Nutritional ketosis as treatment for alcohol withdrawal symptoms in female C57BL/6J mice

Upon both acute and prolonged alcohol intake, the brain undergoes a metabolic shift associated with increased acetate metabolism and reduced glucose metabolism, which persists during abstinence, putatively leading to energy depletion in the brain. This study evaluates the efficacy of ketogenic treatments to rescue psychiatric and neurochemical alterations during long-term alcohol withdrawal. Female mice were intermittently exposed to alcohol vapor or air for three weeks, during which mice were introduced to either a ketogenic diet (KD), control diet supplemented with ketone ester (KE) or remained on control diet (CD). Withdrawal symptoms were assessed over a period of four weeks followed by re-exposure using several behavioral and biochemical tests. Alcohol-exposed mice fed CD displayed long-lasting depressive-like symptoms measured by saccharin preference and tail suspension, as well as decreased norepinephrine levels and serotonin turnover in the hippocampus. Both KD and KE rescued anhedonia for up to three weeks of abstinence. KD mice showed higher latency to first immobility in the tail suspension test, as well as lower plasma cholesterol levels. Our findings show promising effects of nutritional ketosis in ameliorating alcohol withdrawal symptoms in mice. KD seemed to better rescue these symptoms compared to KE.


Chronic intermittent alcohol vapor exposure
Chronic Intermittent alcohol vapor exposure (CIE) was obtained by vaporizing ethanol (96%), which was then mixed with fresh air, and delivered to inhalation chambers at a rate of 11 L/min, resulting in vapor concentrations of 6-10 mg/L, using custom chambers built after Wang et al. 28 .CIE protocol consisted of a 4-day ON and 3-day OFF cycle of alcohol vapor.ON days were 16 h alcohol vapor (from 19:00 to 11:00 on the following day) followed by 8 h air withdrawal.CIE mice were injected with the alcohol dehydrogenase inhibitor pyrazole (1 mmol/kg, i.p. in a volume of 10 mL/kg body weight) and a loading dose of alcohol (1.6 g/kg) prior placement into the vapor chambers to maintain a high and stable level of intoxication during alcohol exposure 29 .Control air-exposed mice were subjected to the same chamber conditions with air instead of alcohol vapor and were administered pyrazole in saline before being placed into the control chambers.The housing conditions in the inhalation chambers were similar to those in the colony room (bedding, enrichment, food and water, etc.).
Experimental diets were administered starting from the third alcohol exposure cycle (Fig. 1).For feasibility and internal replication, mice were tested in three consecutive cohorts balanced for treatment (i.e., randomized block design) with each group represented in all cohorts (n = 12 per group).Mice were randomly assigned to one of 6 groups: • alcohol-exposed and CD (CIE-CD) • alcohol-exposed and KD (CIE-KD) • alcohol-exposed and KE (CIE-KE) • air-exposed and CD (air-CD) • air-exposed and KD (air-KD) • air-exposed and KE (air-KE) The experimenter was blinded to experimental group for all behavioral tests and sample analyses.

Operant oral alcohol self-administration (OSA)
Mice were trained to self-administer a 20% alcohol solution in operant-conditioning chambers (ENV-307A, Med Associates Inc, St Albans, VT, USA) prior to CIE, as previously described 30 .Experimental sessions were performed at the first day of each withdrawal window and then once a week in the long-term abstinence.

Nociception
The hot-plate test, a supraspinal thermal pain assay, was used to measure pain sensitivity 4 h after the last alcohol vapor exposure.Each mouse was placed on a horizontal metal surface (Hot Plate Analgesia Meter, Harvard Apparatus Limited, Holliston, MA, USA) preheated to 52 °C and was confined to the plate with a tall Plexiglas cylinder.The testing room was set to low illumination (≈ 25 lx; Fisherbrand Traceable dual-range light meter, Traceable, Webster, TX, USA).The latency to first licking of hind paws or jumping was recorded.In the absence of a paw licking or jumping response, a 60 s cut-off was used to prevent tissue damage.

