Acute exposure to small amounts of alcohol boosts glymphatic function

Awake, behaving C57Bl6 mice received low, intermediate, and high doses of alcohol (0.5, 1.5 and 4 g/kg [intraperitoneally, I.P.], respectively) 15 min before injection of CSF tracers into the cisterna magna compartment via a cannula implanted 24 hrs earlier. The brains were quickly harvested at 30 min after the tracer injection and immersion-fixed followed by preparation of coronal vibratome sections. Microscopic examination revealed robust tracer influx along paravascular pathways surrounding predominantly cortical arterioles and the anterior choroidal artery in all coronal slices analyzed (+1.1, 0, −1, −2, −3, −4 mm from bregma) (Fig. 1A,B). Tracer influx was also noted in and around the hippocampal formation (Fig. 1C). On average, the low-dose alcohol group (0.5 g/kg) exhibited a 39.8 ± 2.8% increase in CSF tracer influx compared to vehicle controls (2-way ANOVA p < 0.05 for all coronal series, one-way ANOVA for average, p < 0.001) (Fig. 1E,F). In contrast, intermediate (1.5 g/kg) and high acute alcohol doses (4 g/kg) suppressed glymphatic function by a mean of 33.5 ± 3.0% and 28.0 ± 6.0%, respectively (both p < 0.001 for all series and average) (Fig. 1E,F). Interestingly, the suppression of glymphatic influx in the intermediate and high dose of alcohol were most pronounced along the cortical vessels, with lesser reduction in tracer influx via thalamic or hypothalamic influx pathways (Fig. 1B–D). The suppressive effects of alcohol on cortical CSF tracer influx were present across the rostral to caudal axis of the brain. Taken together, these observations show that a low acute dose of alcohol improves glymphatic function, whereas higher acute doses of alcohol have a suppressive effect on glymphatic function assessed with the CSF tracer. The acute glymphatic suppression from moderate and high dose alcohol may be due simply to lowered cardiac output from alcohol intoxication24 resulting in reduced pulse pressure, which has previously been identified as a driver of CSF flux along the periarterial channels25.

Figure 1 Low or high alcohol doses have opposite effects on CSF tracer penetration. (A) Representation of the coronal brain slices used in analysis to assess glymphatic function, with anterior-posterior distance indicated in mm in relation to bregma. (B) Coronal sections of mouse brain at 30 minutes after cisterna magna injections of Alexa-647-conjugated bovine serum albumin (BSA-647) in awake mice. Scale bars: 1 mm. (C) Tracer influx in the cortex in two different representative mice. Scale bars: 100 µm. (D) Influx in the hippocampus at bregma −1 mm. Scale bars: 100 µm. (E) Area covered by tracer influx in coronal brain slices collected 30 minutes after cisterna magna tracer injection in mice given saline or alcohol 15 minutes before they were injected with CSF tracer. X-axis indicates the distance to bregma. CTR, saline control; low, medium, high, 0.5, 1.5 and 4 g/kg ethanol, respectively. 2-way ANOVA compared to control. (F) Average difference compared to control for all brain slices analyzed. One-way ANOVA compared to control. *p < 0.05, **p < 0.01, ***p < 0.001. Bar graphs represent mean and standard error of the mean (SEM) of 7–9 mice per group. Full size image

Chronic low dose alcohol intake increases glymphatic function while chronic medium dose is inhibitory

To assess the effects of chronic alcohol consumption, we next treated mice with low and intermediate doses of alcohol for 30 days and assayed glymphatic function in the awake state shortly after the last alcohol administration (Fig. 2A). The group of mice that received the low chronic dose (0.5 g/kg) exhibited a non-significant trend towards increased CSF tracer influx in coronal slices along the caudal-rostral axis compared with vehicle-treated controls (Fig. 2B,C). When averaging all the slice series, there was a non-significant 6.4 ± 5.2% influx increase in the low dose alcohol group (Fig. 2D). Conversely, prolonged administration of the medium dose (1.5 g/kg) significantly suppressed mean glymphatic function in caudal-rostral slices by 19.2 ± 3.3% (one-way ANOVA, p < 0.001) (Fig. 2C,D). We did not include the high dose in our long-term exposure experiments, as this dose may introduce confounding factors such as hepatic inflammation and steatosis26,27, and in our pilot study, chronic exposure to the high dose of alcohol had a mortality rate of 40%.

