheimer’s disease has been recognized, there is evidence of substantial increased risk for vascular cognitive impairment (VCI) as well, suggesting alcohol consumption impacts the cerebral circulation (Zahr, 2024; Zhang et al., 2022).

Maintaining healthy brain function requires a relatively constant and appropriate level of cerebral blood flow (CBF) to supply oxygen and glucose to meet metabolic demands (Meixensberger et al., 1993; Paulson et al., 1990; Siegel, 1999). The function and structure of the cerebrovasculature are critical in controlling CBF. In the cerebral circulation, one of the most influential vasoconstrictive stimuli is intravascular pressure, which is driven by blood pressure (Bayliss, 1902; Osol et al., 2002). Cerebral arteries and arterioles exist in a state of partial constriction, known as myogenic tone, allowing for vasodilation or vasoconstriction to locally control CBF. Under pathological conditions where myogenic tone is either increased or decreased, the downstream brain regions are at risk for hypoperfusion- or hyperperfusion- injury, respectively (Osol et al., 2002). The endothelium of cerebral arteries plays an important role in cerebral hemodynamics by producing vasoactive stimuli that influence lumen diameter, vascular resistance and blood flow (Andresen et al., 2006; Faraci and Heistad, 1990). Cerebral endothelial cells are delicate, and damage can occur under many pathological conditions, with endothelial dysfunction being a prominent contributor to cerebrovascular disease and VCI.

VCI involves a spectrum of cognitive dysfunction due to cerebrovascular disease, with its most severe form being vascular dementia (2022; Iadecola et al., 2019). Atrophy of gray matter structures such as the hippocampus, a deep brain region critically involved in memory, occur as part of the natural aging process primarily due to hypoperfusion (Chao et al., 2010; Gattringer et al., 2012; Glodzik et al., 2019). White matter hyperintensities, as detected on MRI, often occur as a result of aging, and are a hallmark of cerebral small vessel disease (Pantoni, 2010). Importantly, people with alcohol use disorder have hippocampal atrophy and increased incidence of white matter hyperintensities compared to individuals who do not, suggesting a direct link between chronic alcohol consumption and cerebrovascular disease (Pfefferbaum et al., 2024; Xiong et al., 2025; Zahr, 2024). How alcohol damages the cerebral circulation, including that supplying the hippocampus, is unclear, but could increase the risk for alcohol-related cognitive dysfunction.

In the current study, we investigated how ethanol vapor exposure affected cerebrovascular function and structure in rats. We compared the impact of a single ethanol vapor exposure and repeated cycles of exposure and withdrawal to air exposed controls and assessed endothelial function and myogenic tone ex vivo of posterior cerebral arteries (PCAs) that are upstream of the hippocampus using pressure myography. Structural and biomechanical properties were determined to understand the potential impact on cerebrovascular remodeling. Alcohol use is increasing specifically in women, and women are more sensitive to alcohol-related brain atrophy (Mann et al., 2005; Mann et al., 1992; White, 2020). Therefore, we specifically focused on female rats in this study.

2. Materials and Methods

2.1. Animals

Experiments were performed using adult (~12-month-old) female Wistar rats that were purchased from Charles River (N=24). Animals were housed in the Binghamton University – SUNY animal facility, an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited facility on a 12-h light/dark cycle and allowed access to food and water ad libitum. Rats were housed in groups with environmental enrichment and underwent brief (less than 24hr) single housed conditions prior to terminal experiments due to the nature of ethanol exposures. Female rats were focused on in this study, as evidence suggests that alcohol use is rising exclusively in women, and that women are more sensitive to alcohol-related brain atrophy compared to men (Mann et al., 2005; Mann et al., 1992; White, 2020). All procedures were approved by the Binghamton University – SUNY Institutional Animal Care and Use Committee (protocol #921–24) and conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All euthanasia occurred under isoflurane anesthesia.

2.2. Single and Repeated Ethanol Exposures

To understand the effects of ethanol exposure on PCA function, and whether there are differences between a single and repeated exposures, animals either received a one-time ethanol vapor exposure, 10 repeated ethanol vapor exposures, or air exposure prior to terminal experiments (n=8/group; Figure 1) (Kelley et al., 2024). In the single exposure group, rats were exposed to ethanol vapor for 12 hours at the onset of the dark cycle. For repeated intermittent ethanol exposure, rats were exposed to ethanol vapor for 12 hours each day at the onset of the dark cycle for 10 consecutive days. Ethanol vapor on days 1 – 9 were each followed by 12 hours of exposure to room air (withdrawal period). The air-exposed control group was placed in chambers identical to the ethanol vapor chambers and followed a timeline identical to the repeated ethanol exposure group, but with only air flow. Food pellets were replaced after each air or ethanol exposure. Ethanol concentration in vapor chambers was adjusted systematically to maintain blood ethanol concentrations at approximately 200 mg/dL.