Anxiety-like behaviors
Acute anxiety-like symptoms were assessed by the light-dark transition test on withdrawal days 1 and 5, and was conducted in activity chambers fitted with beam-break movement detection systems (OFA-510, Med Associates), as previously described 17 .The light side had low illumination (≈ 40 lx) not anxiogenic alone 31 , allowing detection of anxiogenic-like effects of alcohol withdrawal 32,33 .The voluntary time spent in the lit area, i.e., the total time spent in the lit area minus the latency to enter the dark area, the total number of crossings between light and dark area and the total distance travelled were analyzed.
Long-lasting anxiety-like symptoms were assessed by the elevated zero maze on withdrawal days 19 and 26, as previously described 34

Depressive-like behaviors
Depressive-like symptoms were assessed using the tail suspension test on withdrawal days 8 and 15 35 .Depressive-like behavior is exhibited by a decrease in the latency to the first episode of immobility and an increase in immobility time.Mice were suspended upside down by taping their tails to a metal bar.The mice were positioned to prevent them from climbing or reaching nearby surfaces and from seeing each other.A hollow cylinder 4 cm long was placed around the tail to prevent them from climbing it.The behavioral room was set to low illumination (≈ 25 lx).The test lasted 6 min and was recorded for later scoring.The latency to the first immobility bout and the duration of immobility were manually scored for the total duration of the test.The saccharin preference test was also used to assess anhedonia on withdrawal days 6-7, 13-14, 20-21, and 27-28.We used saccharin rather than sucrose to avoid potential confounders related to caloric content or diet composition.A decrease in sucrose or saccharin intake and preference over water is taken as a putative sign of anhedonia in rodents 36 .Mice were tested in a fluid-intake automated monitoring system (HM-2 system, MBRose, Faaborg, Denmark).The system was in the colony room and consisted of cages very similar to the animals' regular home cages, with two 11 cm long channels allowing only one mouse at a time access to each bottle.Each channel was equipped with two pairs of microchip readers (one pair at each end) and continuously recorded fluid intake for individual mice 37 .The system was equipped with a scale under each bottle, allowing precise recording of any fluid intake only when a mouse was present in the channel and controlling for spillage by recording any weight added to a drip receptacle under the bottles.Mice were habituated to the system for 48 h prior to alcohol exposure and given a free choice between two bottles, one with 0.1% (w/v) saccharin solution and another with tap water, regularly changing the bottles' positions to avoid side preferences.The 48-h test sessions used the same conditions.Saccharin preference was calculated as saccharin-solution intake divided by total volume of liquids consumed.No preference was considered when saccharin preference was < 65%.

USVs
It has been suggested that rodent USVs reflect the stressful nature of drug withdrawal and the anticipatory positive affect of rewarding stimuli, including alcohol 38 .Ultrasonic calls were recorded with an Avisoft UltraSound-Gate 416 Hb recording interface (Model CM16-CMPA,Avisoft Bioacoustics, Glienicke/Nordbahn, Germany) during the tail suspension test, and in the first hour of two progressive ratio (PR) OSA sessions.USV calls were recorded and analyzed as previously described 30 .

Blood measures
At multiple timepoints, blood samples were collected on ice, centrifuged at 3000 rpm for 10 min and plasma was stored at − 20°.Blood alcohol levels (BALs) were determined at the end of each alcohol exposure cycle using an Analox GL5 or GL6 analyzer (Analox Instruments, Stokesley, UK).Blood glucose levels were assessed using strips and FreeStyle Precision Neo analyzer (Abbott), Fridays between 11:00 and 15:00, after the behavioral test if any occurred on that day.Blood βHB levels were similarly assessed using strips and FreeStyle Precision Neo analyzer (Abbott), typically Tuesday and Fridays between 11:00 and 15:00 (in such cases, sampling was delayed until after behavioral testing).Animals were sacrificed 2-4 h after the last cycle of alcohol exposure, and trunk blood was collected in EDTA-coated tubes on ice, centrifuged at 3000 rpm for 10 min and plasma was stored at − 20°.Cholesterol and triglyceride levels were measured from the terminal plasma using a GL5 analyzer.