Figure 2 Withdrawal from long term alcohol exposure increases glymphatic function. (A) Timeline diagram of experiment. Arrows on the bottom indicate injections of alcohol (EtOH) or saline. This experiment was performed 15 minutes after the last of 30 days of daily injections. (B) Tracer influx in the cortex 30 minutes after cisterna magna injections of Alexa-647-conjugated bovine serum albumin (BSA-647) in awake mice with chronic alcohol exposure. Scale bars: 100 μm. (C) Area covered by tracer influx in coronal brain slices 30 minutes after cisterna magna injection. X-axis indicates the distance to bregma. 2-way ANOVA compared to control. (D) Average difference compared to control condition for all coronal brain slices analyzed. One-way ANOVA compared to control. (E) Timeline diagram of experiment. Arrows on the bottom indicate alcohol (EtOH) or saline injections. This experiment was performed 24 hours after the last of 30 daily tracer injections. (F) Tracer influx in the cortex 30 minutes after cisterna magna injections of Alexa-647-conjugated bovine serum albumin (BSA-647) in awake mice with 24 hours withdrawal from chronic alcohol treatment. Scale bars: 100 μm. (G) Quantifications of tracer influx in mice treated with alcohol or saline for 30 days, measured 24 hours after the last treatment. 2-way ANOVA compared to control. (H) Average difference compared to control for all coronal brain slices analyzed. One-way ANOVA compared to control. CTR, saline; low, 0.5 g/kg ethanol; medium, 1.5 g/kg ethanol. *p < 0.05, **p < 0.01, ***p < 0.001. Bar graphs represent mean and standard error of the mean (SEM) of 7–11 mice per group. Full size image

To assess whether the effects of chronic alcohol on glymphatic function are reversed after withdrawal, we administered alcohol daily for 30 days and then performed glymphatic function assays at 24 hours after the last dose of alcohol (Fig. 2E). Interestingly, the 24-hour withdrawal from low dose chronic alcohol treatment increased glymphatic function by an average of 19.1 ± 6.6% compared to saline (one-way ANOVA, p < 0.05) (Fig. 2F–H). Withdrawal from the intermediate dosage of alcohol for 24 hours was met with a restoration of glymphatic function to normal levels based on two comparisons. We first considered coronal levels at 1 mm intervals extending from −4 to +1.1 mm relative to bregma in the anterior-posterior direction (two-way ANOVA), and next calculated the mean uptake in all slices (one-way ANOVA). Neither of these comparisons revealed any difference in glymphatic function between saline and medium dose alcohol groups at 24 hours after the last of 30 daily alcohol administrations (Fig. 2G,H).

To test whether tracer washout was affected by chronic low and medium alcohol intake, we next quantified tracer accumulation following three hours of circulation28. Since influx of CSF tracers peaks after approximately 30–60 min, the prolonged tracer circulation time can be used to study clearance without invasive intraparenchymal injections28. The CSF tracer signal after three hours was reduced by 22.2 ± 5.3% (p < 0.001) in the low dose and by 40.9 ± 4.0% (p < 0.001) in the medium chronic alcohol dose mice while affected by alcohol (Fig. 3A–C, Supplemental Fig. 1A). At 24-hour withdrawal from chronic treatment, the percent of brain area containing fluorescence signal was on average 17.2 ± 5.3% and 29.9 ± 2.7% lower with low and medium dose of alcohol compared with saline, respectively (p < 0.05 and p < 0.001, respectively) (Fig. 3D–F, Supplemental Fig. 1B). These observations were consistent with increased glymphatic clearance in the low dose alcohol group, since tracer influx at 30 min was higher than the saline group (Fig. 2B), yet the tracer amount was reduced to lower levels at three hours compared with the saline control mice (Fig. 3A–F, Supplemental Fig. 1A,B). Since tracer influx was reduced at 30 min in the chronic moderate alcohol group, the reduced tracer accumulation after three hours accumulation cannot be unambiguously attributed to increased clearance.