Figure 1. Ethanol exposure paradigms.

Figure 1.

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A. Single ethanol exposure consisted of one 24 hour period during which rats were exposed to 12 hours of ethanol vapor exposure during the dark phase of the light cycle. B. Repeated ethanol vapor or air exposure occurred for 10 consecutive days. Rats were euthanized one hour after exposure ended and terminal pressure myography experiments performed.

2.3. Blood Ethanol Concentration

One hour after the cessation of the last air or ethanol exposure, animals were deeply anesthetized with isoflurane (3% in oxygen), body weights were collected and then euthanized by rapid decapitation. Trunk blood was collected and centrifuged at 2500 rpm for 10 minutes at room temperature. Serum was used to immediately quantify blood ethanol concentrations resulting from ethanol vapor exposure using ANALOX (Model Number AM1; Analox Instruments Ltd. London, UK). Remaining serum was aliquoted and stored at −80°C until use.

2.4. Pressure Myography

After euthanasia, brains were rapidly removed and put in ice cold aerated artificial cerebrospinal fluid (aCSF). PCA trees were immediately dissected and tertiary branches mounted, secured to glass cannulas in an arteriograph chamber and pressurized, as previously described (Figure 3A) (Chapman et al., 2013). The proximal cannula was connected to an in-line pressure transducer and a servo-null pressure control system (Living Systems Instrumentation, Inc, Burlington, Vermont, USA). The distal cannula remained closed throughout the experiment to control and manipulate intravascular pressure while avoiding flow-mediated responses. Vessels were transilluminated at a 10X objective on an inverted microscope. Lumen diameter and vascular wall thickness were continuously measured via video microscopy using a video dimension analyzer (Living Systems Instrumentation, Inc, Burlington, Vermont, USA). aCSF was aerated with medical grade blood gas containing 5% CO2, 10% O2, and 85% N2 to maintain pH at 7.4 ± 0.05. Temperature within the arteriograph chamber was maintained at 36.8 ± 0.03°C for the duration of the experiment.

Figure 3. The effect of ethanol exposures on posterior cerebral artery (PCA) function.

Figure 3.

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A. Photo micrograph of a tertiary branch of PCA secured on glass cannulas in an arteriograph chamber for pressure myography. B. PCA segment at 10X magnification and pressurized to 80 mmHg. The boxed inset denotes the region undergoing continuous lumen diameter and wall thickness measurements. C. Lumen diameters and D. calculated percent myogenic tone of PCAs from rats that were either air exposed, underwent a single ethanol exposure or repeated exposures (Rpt’d) across the intravascular pressure range studied. **p<0.01 by one-way ANOVA with a post-hoc Tukey test.

Arteries underwent a one-hour equilibration period at 80 mmHg intravascular pressure which is within the healthy pressure range for tertiary branches of PCA. After equilibration, myogenic reactivity to changes in intravascular pressure was measured by stepwise increases in pressure at 20 mmHg intervals up to 160 mmHg. Lumen diameter and wall thickness were recorded at each pressure once stable. Intravascular pressure was decreased to 80 mmHg where it remained during pharmacological probing of vascular function. Endothelial function was assessed in two ways: 1) Endothelial-derived nitric oxide (NO) that is basally produced in PCA and involved in controlling myogenic tone, vascular resistance and cerebral blood flow was tested using a nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-Arginine methyl ester hydrochloride (L-NAME; 10−5 M) (Andresen et al., 2006; Faraci and Brian, 1994). 2). Function of small and intermediate conductance calcium activated potassium (SKCa/IKCa) channels involved in endothelium-derived hyperpolarization (EDH) were tested using a specific agonist NS309 (10−5 M) (Gannon et al., 2024; Hannah et al., 2011). Lumen diameter and wall thickness were recorded under basal conditions and again after NS309 or L-NAME exposure. At the end of each experiment, PCAs were fully relaxed by replacing aCSF with a zero calcium aCSF buffer, a calcium chelator (EGTA), papaverine (10−4 M) and diltiazem (10−5 M). Passive structural measurements were made within the pressure range of 5 – 200 mmHg. Remaining PCA were frozen at stored at −80°C until use.