High-performance liquid chromatography (HPLC)
Hippocampus, striatum, and cerebellum were dissected from one hemisphere under microscope by an experienced experimenter between 2 and 4 h after the last cycle of alcohol exposure, weighed and directly snap frozen at − 80°C.Hippocampus and striatum samples were each homogenized in 250 µL 0.1 M perchloric acid using an immersion hand disperser, Polytron PT 1200 E (Kinematica Inc., Keyland Court Bohemia, NY, USA).Cerebellum samples were homogenized with the same method in 500 µL milliQ water.Samples were then centrifuged at 14,000 rpm at 4 °C for 20 min and then supernatant collected using a 0.22 µm filter (Avantec, 13CP020AS).

Neuroinflammation assay
Brain samples from one hemisphere, except hippocampus, striatum, and cerebellum, were dissected 2-4 h after the last cycle of alcohol exposure, weighed, and directly snap frozen at − 80 °C.Brain sample weights were around 100-130 mg and were homogenized by MagNa Lyser Instrument (F.Hoffmann-La Roche Ltd, Basel, Switzerland).Brain samples were transferred to MagNa Lyser tubes containing 750 µL of tissue extraction reagent II (FNN0081, Thermo Fisher Scientific, Waltham, MA, USA) and 2 × protease inhibitor cocktail (P8340, Merck KGaA, Darmstadt, Germany).The homogenization protocol consisted of two rounds of 25 s at 6000 rpm, with 90 s break on ice.Samples were then spun at 16,000 rcf at 4 °C for 1 min to remove some of the foam, transferred to Eppendorf tubes and then centrifuged at 16,000 rcf at 4 °C for 20 min.Supernatant was aliquoted and frozen at -80 °C.The total protein concentration was measured by Bradford assay (Merck KGaA, Darmstadt, Germany).The tumor necrosis factor (TNF)-α levels were detected by ELISA (# 88-7324-86, Thermo Fisher Scientific) following manufacturer instructions.Brain samples were loaded 1:4.Final concentrations were adjusted to the brain sample weights and protein concentrations.

Principal component analysis (PCA)
To deconvolute multidimensional phenotypes associated with each treatment and diet, a PCA analysis was performed for each diet 'pair' (CIE and air) with all variables that showed an alcohol effect, i.e., a significant difference between CIE-CD and air-CD mice.We used the number of light/dark crossings on withdrawal day 1, the average of the first 3 saccharin preference tests, the average of the two tail suspension latencies, the NE levels from hippocampus and striatum, and the Mg ++ and Na + levels.Data were standardized and principal components were selected when eigenvalues were > 1.Data were analyzed using Prism (Version 10, GraphPad, San Diego, CA, USA).

Statistical analyses
Group sizes were selected based on pilot studies 30 .Data were analyzed with alcohol exposure and diet as betweensubject factors and cohort as a blocking factor, followed by planned pairwise comparisons with all CIE groups plus the air-CD mice compared to the CIE-CD mice, and air-exposed groups compared to the air-CD mice.In addition, for the ketone levels, KD and KE groups were compared to each other.Bodyweight, BALs, ketone and glucose levels were each analyzed by mixed model repeated measures (MMRM) with time as repeated factor, alcohol exposure (except for BALs) and diet as between-subject factor, followed by planned pairwise comparisons for each withdrawal day, adjusted for multiple comparisons using the Hochberg approach.For the OSA data, a linear curve was fitted to each animal, followed by one-way analysis of co-variance (ANCOVA) on the intercept and slope parameters with baseline as the covariate.Saccharin preference and consumption, light-dark, tail suspension, elevated zero maze, cholesterol, and triglycerides data were analyzed by two-way analysis of variance (ANOVA) with alcohol and diet as between-subject factors, followed by planned pairwise comparisons using the Hochberg approach.Latencies to events were analyzed by cox proportional hazards regression model.HPLC data were analyzed by two-way ANOVA, with alcohol and diet as between-subject factors, followed by planned pairwise comparisons using the Hochberg approach.Neuroinflammation assay was run in triplicates and the median analyzed by two-way ANOVA.HPLC data, ketone and glucose levels were log (10) transformed to satisfy the parametric analysis assumptions.HPLC datapoints were considered as outliers and excluded from analyses when standard deviation differed more than four times from the group mean (three datapoints in total).Statistical analyses were performed using InVivoStat (Version 4.7.0) and R (version 3.6.3).Graphs were plotted using Prism (Version 10, GraphPad).Bodyweight, ketone and glucose levels, BALs, OSA and saccharin data are shown as group means ± SEMs.All other data are presented as individual subjects, either with violin plots representing group means, distribution, and density or as survival curves.Differences were reported as statistically significant by convention for p values < 0.05.