Figure 3 Tracer clearance is improved in alcohol-treated mice. (A) CSF tracer in coronal slices of left brain hemisphere at 180 min after injection in mice chronically treated with saline (CTR), low (0.5 g/kg) and medium (1.5 g/kg) doses of alcohol with the last dose of alcohol given immediately before tracer injection (EtOH), (B) quantification of tracer in slices at the indicated distances to bregma (2-way ANOVA) and (C) average difference compared to control condition for all coronal brain slices analyzed (one-way ANOVA). (D) CSF tracer in coronal slices of left brain hemisphere at 180 min after injection in mice chronically treated with saline (CTR), low and medium dose of alcohol with the last dose of alcohol given 24 hours before tracer injection (withdrawal), (E) quantification of tracer in slices at the indicated distances to bregma (2-way ANOVA) and (F) average difference compared to control condition for all coronal brain slices analyzed (one-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001. Bar graphs represent mean and standard error of the mean (SEM) of 5 saline mice, 10 low and 10 medium alcohol dose mice per group for both EtOH and withdrawal experiments. Full size image

Clearance of tracer from the brain could also be confounded by leakage via the blood-brain barrier (BBB). There are conflicting reports on the effect of chronic alcohol exposure on BBB function. Some reports in laboratory animals have found an increase in BBB permeability due to alcohol intake but only when another challenge, such as food withdrawal or lipopolysaccharide (LPS) was added29,30. Efflux of CSF tracer usually happens by drainage via the peripheral lymphatic system31. However, a defect in the BBB could potentially cause leakage of tracer directly into the blood circulation. To test whether increased dye shunting to blood occurred in the alcohol groups, we evaluated the signal derived from the CM-injected tracer in the livers of mice receiving saline or alcohol treatment (Supplemental Fig. 2A,B). The fluorescence signal was increased compared to the background signal in non-tracer injected mice (Supplemental Fig. 2C), but there was no difference in the dye intensity between the tracer-injected groups, neither for chronic alcohol administration, nor upon withdrawal after chronic administration (p = 0.23 and p = 0.74, respectively) (Supplemental Fig. 2D).

The effect of alcohol on glymphatic function is not mediated by changes in sleep

Since glymphatic function is highly regulated by the sleep cycle20,32 we investigated whether the beneficial effects of low dose alcohol on the glymphatic system in mice was due to changes in average sleep times. We used an immobility-defined sleep assay to non-invasively measure the fraction of sleep versus waking in the saline, low and medium alcohol mouse groups33,34. This assay correlates with 94% accuracy to sleep measured by standard EEG/EMG procedures33,34,35. Following three days of acclimatization to their new home cage, we analyzed sleep during a 24 hour period. The low and medium dose alcohol group did not show any difference in light phase, dark phase or average sleep time compared to the saline group (1-way ANOVA, p = 0.86, p = 0.23 and p = 0.26, respectively) (Fig. 4A–C). Neither could we detect any group differences for the mean number or length of sleep bouts for low or medium alcohol (p = 0.13 and p = 0.89, respectively, not shown). We note that the mice were closely monitored throughout the glymphatic experiments, which span 30 minutes (for influx) and 180 minutes (for clearance), and were not allowed to sleep during the period spanning from tracer injection to brain harvest.