2.5. Immunohistochemistry

To assess vascular inflammation, we measured inducible (i)NOS expression in tertiary branches of PCA using immunohistochemistry. Frozen PCAs were rapidly thawed in 0.1M PBS (phosphate-buffered saline, calcium- and magnesium-free, pH 7.4). Tissue was fixed in 4% paraformaldehyde (Thermo Fisher, Waltham, MA, USA) for 30 min at 4 °C and subsequently washed with PBS. Samples were dried onto slides for 30 min at room temperature, rehydrated in PBS, and subjected to antigen retrieval in warm citrate buffer (pH 6.0; C9999, Sigma-Aldrich, Saint Louis, MO, USA) for 20 min. Tissue sections were blocked and permeabilized in PBS containing 10% normal goat serum (005–000-121, Jackson Immuno Research, West Grove, PA, USA) and 0.3% Triton X-100, followed by overnight incubation (16–18 h) at 4 °C with rabbit anti-iNOS primary antibody (1:100; 15323, Abcam, Waltham, MA, USA). The following day, sections were incubated with goat anti-rabbit Alexa Fluor 568 secondary antibody (1:500; A-11036, Invitrogen, Waltham, MA, USA). Slides were then mounted using ProLong Diamond Antifade Mountant (P36961, Thermo Fisher Scientific, Waltham, MA, USA) and coverslipped with #1.5 coverslips. Z-stack images were acquired at 2 μm intervals using a Leica DMi8 TCS SP8 confocal microscope equipped with a 20× oil immersion objective (PL APO 20x, NA 0.75). Image analysis was performed in ImageJ. Regions of interest (ROIs) were manually traced around vessel walls on single-plane images, and thresholding was applied within the ROI. Pixel density was defined as integrated density normalized to ROI area (integrated density/area).

2.6. Serum Biomarkers

Circulating factors involved in inflammation, endothelial dysfunction, and vascular remodeling were measured using enzyme-linked immunosorbent assays (ELISAs). Commercially available ELISA kits were used to measure serum levels of tumor necrosis factor alpha (TNFα; undiluted, RTA00–1, R&D Systems Inc, Minneapolis, MN, USA), interleukin-1 beta (IL-1β; undiluted, RAB0277, Sigma Aldrich, Saint Louis, MO, USA), intercellular cell adhesion molecule-1 (ICAM-1; 50-fold dilution, RIC100, R&D Systems Inc, Minneapolis, MN, USA), 8-isoprostane (8-iso; 2-fold dilution, 516351, Cayman Chemical, Ann Arbor, MI, USA), and matrix metalloproteinase-9 (MMP-9; undiluted, RMP900, R&D Systems Inc, Minneapolis, MN, USA).

2.7. Drugs and Solutions

NS309, L-NAME, papaverine, and diltiazem were all purchased from Sigma Aldrich (Saint Louis, MO, USA. All compounds used to make aCSF were also purchased from Sigma Aldrich (Saint Louis, MO, USA) and Fisher Scientific (Fair Lawn, NJ, USA). NS309 was reconstituted, aliquoted and stored at −20°C. Stock solutions of L-NAME, papaverine, and diltiazem were made weekly and stored at 4°C until use. Ethanol (EthylAlcohol, 190 Proof, 95%) was purchased from the Science Store at Binghamton University. aCSF contained (mM): NaCl 122.0, NaHCO3 26.0, NaH3PO4 1.25, KCl 3.0, MgCl2 1.0, and CaCl2 2.0. aCSF was made weekly and stored at 4°C without glucose. Glucose (4.0 mM) was added immediately prior to each experiment. aCSF containing zero calcium was made similarly, replacing CaCl2 with 0.5 mM EGTA.

2.8. Data Calculations and Statistical Analysis

Results are presented as mean ± standard error of the mean. Percent tone of isolated arteries was calculated using the equation: Percent tone = (1-[φactive/φpassive]) × 100%, where φactive is the lumen diameter in active conditions and φpassive is the maximum lumen diameter in fully relaxed conditions at each intravascular pressure studied. Percent reactivity to NS309 was calculated using the following equation: Percent reactivity = [(φdose – φbaseline)/(φpassive – φactive)] × 100%, where φdose is the lumen diameter of the vessel after administering NS309, φbaseline is the lumen diameter of the vessel before administering any NS309. Percent constriction to L-NAME was calculated using the following equation: Percent constriction = [1-(φdrug/φbaseline)] × 100%, where φdrug is the lumen diameter of the vessel after administering the drug, and φbaseline is the lumen diameter of the vessel before administering the drug. For wall tension, wall stress and wall strain, all diameter and wall thickness measurements were converted to cm. Wall tension was calculated by converting pressure (mmHg) into dynes/cm2 × (φinner / 2). Wall stress was calculated by the equation: Wall Stress = wall tension / wall thickness. Wall strain was calculated by the equation: Wall Strain = (φpassive – φ5) / φ5; where φ5 is the passive diameter at 5 mmHg. Percent distensibility was calculated using the equation: % Distensibility = [(φpassive – φ5) / φ5] × 100%. All statistical analyses were completed using GraphPad 8 (GraphPad Software Inc., La Jolla, CA, USA). Differences between groups were determined using an ordinary one-way ANOVA with a Tukey’s post hoc test to correct for multiple comparisons. Relationships between blood ethanol level and vascular function, iNOS expression and myogenic tone or distensibility were determined using linear regression analyses with Pearson’s correlations. Rats were randomized to treatment group; their ages evenly distributed between groups and experiments and analyses completed in a blinded fashion.