Ethics declaration
The animal study was reviewed and approved by Animal Experiments Inspectorate under the Danish Ministry of Food, Agriculture, and Fisheries.

Nutritional ketosis mitigated long-lasting depressive-like symptoms during alcohol withdrawal
CIE-CD displayed long-lasting depressive-like symptoms, assessed by tail suspension test, which were ameliorated by KD (Fig. 3a-d).The two timepoints were analyzed separately, since it is known that mice can habituate 35 .Cox proportional hazards regression analyses revealed that CIE-CD mice had significantly shorter latencies to first immobility compared to air-control mice on both the 8th and 15th withdrawal days (p = 0.008 and p = 0.03 respectively; Fig. 3a,b), interpreted as more depressive-like behavior.CIE-KD mice had a higher latency to first immobility compared to CIE-CD mice (p = 0.03) on withdrawal day 8 (Fig. 3a).On withdrawal day 15, both CIE-KD and CIE-KE showed a trend (p = 0.08 and p = 0.07 vs. CIE-CD) (Fig. 3b).Two-way ANOVA similarly revealed a significant overall effect of alcohol and diet (F 1,55 = 4.2, p = 0.045 and F 2,55 = 7.6, p = 0.001 respectively) and a trend for alcohol-diet interaction (F 2,55 = 2.6, p = 0.085) for time spent immobile on withdrawal day 8 (Fig. 3c).Post hoc analysis indicated that CIE-KD and KE mice showed lower time spent immobile compared to CIE-CD (p = 0.0004 and p = 0.07 respectively).However, air-CD mice did not differ significantly from CIE-CD mice.There was a significant overall effect of alcohol (F 1,51 = 4.2, p = 0.045) for time spent immobile on withdrawal day 15, but no post hoc differences (all p > 0.4) (Fig. 3d).There were no other statistically significant differences in any parameter in the air-control mice at either timepoint (all p > 0.2).