Figure 4 Long term exposure to 1.5 g/kg alcohol does not affect sleep but impairs motor and learning skills. Immobility-defined sleep duration in saline (CTR), 0.5 g/kg (low) and 1.5 g/kg (medium) alcohol treatment groups did not show any difference between the groups for (A) light phase sleep, (B) dark phase sleep or (C) mean sleep duration. Alcohol was administered at 9 am, three hours into the light phase. Latency to fall of the Rota-Rod (D) at 24 hours after withdrawal from 30 days chronic alcohol exposure or (E) with acute alcohol challenge following 30 days of chronic exposure. 2-way ANOVA with Tukey test compared to saline treatment or low dose alcohol. *p < 0.05, **p < 0.01 compared to saline. #p < 0.05 compared to low dose. Novel object test at (F) 24 hours after withdrawal from chronic alcohol exposure and (G) acutely affected by alcohol following 15–28 days of chronic exposure. New objects had never previously been seen by the mouse, and old objects had been introduced to the mouse for first examination for ten minutes at a time one hour prior to testing on the same experimental day. Student’s t-test comparing percentage time spent exploring new and old object. CTR, saline; low, 0.5 g/kg ethanol; medium, 1.5 g/kg ethanol. Ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Bar graphs represent mean and standard error of the mean (SEM) of 4 mice per group for sleep analysis, 11–21 mice per group for novel object test and plots represent mean and SEM of 15–25 mice for Rota-Rod test. Full size image

24 hours of withdrawal from chronic binge-level exposure alcohol does not entirely restore motor function

Comparisons of preclinical studies of alcohol toxicity are confounded by factors influencing ethanol elimination, notably including food intake, which was not controlled in this study36. However, we did investigate the effects of alcohol on motor performance of the mice, using a Rota-Rod with a rotation rate increasing from 5 to 40 rpm37. Results of two trials were averaged and repeated three times on separate days. We first performed the withdrawal trials in the mice with chronic alcohol treatment followed by 24 hours withdrawal; the mean Rota-Rod performance in saline-treated mice was then 165.9 ± 13.0 seconds (Fig. 4D). The saline and low alcohol groups showed improvement in the second trial, the saline being able to remain on the rotating rod for 246.3 ± 13.3 seconds on day 2, but motor performance did not improve after withdrawal in the medium dose group. The latency to fall off the Rota-Rod upon withdrawal was 67.2 ± 15.3 seconds briefer in the medium dose alcohol group than in the saline-treated group (p < 0.05). Overall, the medium-dose group showed a 27.3 ± 6.2% decrease in time to failure in comparison to the saline group. All groups showed an improvement in motor performance between days two and three, and there were no significant group differences on day three. Performance by the low-dose and control groups did not differ in any trial.

We then assessed mice on the Rota-Rod after chronic alcohol exposure with final alcohol administration at 15 min before the trial. The saline-treated group stayed on the rod for 384.8 ± 29.2 seconds at day 1, 345.2 ± 30.3 seconds at day 2, and 406.9 ± 34.5 seconds at day 3. While there was no difference between the saline and the low dose group in any of the trials, performance differed between the low and the medium dose group on day 2, when the medium group fell off the rod 128.8 ± 38.3 seconds sooner (p < 0.05), corresponding to a 37.3 ± 11.2% decrease in Rota-Rod endurance. On day 3, the mice that had received the medium dose of alcohol fell off the rod 147.1 ± 17.6 seconds before the saline-treated mice (p < 0.01) (Fig. 4E), corresponding to a 31.7 ± 7.6% decrease in comparison to the saline group. Thus, the low dose of alcohol did not affect motor skills after acute alcohol challenge, while the medium dose perturbed motor skill with acute alcohol challenge and upon 24 hours of withdrawal.

Chronic administration of alcohol impairs learning and memory both under the influence and during withdrawal

After chronic alcohol administration, the mice also underwent a novel-object test for learning and memory. Each group of mice was randomized into three sub-groups and left to explore two objects in an open field. In the test phase, the mice were presented with one familiar and one novel object, and their interactions recorded.