3. Results

A single exposure and repeated exposures to ethanol vapor significantly elevated blood ethanol concentrations, as expected (F(2,21) = 11.61, p = 0.034, Figure 2A). Repeated ethanol vapor exposure resulted in a reduction in body weight compared to a single exposure and air exposed rats (F(2,21) = 4.01, p = 0.0004, Figure 2B), however, there was no correlation between body weight and blood ethanol levels (Figure 2C).

Figure 2. Blood ethanol levels and body weights.

Figure 2.

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A. Blood ethanol (EtOH) concentrations at time of euthanization one hour into acute withdrawal. B. Body weights at time of euthanasia. C. The relationship between blood EtOH levels and body weight. *p<0.05, **p<0.01 by one-way ANOVA with a post-hoc Tukey test.

To understand how ethanol exposure effects the cerebral circulation and compare effects of a single exposure to repeated exposure and withdrawal, we assessed the function and structure of freshly isolated PCAs from female rats that were either exposed to ethanol for a 12-hour period or underwent repeated exposure to ethanol vapor or air for 10 days using ex vivo pressure myography. Figure 3B shows a transilluminated, cannulated PCA with a lumen diameter of ~ 140μm at an intravascular pressure of 80 mmHg in an arteriograph chamber. Two PCAs from the single ethanol exposure group were excluded from functional assessments due to damage sustained during cannulation. Lumen diameters of PCAs from both groups of ethanol exposed rats were larger than arteries from control rats exposed to air (F(2,12) = 10.71, p = 0.002, Figure 3C) that was associated with lower myogenic tone, although this did not reach statistical significance at any pressure studied (Figure 3D). Lumen diameters of PCAs from all groups remained relatively stable with consistent myogenic tone across the intravascular pressure range studied, however, diameter of PCAs from air exposed control rats increased and myogenic tone decreased in response to intravascular pressure of 160 mmHg that was not statistically significant (Figure 3C and 3D).

When intravascular pressure was returned to 80 mmHg, there was a differential effect of ethanol exposure on myogenic tone (Figure 4A). PCAs from rats with a single ethanol exposure had higher myogenic tone compared to PCAs from rats with repeated exposure that demonstrated a reduction in tone (F(2,19) = 3.82, p = 0.0403, Figure 4A). This differential response was also apparent in the correlations between blood ethanol concentrations and myogenic tone (Figure 4B). There was a positive correlation between blood ethanol concentrations and tone in PCAs after a single exposure, such that higher ethanol levels at time of euthanasia were associated with more myogenic tone. In contrast, PCA from rats undergoing repeated exposure to blood ethanol vapor had a negative correlation, with higher blood ethanol levels being associated with lower myogenic tone (Figure 4B).

Figure 4. Percent tone of posterior cerebral arteries (PCAs).

Figure 4.

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A. Myogenic tone of PCAs from rats that received a single ethanol exposure compared to rats undergoing repeated exposures (Rpt’d). Intravascular pressure is 80 mmHg. B. Correlations between blood ethanol (EtOH) levels and percent myogenic tone of PCAs. *p<0.05 by one-way ANOVA with post-hoc Tukey test.

The cerebral endothelium releases vasoactive substances that mediate lumen diameter and tone and therefore are actively involved in local control of cerebral blood flow. In the pial circulation, including PCAs, the potent vasodilator NO is basally produced through activity of endothelial NOS that counters myogenic tone (Andresen et al., 2006). To determine whether ethanol exposure affected NO production and endothelial function, we used the NOS inhibitor LNAME. Figure 5A shows the degree of vasoconstriction of PCAs in response to NOS inhibition. PCAs from air exposed control rats constricted ~ 30%, demonstrating that NO was being basally produced in healthy PCA. Ethanol exposure, regardless of duration, resulted in substantial reduction in vasoconstriction upon NOS inhibition, suggesting impaired endothelial function (F(2,19) = 6.07, p = 0.0091, Figure 5A). There were no correlations between blood ethanol concentrations and vasoconstriction in response to NOS inhibition with LNAME (Figure 5B). Two PCAs from air exposed rats were unable to be included in LNAME or NS309 exposures due to development of pressure leaks and subsequent damage to vascular wall, and were excluded from the remaining functional assessments.