KD had modest effects on alcohol-induced anxiety-like symptoms during alcohol withdrawal
CIE-CD mice displayed acute anxiety-like symptoms measured in the light-dark transition test, which tended to be rescued by KD (Fig. 4a-f).The two timepoints were analyzed separately, since it is known that mice can habituate to anxiogenic effects in the test.For withdrawal day 1, two-way ANOVA revealed a significant overall alcohol and diet effect for number of light/dark crossings (F 1,63 = 16.1, p = 0.0002 and F 2,63 = 3.3, p = 0.04) and distance travelled (F 1,63 = 16.9, p = 0.0001 and F 2,63 = 11.3,p < 0.0001) and a trend for alcohol effect for voluntary time spent in the light area (F 1,63 = 3.5, p = 0.06) (Fig. 4a,c,e).Post hoc analyses indicated that CIE-CD mice had significantly lower light/dark crossings compared to air-CD mice (p = 0.004) and a trend for lower distance travelled (p = 0.09).In addition, CIE-KE mice travelled less than CIE-CD mice (p = 0.046) and air-KE tended to travel less than air-CD (p = 0.085).For withdrawal day 5, there was a small overall effect of alcohol for voluntary time spent in the light area (F 1,62 = 4.1, p = 0.047), diet and alcohol-diet interaction for distance travelled (F 2,62 = 9, p = 0.0004 and F 2,62 = 2.9, p = 0.06 respectively) and alcohol-diet interaction for the number of light/dark crossings (F 2,62 = 3.3, p = 0.045) (Fig. 4b,d,f).Post hoc analyses indicated that both air-KD and air-KE ambulated significantly less on withdrawal 5 compared to air-CD (p = 0.04 and p = 0.0004 respectively).There were no other statistically significant differences in the number of light/dark crossing and in the voluntary time spent in the light area in the air-control mice at either timepoint (all p > 0.2).CIE-CD mice did not display long-lasting anxiety-like symptoms as measured in the zero maze (Fig. 4g-l).Data from the 2nd and 3rd cohorts were included in the analyses, while the 1 st cohort was excluded due to different light conditions.The two timepoints were analyzed separately, since it is known that mice can habituate.On withdrawal day 19, two-way ANOVA revealed no main effects or interactions for the time spent in the open arms or the number of SAPs, but there was a trend for an alcohol-diet interaction for the number of open entries (F 2,41 = 2.9, p = 0.07) (Fig. 4g,i,k).On withdrawal day 26, two-way ANOVA revealed a significant overall