24 hours after the last alcohol or saline administration, the saline group spent a significantly longer time exploring the new object compared to the familiar object (p < 0.001), similar to results in the low dose alcohol group (p < 0.01) (Fig. 4F). In contrast, the medium alcohol dose group did not spend significantly more time exploring the novel object in the withdrawal phase (p = 0.35). In the experiments with acute alcohol treatment after chronic exposure, the mice in the saline and low alcohol dose groups still spent more time exploring the new object than the old object (p < 0.01, and p < 0.05, respectively) (Fig. 4G). However, the medium dose group did not show any increased interest in the novel object, suggesting that these mice suffer from learning impairment both under the influence and during alcohol withdrawal (p = 0.43).

Alcohol exposure produces different changes in GFAP and AQP4, depending on the dose

A range of conditions in which astrocytes acquire a reactive phenotype, characterized by hypertrophy of GFAP-positive processes, have in several prior publications been associated with reduced glymphatic function38,39,40. We evaluated GFAP immunostainings of mice chronically treated with low or medium alcohol doses in comparison to control groups. Chronic exposure to the medium dose of alcohol did not change astrocytic GFAP-positive processes in the cortex (Fig. 5A,B). However, chronic exposure to the medium alcohol dose upregulated GFAP immunostaining-intensities in the corpus callosum (Fig. 5C,D) and hippocampus (Fig. 5E,F) (p < 0.05 and p < 0.05, respectively). Interestingly, treatment with the low dose of alcohol significantly decreased GFAP immuno-staining in cortex, corpus callosum, and hippocampus (p < 0.01, p < 0.05, p < 0.05, respectively) (Fig. 5A–F).

Figure 5 Chronic administration of 0.5 g/kg alcohol decreases GFAP, while 1.5 g/kg alcohol increases astrogliosis. Immunostaining for GFAP (astrocyte marker, magenta) and DAPI (white) and quantifications thereof in control mice and in mice chronically treated with alcohol in cortex (A,B), corpus callosum (C,D) and hippocampus (E,F). CTR, saline; low, 0.5 g/kg ethanol; medium, 1.5 g/kg ethanol. *p < 0.05, **p < 0.01, one-way ANOVA with Tukey test. Scale bars, 100 µm. Bar graphs represent mean and standard error of the mean (SEM) of 3–4 mice per group. Full size image

We next investigated the astrocyte-specific water channel AQP4, which is located specifically in the plasma membrane of astrocyte end-feet41,42,43. AQP4 has previously been shown to play a crucial role in glymphatic function18,20,38,44. Chronic daily exposure to medium alcohol doses increased the general level of parenchymal AQP4 in astrocytes of the cortex and corpus callosum (p < 0.001 and p < 0.05, respectively) (Fig. 6A–D), but AQP4 expression was unaffected in the hippocampus (Fig. 6E,F). There were no corresponding effects of low dose of alcohol relative to the saline group.

Figure 6 Chronic administration of medium dose alcohol increases AQP4 expression and reduces AQP4 polarity. Immunostaining for AQP4 (blue) and DAPI (white) and quantifications thereof in control mice and mice with chronic alcohol treatment in cortex (A,B), corpus callosum (C,D) and hippocampus (E,F). Line-plots of AQP4 immuno-intensity across arterioles (G) in the cortex, and calculation of the peak-to-baseline level (AQP4 polarization index) (H). Line-plots of AQP4 immuno-intensity across capillaries (I) in the neocortex and calculation of the AQP4 polarization index (J). CTR, saline; low, 0.5 g/kg ethanol; medium, 1.5 g/kg ethanol. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey test. Scale Bars, 100 µm. Bar graphs and plots represent mean and standard error of the mean (SEM) of 3–4 mice per group. Full size image

An analysis of the AQP4 polarization index38 showed that the polarization (peak intensity value at the end-feet divided by the tissue baseline intensity) was decreased in the medium alcohol group (p < 0.05), while there was no significant change in the low alcohol group (Fig. 6G,H). The reduction in polarity in the medium alcohol group was attributable to an increase in the baseline of AQP4 expression, i.e. the parenchymal expression (p < 0.001) (Fig. 6G). AQP4 polarization around capillaries did not differ in between the groups (Fig. 6I,J).

