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Antioxidants 13 (10): 1164 (2024)
University of British Columbia. Life Sciences Institute
Gladen-Kolarsky, Noah
Monestime, Olivia
Bollen, Melissa
Choi, Jaewoo
Yang, Liping
Magaña, Armando Alcazar
Maier, Claudia S.
Soumyanath, Amala
Gray, Nora E.
Withania somnifera (WS), also known as ashwagandha, is a popular botanical supplement used to treat various conditions including memory loss, anxiety and depression. Previous studies from our group showed an aqueous extract of WS root (WSAq) enhances cognition and alleviates markers for depression in Drosophila. Here, we sought to confirm these effects in the 5xFAD mouse model of β-amyloid (Aβ) accumulation. Six- to seven-month-old male and female 5xFAD mice were treated with WSAq in their drinking water at 0 mg/mL, 0.5 mg/mL or 2.5 mg/mL for four weeks. In the fourth week of treatment, spatial memory, anxiety and depressive-like symptoms were evaluated. At the conclusion of behavioral testing, brain tissue was harvested, immunohistochemistry was performed, and the cortical expression of antioxidant response genes was evaluated. Both concentrations of WSAq improved spatial memory and reduced depressive and anxiety-related behavior. These improvements were accompanied by a reduction in Aβ plaque burden in the hippocampus and cortex and an attenuation of activation of microglia and astrocytes. Antioxidant response genes were upregulated in the cortex of WSAq-treated mice. Oral WSAq treatment could be beneficial as a therapeutic option in AD for improving disease pathology and behavioral symptoms. Future studies focused on dose optimization of WSAq administration and further assessment of the mechanisms by which WSAq elicits its beneficial effects will help inform the clinical potential of this promising botanical therapy.
Citation: Gladen-Kolarsky, N.;Monestime, O.; Bollen, M.; Choi, J.;Yang, L.; Magaña, A.A.; Maier, C.S.;Soumyanath, A.; Gray, N.E. Withaniasomnifera (Ashwagandha) ImprovesSpatial Memory, Anxiety andDepressive-like Behavior in the5xFAD Mouse Model of Alzheimer’sDisease. Antioxidants 2024, 13, 1164.https://doi.org/10.3390/antiox13101164Academic Editor: Soliman KhatibReceived: 14 August 2024Revised: 14 September 2024Accepted: 20 September 2024Published: 25 September 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).antioxidantsArticleWithania somnifera (Ashwagandha) Improves Spatial Memory,Anxiety and Depressive-like Behavior in the 5xFAD MouseModel of Alzheimer’s DiseaseNoah Gladen-Kolarsky 1, Olivia Monestime 1 , Melissa Bollen 1,2, Jaewoo Choi 2,3, Liping Yang 2,4, ArmandoAlcazar Magaña 2,4,5 , Claudia S. Maier 2,3,4, Amala Soumyanath 1,2 and Nora E. Gray 1,2,*1 Department of Neurology, Oregon Health and Science University, Portland, OR 97239, USA2 BENFRA Botanical Dietary Supplements Research Center, Portland, OR 97239, USA;armando.alcazarmagana@ubc.ca (A.A.M.)3 Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA4 Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA5 Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z4, Canada* Correspondence: grayn@ohsu.eduAbstract: Withania somnifera (WS), also known as ashwagandha, is a popular botanical supplementused to treat various conditions including memory loss, anxiety and depression. Previous studiesfrom our group showed an aqueous extract of WS root (WSAq) enhances cognition and alleviatesmarkers for depression in Drosophila. Here, we sought to confirm these effects in the 5xFAD mousemodel of β-amyloid (Aβ) accumulation. Six- to seven-month-old male and female 5xFAD micewere treated with WSAq in their drinking water at 0 mg/mL, 0.5 mg/mL or 2.5 mg/mL for fourweeks. In the fourth week of treatment, spatial memory, anxiety and depressive-like symptoms wereevaluated. At the conclusion of behavioral testing, brain tissue was harvested, immunohistochemistrywas performed, and the cortical expression of antioxidant response genes was evaluated. Bothconcentrations of WSAq improved spatial memory and reduced depressive and anxiety-relatedbehavior. These improvements were accompanied by a reduction in Aβ plaque burden in thehippocampus and cortex and an attenuation of activation of microglia and astrocytes. Antioxidantresponse genes were upregulated in the cortex of WSAq-treated mice. Oral WSAq treatment could bebeneficial as a therapeutic option in AD for improving disease pathology and behavioral symptoms.Future studies focused on dose optimization of WSAq administration and further assessment of themechanisms by which WSAq elicits its beneficial effects will help inform the clinical potential of