Discussion
Long-lasting alcohol withdrawal symptoms are characterized by complex and subtle neuronal mechanisms and are key contributors to relapse.In the present study we assessed the efficacy of nutritional ketosis, in the form of KD and KE supplementation, in ameliorating long-lasting alcohol withdrawal symptoms.We recently reported that the C57BL/6JRj mouse strain is more suitable than the C3H/HeNRj strain to study long-term alcohol withdrawal, in female mice, and we established a battery of behavioral and biochemical tests, which we implemented in the present report 30 .Both KD and KE reduced the severity of several alcohol withdrawal symptoms and they also showed a trend towards improving neurochemical alterations caused by chronic alcohol exposure, followed by long-term abstinence and re-exposure.Indeed, both KD and KE ameliorated long-lasting depressive-like symptoms, whereas only KD partially improved early anxiety-like disturbances.Depression-and anhedonia-like disturbances are among the most challenging and long-lasting alcohol withdrawal symptoms [40][41][42] .We detected clear depression-and anhedonia-like symptoms following chronic intermittent alcohol exposure for almost the entire duration of abstinence, which is consistent with previous reports 30,[43][44][45] .
In the present study, we show for the first time that both KD and KE provided lasting and effective reduction of these symptoms in female mice.The effects are consistent with anti-depressive-like effects of nutritional ketosis in other rodents models used to induce depressive-like behaviors 46,47 .We hypothesize that these anti-depressants effects are driven by restoration of neurotransmitters homeostasis, specifically GABA and glutamate 18,48 .
CIE-CD mice displayed early anxiety-like symptoms in the light-dark test, but no long-lasting effects the same test or in the zero maze.We previously showed that KD rescued these acute anxiety-like disturbances, however here we only had a slight tendency for improvement 17 .This could be due to the decreased distance travelled by both CIE-CD and CIE-KE mice compared to air-CD mice on withdrawal day 1, which was further evident for the KE since air-KE mice travelled significantly less than air-CD on both withdrawal days.This could be a confounder, but, even though hypolocomotion has been often described during early alcohol withdrawal, the most consistent and pharmacologically validated parameter for the light/dark test was the number of light/dark transitions 49,50 .Long-lasting anxiety-like symptoms have proven challenging to measure in mice, with only one study describing them for up to three weeks following a 30-day (but not 14-day) binge drinking model in male C57BL/6J mice, in the elevated plus maze 44,51 .For the zero maze, lack of statistical power may have obscured mild effects in the present study since only two cohorts were tested.Both on withdrawal days 19 th and 26 th , CIE-CD showed marginally reduced time spent in the open arms compared to CIE-KD and air-CD.It cannot be excluded that different timepoints and larger sample size could identify prolonged anxiety-like disturbance and effects of KD.Anxiolytic effects of nutritional ketosis have been recently described by Ari and colleagues 52 , who showed reduced anxiety-like symptoms upon KE administration in rats.Moreover, another recent study reported a dosedependent anxiolytic effect of KE in female rats, with ketone levels in the range of 3 mM ameliorating anxiety-like symptoms in the light/dark test, while ketones within 4-5 mM, did not induce any change 53 .
A previous study reported the association between anticipatory 50 kHz USVs and the escalation of alcohol intake in dependent rats 38 .Following on our previous report, we found that a higher percentage of CIE-CD mice emitted USVs during OSA compared to air-CD, and they also emitted more USVs in total 30 .Interestingly, only one CIE-KD mouse and 2 CIE-KE mice emitted USVs.This might indicate that increased USVs emissions during alcohol withdrawal could reflect a general distress, which seems to be alleviated by nutritional ketosis.A recent study showed that a KD restored a neurodevelopmental communication deficit associated with decreased USVs in a rat model of intractable infantile spasm syndrome 54 .Very few mice emitted USVs during the tail suspension test, but this could be due to the short testing time.
CIE-CD mice did not escalate alcohol OSA and CIE-KD and CIE-KE mice did not display any difference in the present study.Similarly, CIE-CD mice did not show any signs of hyperalgesia.We recently reported that alcohol-exposed C57BL/6JRj mice did not escalate alcohol OSA using the same protocol, but they decreased their alcohol OSA more slowly compared to the control mice and they showed hyperalgesia 30 .This is in contrast to higher self-administered alcohol in vapor-exposed male rats relative to air-control, which was decreased by a history of KD 18 .As previously discussed, the long PR experimental sessions used could have masked the alcohol OSA escalation and produced high variability.It is possible that alcohol intake (more apparent using a fixedratio schedule of reinforcement), but not "motivation" or "seeking" is decreased by KD, consistent with a report showing that KD-treated mice responded for alcohol rewards but did not drink them 55 .It has been previously reported that male mice escalated their OSA following chronic alcohol exposure including using exposure by vapor inhalation and intragastric infusion 56,57 .Only one study has shown increased alcohol OSA in female mice, which however were not previously exposed to alcohol 58 .
Anti-inflammatory effects of KD have been previously described in a variety of preclinical rodent assays 22,27,46,59 .Conversely, alcohol has been shown to increase neuroinflammation 60,61 , and we therefore wanted to test whether ketosis ameliorated alcohol-induced neuroinflammation as part of its protective mechanism.We and others previously reported higher TNF-α central levels following alcohol exposure in mice 30,61 .In this study however, we did not detect a similar effect.However, a clinical trial found that switching from an American standard diet to an isocaloric KD increased glucose, total and low density lipoprotein cholesterol, and peripheral inflammation markers 62 .Thus, further investigations of different sample types and at multiple timepoints are needed to elucidate any potential anti-or pro-inflammatory effects of nutritional ketosis.