Not unexpectedly, acute alcohol exposure did not change the level of GFAP or AQP4 compared to the saline control group for neither cortex, corpus callosum or hippocampus (p = 0.99, p = 0.52, and p = 0.77 for GFAP and p = 0.44, p = 0.27, and p = 0.36 for AQP4, respectively, data not shown) suggesting that the effect of acute alcohol exposure on glymphatic function is not mediated by a change in GFAP or AQP4 expression. However, the inhibitory effects of chronic medium alcohol exposure on glymphatic function may be in part mediated by the increased reactive gliosis and mislocation of AQP4 water channels in astrocytes38,39. Conversely, the beneficial effects of low doses of alcohol on glymphatic activity might point to a novel mechanism whereby decreased GFAP expression facilitates glymphatic system function. On the other hand, it is also possible that the decrease in GFAP is not causally linked to the increase in glymphatic activity.

Low doses of alcohol exposure change the cerebral cytokine profile

Gliosis often goes hand in hand with neuroinflammation45. The up- and downregulation of GFAP in astrocytes exposed to prolonged medium and low levels of alcohol, respectively, led us to ask whether alcohol affects the inflammatory status of the brain. Depending on the dose, alcohol has different effects on systemic inflammation with a tendency to a decrease in peripheral inflammation with light intake and conversely an increase in inflammation in chronic high intake46,47. However, there are no reports describing the effect of prolonged exposure to low doses of alcohol on the CNS cytokine profile. We therefore treated groups of mice with low and medium doses of alcohol for four weeks and collected the brains after a brief transcardial perfusion with PBS to avoid contaminating the brain samples with systemic cytokines. Interestingly, the blinded cytokine analysis of the brain showed that interleukin (IL)-9 was elevated to 228.2 ± 26.1% of saline values (p < 0.05) in the low dose alcohol group, but unchanged in the medium dose alcohol group (p = 0.78) (Fig. 7A). IL-9 is a pleiotropic cytokine expressed by thymocyte helper 2 (Th2) or T regulatory (Treg) cells; its expression can be induced by the anti-inflammatory cytokines TGFb and IL-4, but is typically reduced by classic pro-inflammatory cytokines such as interferon gamma (IFNγ) and IL-2348. Since IL-9 is produced by and acts on T cells and since no cell type native to the brain express IL-9 receptors, it is not likely to affect the brain directly49,50. The analysis of the medium alcohol dose group depicted a general pattern of down-regulation of pro-inflammatory cytokines IFNγ (−49.1 ± 2.6%; p < 0.01), and IL-12-p40 (−55.3 ± 9.3%; p < 0.05) as well as the anti-inflammatory cytokine IL-13(−92.1 ± 1.4%; p < 0.05) compared with saline treated mice (Fig. 7B–D). IL-12 acts upstream of the IFNγ pathway by inducing IFNγ secretion from natural killer (NK) cells and T cells51. IL-13, which showed a modest but significant decrease of 7.9 ± 1.4% (p < 0.05) in the medium alcohol group dose, is expressed by microglia52. The average level of many cytokines was lower in the medium dose alcohol group compared to the low dose alcohol group: IFNγ, IL-4, IL-7, IL-9, IL-12 p40, IL-12 p70, IL-13, IL15, leukemia inhibitory factor (LIF), and CCL5 (Fig. 7E–M). These cytokines are not classical astrocyte cytokines45, suggesting that chronic alcohol exposure does not affect astrocytes’ inflammatory state directly53, but decreases the general level of cytokines in the brain. We did not detect any differences between the groups in the following cytokines or growth factors: eotaxin, G-CSF, GM-CSF, IL-1a, IL-1b, IL-2, IL-3, IL-5, IL-10, IL-17, LIX, MCP-1, M-CSF, MIG, MIP-1a, MIP-1b, MIP-2, VEGF, IL-11, TGFb, MCP-1 (not shown). Thus, we find that the low alcohol dose did not induce significant changes in the cytokine profile except for IL-9, while chronic exposure to 1.5 g/kg alcohol per day was linked to a distinct cytokine pattern, with prominent downregulation of cytokines.