Figure 5. Constriction of posterior cerebral arteries (PCAs) in response to NOS inhibition.

Figure 5.

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A. Percent constrictions of PCAs from rats that were exposed to either air, a single ethanol exposure or repeated ethanol exposure (Rpt’d) in response to NOS inhibition with LNAME. B. Correlation between percent constriction to LNAME and blood ethanol (EtOH) concentrations. **p<0.01 by one-way ANOVA with a post-hoc Tukey test.

EDH is another primary vasodilatory pathway that involves SKCa and IKCa channels, calcium-activated potassium channels expressed in endothelial cells (Hannah et al., 2011). In contrast to NO that is basally produced in PCAs, SKCa and IKCa, channels are expressed but not basally active (Cipolla et al., 2009). To understand additional aspects of endothelial function that may be compromised by ethanol exposure, we assessed vasodilation to SKCa and IKCa, channel activation with agonist NS309. Figure 6A shows vasoreactivity, the degree of vasodilation as it relates to the maximal dilation possible, of PCA in response to NS309. PCA from air exposed control rats underwent ~90% vasoreactivity upon SKCa and IKCa, channel activation, demonstrating healthy endothelial function. PCA from rats exposed once to ethanol vapor had impaired SKCa and IKCa channel function and substantially decreased vasoreactivity, however, PCA from the repeated exposure group had decreased vasoreactivity that did not reach statistical significance and was variable (F(2,17) = 3.88, p = 0.0409, Figure 6A). There were no correlations between blood ethanol levels and reactivity to NS309 in either exposure group (Figure 6B). A summary of these findings and corresponding correlations are displayed in Table 1.

Figure 6. Vasoreactivity in response to small and intermediate conductance calcium activated potassium channels (SKCa and IKCa) channels.

Figure 6.

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A. Reactivity of posterior cerebral arteries (PCAs) from air exposed, single ethanol exposed or repeatedly exposed (Rpt’d) rats in response to pharmacological activation of SKCa and IKCa channels via NS309. B. Correlation between reactivity to NS309 and blood ethanol (EtOH) levels. *p<0.05 by one-way ANOVA with a post-hoc Tukey test.

Table 1.

Summary table of main findings and correlations between blood ethanol concentrations (BEC), body weight and endothelial function.

Air Exposure Single Exposure Repeated Exposure
BEC (mg/dL) 14 ± 1 191 ± 43 ** 229 ± 39 **
Body weight (g) 330 ± 14 332 ± 17 284 ± 8
Myogenic Tone (%) 32.2 ± 2.2 36.6 ± 2.9 24.7 ± 3.6#
Constriction to L-NAME (%) 31.9 ± 4.8 9.9 ± 4.5 ** 16.4 ± 3.9
Reactivity to NS309 (%) 86.5 ± 4.1 35.4 ± 15.6 * 57.2 ± 13.0
Correlations Single Exposure Repeated Exposure
BEC vs. r2 p-value r2 p-value
Body Weight (g) 0.01 0.86 0.03 0.71
Myogenic Tone (%) 0.55 0.09 0.16 0.33
Constriction to L-NAME (%) 0.08 0.58 0.03 0.67
Reactivity to NS309 (%) 0.10 0.44 0.24 0.32
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Data are mean ± SEM.

*

p<0.05,

**

p<0.01 vs. Air,

#

p<0.05 vs. Single Exposure by one-way ANOVA with post-hoc Tukey test.

Structural remodeling of the cerebrovasculature, including PCAs, can occur under pathological conditions and affect cerebral hemodynamics. Under fully relaxed conditions, lumen diameter of PCAs (Figure 7A) and vascular wall thickness (Figure 7B) were similar in size between ethanol and air exposed groups of rats. However, PCA from rats that were repeatedly exposed to ethanol vapor were substantially stiffer than PCA from rats that had a single exposure or were air exposed, demonstrated by the leftward shift in the stress-to-strain curve (Figure 7C). Passive structural measurements were unable to be collected from one PCA from the single ethanol exposure group due to a clog in the glass cannula. These data suggest that while overt structural remodeling did not occur, as there were no changes in lumen diameter or wall thickness, composition of the vascular wall shifted towards a less distensible state after repeated ethanol exposure.

Figure 7. Structural and biomechanical properties of posterior cerebral arteries (PCAs).

Figure 7.