There is extensive evidence indicating alterations of dopaminergic, noradrenergic, and serotonergic signaling following alcohol exposure in both rodents and humans [63][64][65][66][67] .We did find lower NE levels in the hippocampus and in the striatum of CIE-CD mice compared to air-CD mice, consistent with a previous study in the frontal cortex and hippocampus of adult female rats 68 .The effects of alcohol on noradrenergic signaling are complex and dependent on timing, dose and route of administration but its dysregulation is a main factor of the pathophysiology of AUD 65,69 .We also detected marginally lower 5-HIAA/5-HT ratio in the hippocampus of CIE-CD mice compared to air-CD mice, which was moderately rescued by KE.The 5-HIAA/5-HT ratio is used as an estimate of serotoninergic activity and can provide useful information about the catabolic rate but has also important limitations.We found no other significant alterations in the concentrations of MOPEG, DOPAC, DA, 5-HT, 5-HIAA, and DOPAC/DA ratios in either the striatum or hippocampus of CIE-CD mice compared to air-CD mice.In exploratory studies, we previously described moderate cation imbalances in the cerebellum of alcohol-exposed mice 30 .Here, we found lower Mg ++ and Na + levels after CIE, similar to what has been reported in peripheral clinical samples 30,70,71 .
Both KD and KE yielded sustained elevated blood βHB levels, reaching nutritional ketosis, i.e., blood ketone levels above 0.5 mM.KE produced higher βHB levels than KD after the first week and throughout the testing period.This was however in contrast with our previous study where KD rather than KE reached blood ketone levels up to 5 mM 17 .Sex and vendor differences as well as timing of blood collection could explain these divergences.Nutritional ketosis, achieved by either KD or KE supplementation, can elicit several metabolic effects such as lower plasma glucose levels and lower bodyweight gains in both rats and humans 15,19,62,72 .However, nutritional ketosis induced variable total and LDL cholesterol levels in both rodents and humans during physiological conditions 19,62,[72][73][74] .The report by Clarke and colleagues 19 is the only one investigating both sexes in rats showing a slight cholesterol increase after KE administration (30% calories from a ketone monoester).Meidenbauer and colleagues 74 also showed increased cholesterol as well as triglycerides following KD ad libitum in male mice, but the amount of fat in the KD was 70%.Kemper and colleagues instead 73 , reported that KE supplementation (30% calories from the βHB ester) lowered plasma total cholesterol in male rats and mevalonate, a liver cholesterol synthesis biomarker, in humans.In the present study, we showed for the first time that not only KD but also KE reduced plasma glucose levels and KE also reduced weight gain at specific timepoints in female mice during alcohol exposure 73 and withdrawal.There were differences in glucose levels over time, specifically for the CIE-KD mice.This could be related to changing conditions over time, such as alcohol exposure (first and last timepoint).Contrary to some previous studies in rodents and humans during physiological conditions, we found lower total cholesterol levels following KD administration, independently of alcohol exposure, and no changes in triglycerides 19,62 .The KE supplementation did not affect cholesterol or triglycerides levels.It has been hypothesized that substituting carbohydrate calories with either KD or KE could reduce the availability of acetyl-CoA for lipogenesis 73 .Together, our findings are consistent with good tolerability and low risk of deleterious effects of KD, with possibly higher risk of weight loss for the KE.
We conducted three separate PCA for each diet pair to better visualize and summarize the multiple dimensions associated with alcohol exposure and nutritional ketosis.There was a clear separation only between the alcohol and air exposed mice fed CD, whereas the PCA for the KD and KE pairs revealed more similar behavioral and biochemical patterns, suggesting that both KD and KE reversed at least some the alterations induced by alcohol.However, the air-KE had a different pattern compared to air-CD and air-KD mice, which could indicate a potential for aversive side effects.
We are aware of some limitations of this study.First, only female mice were included.Second, the timing of start of the experimental diets might be important for the outcome, and effects may not generalize to shorter diet exposure before onset of withdrawal.We previously described reduced alcohol withdrawal induced by nutritional ketosis when the diets were administered from the beginning of alcohol exposure and also when started at abstinence.For the latter, only KE was effective, likely due to a faster onset of blood βHB elevation relative to KD 17 .In the present study, diets were introduced in the last week of alcohol exposure, to mimic a possible clinical design.Lastly, the biochemical endpoints were measured at a single timepoint, after the final alcohol re-exposure.The time needed to process all samples caused the first mice to be terminated 2 h after the last alcohol exposure, and the last, 4 h, which may have introduced variability in the results.
In conclusion, our findings show that the beneficial effects of nutritional ketosis in ameliorating acute alcohol withdrawal symptoms in mice are also evident for long-term symptoms.This could have a positive impact in preventing relapse in AUD individuals.In addition, some of the biochemical endpoints measured were also partially ameliorated by nutritional ketosis after re-exposure.Overall, compared to KE, KD seemed to better relieve alcohol withdrawal symptoms, despite producing lower βHB levels.This indicates that high ketone levels alone are not necessary or sufficient to produce better effects, rather it seems that providing fatty acids to fuel the entire metabolic pathway produces multifactorial beneficial effects.Thus, the underlying mechanisms are yet to be fully identified.Notably, the ketogenic manipulations alone did not have any effect on behavioral or biochemical measures other than reduced cholesterol levels and reducing the distance travelled at a specific timepoint, demonstrating their safety and tolerability 24,25,75 .Our results also extend previous findings from male to female rodents [16][17][18] .These results further support promising beneficial effects of nutritional ketosis for the treatment of alcohol withdrawal symptoms.Given the apparent differences between the KD and KE effects, further studies are needed to better understand the underlying mechanisms of action and refine ketosis-based treatments.
. The behavioral room was set to medium illumination (≈ 300 lx).The parameters were manually scored in real time and included the time spent on the open areas, the number of entries into the open areas and the number of stretched-attend postures (SAPs).

Figure 1 .
Figure 1.Timeline for alcohol exposure, abstinence, and behavioral assays.Alcohol exposure is highlighted in white and withdrawal periods in black.Behavioral tests are indicated by the colored bubbles, with the testing day specified inside, counted as days of forced abstinence within each specific withdrawal period.Blood for BAL measurement was collected at the end of each alcohol exposure cycle, i.e., at the end of each white arrow.Oral self-administration (OSA).

Figure 2 .
Figure 2.Both KD and KE induced nutritional ketosis and reduced blood glucose levels at specific timepoints, and KD lowered cholesterol levels independently of alcohol exposure.Blood ketone levels (a) and glucose levels (b) were measured on multiple days.Bodyweight in grams during the whole experiment (c).BALs for the alcohol-exposed mice (d).Plasma total cholesterol levels in CIE (e) and plasma triglycerides levels (f).Data are shown as group means, with the shaded area representing ± SEMs and the filled symbols representing a significant effect (a-d).Data are shown as individual subjects, horizontal line in violin plots represents group means (e-f).n = 10-12 (5-12 for panel d).*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.# and ##p < 0.05 and p < 0.01 (KD vs. KE).Blood alcohol levels (BALs).

Figure 3 .
Figure 3. Nutritional ketosis ameliorated depressive-like disturbances induced by alcohol exposure.The latencies in seconds to first immobility (a, b) and the % of time spent immobile (c, d) were assessed with the tail suspension test on withdrawal days 8 and 15.The % of saccharin preference (e) and the saccharin solution intake (f) were assessed on multiple withdrawal days.Latencies are shown as the % of mice showing a response as a function of time (a, b) and the filled symbols represent a significant effect.Data are shown as individual subjects, horizontal line in violin plots represents group means (c, d).Data are shown as group means, with the shaded area representing ± SEMs and the filled symbols representing a significant effect (e, f).Note the truncated ordinate in panel e. n = 8-12.*p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 4 . 5 Figure 5 .Figure 6 .
Figure 4. KD had modest effects on anxiogenic-like behaviors during alcohol withdrawal.The number of light/ dark crossing (a, b), the % of time spent voluntarily in the lit area (c, d), and the distance travelled (e, f) were assessed with the light/dark test on withdrawal days 1 and 5.The % of time spent in the open arms (g, h), the number of entries into open arms (i, j), and the number of stretched-attend postures (SAPs) (k, l) were assessed with the elevated zero maze on withdrawal days 19 and 26.Data are shown as individual subjects, horizontal line in violin plots represents group means.Note the truncated ordinate in panel g.n = 8-12.*p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 7 .
Figure 7. PCA reveals differential clustering between each diet pair.Principal component scores for the CD (a), KD (b), and the KE (c) pairs.Data are shown as individual principal component scores for each subject.n = 8-11.Principal component (PC).

Table 1 .
Summary of behavioral and biochemical effects KD and KE in alcohol-exposed mice.Only assays where there was a significant effect of alcohol are listed.+ denotes a significant improvement compared to CIE-CD mice; (+) denotes a trend or mixed results; Ø indicates no effect.