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A. Lumen diameters and B. vascular wall thickness of PCAs from rats that were either exposed to air, a single ethanol exposure or repeated ethanol exposures (Rpt’d) under fully relaxed conditions across the intravascular pressure from 5 – 200 mmHg. C. Stress-to-strain curves of PCAs under fully relaxed conditions as a measure of vascular stiffness.

To understand potential mechanisms by which endothelial dysfunction and structural remodeling were occurring after ethanol exposure, we measured iNOS expression in PCA, serum levels of pro-inflammatory cytokines and markers of oxidative stress and vascular remodeling. Rats repeatedly exposed to ethanol vapor had higher levels of iNOS expression in the vascular wall of PCA compared to rats that underwent a single vapor exposure (F(2,18) = 4.07, p = 0.0359, Figure 8A). PCA iNOS expression negatively correlated with myogenic tone (Figure 8B), but not PCA distensibility (intravascular pressure of 200 mmHg) (Figure 8C). Circulating levels of oxidative stress marker 8-isoprostane were higher in rats repeatedly exposed to ethanol vapor compared to those exposed to ethanol vapor a single time (Table 2). One sample in each group was excluded from the 8-isoprostane ELISA due to being statistical outliers (> 2 SD above the mean). There were no differences in circulating ICAM-1, IL-1β or MMP-9 between groups (Table 2), and serum TNFα was below the detection limit of the assay.

Figure 8. Vascular inflammation in posterior cerebral arteries (PCA) and its relationship to myogenic tone and vascular distensibility.

Figure 8.

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A. Representative images of inducible nitric oxide synthase (iNOS) expression in tertiary PCA from air exposed (left), single ethanol exposed (middle) or repeatedly exposed (Rpt’d; right). B. Quantified iNOS expression in PCA. C. Correlation between iNOS expression and myogenic tone of PCA at 80 mmHg intravascular pressure. D. Correlation between iNOS expression and distensibility of fully relaxed PCA at intravascular pressure of 200 mmHg. *p<0.05 by one-way ANOVA with a post-hoc Tukey test.

Table 2.

Serum levels of proinflammatory cytokines and markers of oxidative stress and vascular remodeling.

Air
(n=8)		 Single
(n=7)		 Repeated
(n=8)
IL-1β (pg/mL) 288 ± 147 527 ± 325 138 ± 100
ICAM-1 (pg/mL) 1676 ± 98 1451 ± 61 1743 ± 150
8-isoprostane (pg/mL) 88 ± 4 57 ± 10 133 ± 28*
MMP-9 (pg/mL) 5.2 ± 0.5 5.6 ± 0.2 5.6 ± 0.9
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Data are mean ± SEM.

*

p<0.05 vs. Single by one-way ANOVA with post-hoc Tukey test.

4. Discussion

Incidence of alcohol consumption, binge drinking and chronic alcohol use have increased since the beginning of the COVID-19 pandemic, and are projected to continue to rise in adults through 2040 (Ayyala-Somayajula et al., 2025; Capasso et al., 2021; Leventhal et al., 2022; Platt et al., 2021). People who engage in chronic alcohol consumption and have alcohol use disorder have increased incidence of white matter hyperintensities in the brain, suggesting the cerebral vasculature may play a causative role in hippocampal atrophy and alcohol-related dementia (Pfefferbaum et al., 2024; Xiong et al., 2025). Here, we show for the first time that ethanol vapor exposure in female rats damages critical aspects of cerebral endothelial function and has a differential effect on myogenic tone based on exposure level. Further, repeated intermittent ethanol vapor exposure increased cerebrovascular stiffness after only 10 days, suggesting that repeated cycles of ethanol intoxication and withdrawal accelerate vascular aging. These findings demonstrate the rapid damage that ethanol has on the cerebrovasculature, particularly after repeated exposures, and supports a putative role for the cerebral circulation in the pathogenesis of ethanol-related brain injury and dementia that is more severe in women (Mann et al., 2005; Mann et al., 1992; White, 2020).

The brain requires a constant blood supply to maintain healthy function that is largely controlled by the lumen diameter of cerebral arteries, including the PCA. Even relatively small changes in lumen diameter have a robust effect on cerebrovascular resistance and therefore blood flow (Faraci and Heistad, 1990; Wheeler and Brenner, 1995). Impaired endothelial function often causes vasoconstriction due to the loss of the vasodilatory influence of endothelial (e)NOS-derived NO (Iadecola and Davisson, 2008). In the current study, PCAs from rats that underwent a single ethanol exposure had increased myogenic tone and a blunted vasoconstrictive response to NOS inhibition, indicating decreased generation of NO. Previous studies have shown that exposure of brain endothelial cells to ethanol causes local oxidative stress that reduces the bioavailability of NO, and acute exposure of pial arteries to ethanol impairs NO synthesis in-vivo (Haorah et al., 2005; Mayhan and Didion, 1995). The current study is in support of these previous findings, and advances our understanding of how acute, systemic ethanol exposure impairs endothelial function of the pial circulation upstream of the hippocampus.

In contrast to a single ethanol exposure, repeated intoxication and withdrawal cycles resulted in elevated serum 8-isoprostane, increased iNOS expression in PCA and decreased myogenic tone. Increased expression of iNOS causes overproduction of NO, resulting in vasodilation and decreased myogenic tone (Hemmrich et al., 2003; Turner et al., 2024). In the current study, PCAs from repeatedly exposed rats had greater vasoconstriction to NOS inhibition, and although this did not reach statistical significance, it suggests higher levels of NO being produced than in vessels after one exposure. Further, iNOS, which is not expressed under healthy conditions, impairs SKCa and IKCa channel function (Villalba et al., 2014), which was also a consequence of repeated ethanol exposure. There were no increases in circulating proinflammatory cytokines TNFα, IL-1β or ICAM-1 after repeated ethanol exposure, suggesting that the one-hour withdrawal window missed the peak inflammatory response, and/or vascular changes may be driven by localized oxidative stress rather than systemic inflammation. Regardless, repeated exposure to ethanol vapor appears to cause cerebrovascular inflammation and endothelial cell dysfunction. These effects could have deleterious consequences, particularly in the setting of chronic alcohol use and in individuals with pre-existing cerebrovascular disease.

Vascular inflammation and diminished myogenic tone impair multiple protective actions of the cerebral circulation, including CBF autoregulation (Reeve et al., 2024). CBF autoregulation is the innate property of the brain to maintain relatively constant blood flow in the face of changes in cerebral perfusion pressure between ~ 60 – 160 mmHg (Armstead, 2016; Harper, 1966). A primary mechanism by which this occurs is through the ability of cerebral arteries to increase myogenic tone in response to increased blood pressure and vice versa. By rapidly adjusting vascular resistance, blood flow remains unchanged and the delicate microcirculation remains protected from excessively high perfusion pressure (Faraci and Heistad, 1990). Our findings of impaired myogenic tone in PCAs from rats that were repeatedly exposed to ethanol vapor suggests autoregulatory mechanisms are impaired. As alcohol consumption rapidly stimulates the sympathetic nervous system and increases blood pressure, impaired autoregulation would result in increased cerebral perfusion (Andrade et al., 2009; Ireland et al., 1984; Randin et al., 1995). In fact, CBF was higher in college students with a history of alcohol use one hour after an alcohol challenge compared to placebo controls, as measured via ASL MRI (Tolentino et al., 2011). Further, social drinkers had increased CBF in temporal regions one hour after consuming alcohol, as measured using PET, suggesting involvement of the PCA (Volkow et al., 1988). Thus, our findings that repeated ethanol exposures causes iNOS expression in PCA and reduced myogenic tone suggest autoregulation is impaired, resulting in a more linear relationship between alcohol-induced hypertension and CBF. Given that the PCA is upstream of the hippocampus, a brain region known to be highly susceptible to injury and critically involved in memory, prolonged PCA dysfunction and unregulated blood flow associated with chronic alcohol consumption may be a mechanism of hippocampal injury and alcohol-related dementia, however, future studies are needed to investigate this further.

Here, we show for the first time that repeated cycles of ethanol exposure and withdrawal over 10 days increase PCA stiffness, providing evidence that relatively brief ethanol exposure changes the composition of the vascular wall of large cerebral arteries to decrease elasticity and promote stiffness. The elasticity of arteries, including pial arteries in the brain, acts to absorb pulse pressure thereby protecting the smaller arterioles and microvasculature from pressure-induced damage (Reeve et al., 2024). Increased vascular stiffness results in greater cerebral pulsatility being transmitted deeper into the microcirculation that damages the endothelium, and higher pulsatility in pial arteries is associated with progression of cerebral white matter lesions (Birnefeld et al., 2020; Kneihsl et al., 2020; Saji et al., 2015; Saji et al., 2016). There are several mechanisms involved in increased cerebrovascular stiffness, including a role for iNOS. iNOS-mediated nitrosative stress increases cerebrovascular stiffness, promotes cell senescence and contributes to VCI (Yin et al., 2022). Interestingly, PCA stiffness did not correlate with iNOS expression in the current study, and future studies are needed to determine collagen:elastin ratios in the vascular wall that may contribute to this increased vascular stiffness. Regardless, the repeated ethanol exposure-induced vascular stiffness could contribute to greater incidence of white matter lesions on MRI present in individuals who chronically consume alcohol or have alcohol use disorder (Pfefferbaum et al., 2024; Xiong et al., 2025).

Excessive and prolonged alcohol consumption increases the risk of chronic hypertension, particularly in women, which could contribute to PCA dysfunction in the current study (Cecchini et al., 2024; Del Giorno et al., 2022; Hwang et al., 2022). However, there was no overt ethanol-induced vascular remodeling that would be expected to occur if repeated ethanol exposure caused chronic hypertension (Iadecola and Gottesman, 2019). It is possible that the increase in PCA stiffness may be the early stages of the remodeling process, and a more severe and chronic exposure paradigm may cause hypertension and result in remodeling of the cerebrovasculature. Alternatively, ethanol-induced cerebrovascular stiffness may be unique, involve multiple pathological or inflammatory processes, and/or be unrelated to hypertension. Whether the ethanol exposure paradigm used in the current study increases blood pressure, the extent to which this occurs and for how long it persists remains unclear, but necessary to better understand the potential role of blood pressure in ethanol-induced increased PCA stiffness.

Estrogen and progesterone impact cerebral endothelial cell function, myogenic tone and vascular stiffness, including through NO signaling (Krause et al., 2006). In the current study, estrous cycles were not assessed at the time of terminal experiments. Therefore, we are unable to determine if estrous cycle contributed to ethanol-associated changes in myogenic tone or PCA stiffness. However, rats were all housed similarly and randomly assigned to group, making it unlikely that rats receiving a single versus repeated ethanol exposure were cycling differently that could skew the PCA data. Thus, it seems more likely that the differential effects of ethanol exposure on myogenic tone and PCA stiffness were due to the degree of ethanol exposure and intoxication and withdrawal cycles rather than estrous cycle alone.

Alcohol consumption is projected to continue increasing, and women are particularly at risk for alcohol-related cardiovascular disease and brain injury. The current study was focused on understanding how single or repeated ethanol vapor exposures directly affected the cerebrovasculature in females. Therefore, PCA function and structure were studied one hour into acute ethanol withdrawal. A limitation of the current study is the acute nature of vascular assessments. Future studies are needed to determine whether ethanol-induced cerebrovascular dysfunction and increased stiffness are long-lasting, whether CBF autoregulation is impaired, and any subsequent neuronal injury and cognitive dysfunction. Further, it is unclear whether the changes observed in vascular function and structure were driven by intermittent intoxication, intermittent withdrawal-related stress, or a combination of the two and whether they are reversible upon ethanol abstinence. While the findings from the current study provide evidence that repeated ethanol intoxication and withdrawal cycles cause cerebrovascular inflammation, endothelial dysfunction and increased stiffness, future studies are needed to understand how these changes may progress or persist, and whether repeated ethanol exposure increases the susceptibility to alcohol-related brain injury and dementia later in life.

Overall, our findings of ethanol-induced inflammation, endothelial dysfunction and increased cerebrovascular stiffness suggest that repeated ethanol exposures accelerate cerebrovascular aging that may contribute to development/progression of white matter lesions and cognitive decline in otherwise healthy individuals. Furthermore, people with preexisting cerebrovascular disease, such as those with chronic hypertension and VCI may be more susceptible to the damaging effects of chronic alcohol consumption on the cerebral circulation. In summary, the current findings advance our understanding of the detrimental effects of repeated ethanol exposure on cerebrovascular function and structure.

Highlights.

  • One EtOH vapor exposure in female rats impairs cerebrovascular endothelial function

  • NO bioavailability is reduced in cerebral arteries during acute EtOH withdrawal

  • Repeated EtOH exposure causes cerebrovascular inflammation, impairing myogenic tone

  • Repeated EtOH exposure increases pial artery stiffness, accelerating vascular aging

Funding

This work was supported by the NIH National Institute of Neurological Disorders and Stroke and National Institute on Aging 1R01 NS127284 (AC), National Institute on Alcohol Abuse and Alcoholism R01 AA028228 and P50 AA017823 (ANK) and Binghamton University – State University of New York (AC). Funding sources were not involved in study design, collection, analysis or interpretation of data, in the writing of the manuscript, or in the decision to submit the article for publication.

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability

All datasets generated and/or analyzed during the current study are publicly available within this manuscript and in the Harvard Dataverse repository https://doi.org/10.7910/DVN/6FBOHO.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All datasets generated and/or analyzed during the current study are publicly available within this manuscript and in the Harvard Dataverse repository https://doi.org/10.7910/DVN/6FBOHO.

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