thispromising botanical therapy.Keywords: Alzheimer’s disease; memory; depression; anxiety; ashwagandha; neuroinflammation;oxidative stress; beta-amyloid; 5xFAD mice1. IntroductionAlzheimer’s disease (AD) is the most common cause of dementia, accounting for60–80% of all cases, and is the seventh leading cause of death worldwide [1]. An estimated6.7 million Americans aged 65 and older are living with AD today, which could grow to asmuch as 13.8 million by 2060, barring the development of novel methods to prevent, cure orslow the disease [1]. In addition to the hallmark cognitive impairment associated with AD,studies have shown that the frequency of neuropsychiatric symptoms is also much higherin AD than in the general population, of which the most frequently observed symptomsare increased generalized apathy, anxiety and depression [2].Pathologically, AD is characterized by the accumulation of β amyloid (Aβ) plaquesand neurofibrillary tangles made up of hyperphosphorylated tau, which together con-tribute to synaptic dysfunction and eventual neuronal death [3]. Increased oxidative stressAntioxidants 2024, 13, 1164. https://doi.org/10.3390/antiox13101164 https://www.mdpi.com/journal/antioxidantsAntioxidants 2024, 13, 1164 2 of 15and chronic neuroinflammation are two additional consequences of plaque and tangleaccumulation that are widespread throughout the AD brain [3,4].Chronic neuroinflammation is central to AD progression and is believed to contributeto cognitive impairment [5]. Astrocytes and microglia work in tandem to mediate theinflammatory response to neuronal injury [6]. In early AD, these cells play a neuroprotectiverole, promoting cell repair, but as the disease progresses, they become chronically activatedand produce pro-inflammatory cytokines, resulting in further neuroinflammation andneurotoxicity induced by inflammatory mediators [7–9]. Neuroinflammation is associatedwith reduced synaptic density and impaired cognitive function [10,11] and may also bea link between AD and depression [12,13] as studies have shown that treatments forneuroinflammation can improve endpoints related to depression [12,13].Increased oxidative stress, caused by excess reactive oxygen species (ROS), is an-other early event in AD pathogenesis [14]. Rodent studies have demonstrated a directrelationship between oxidative stress and synaptic dysfunction in AD [15]. Antioxidantcompounds can improve synaptic deficits in both in vitro and in vivo models of AD [16,17].These alterations in synaptic function are thought to be the physiological underpinningof the improved cognitive function observed following antioxidant treatment in mousemodels [18–20]. Although the mechanism is less well defined, increased oxidative stress isalso linked with increased depression in patient populations [21].Significant crosstalk also exists between the pathways mediating neuroinflammation andoxidative damage. Elevated ROS can trigger astrocytes and microglia to release pro-inflammatorycytokines in a coordinated response. These pro-inflammatory cytokines lead to further ROSproduction, as synaptic function and neurotransmission continue to deteriorate [9,22,23]. Becauseof this interconnectedness, there is growing interest in identifying therapeutic interventions thatcan target both inflammatory and antioxidant pathways.Ashwagandha, or Withania somnifera (L.) Dunal (WS), is a traditional Ayurvedic herbwidely used for treating an array of conditions, including memory loss, stress, anxiety,depression, and insomnia [24]. Extracts of WS were shown to improve cognitive functionin various rodent models of neurodegeneration [25–27]. Antidepressive and anxiolyticproperties were also reported in both rodent and clinical studies [24,28–30]. Our group hasshown that an aqueous extract of WS (WSAq) can improve similar endpoints in Drosophilamodels as well [31,32]. We found that WSAq attenuated stress-induced depressive-likesymptoms and improved performance in a phototaxis test in both healthy aged flies, aswell as a fly model of increased oxidative stress [31,32].Here, we evaluate the effect of WSAq in the 5xFAD mouse model of Aβ accumula-tion. The 5xFAD mouse model of AD overexpresses mutant human amyloid precursorprotein (APP) containing the Swedish (K670N, M67IL), Florida (I716V), and London (V717I)Familial Alzheimer’s Disease (FAD) mutations, as well as the human presenilin 1 (PS1)gene with two FAD mutations, M146L and L286V. These mice model major pathologicalhallmarks resulting from Aβ accumulation as young as 2 months of age and develop robustplaque pathology along with severe cognitive impairment by 4–6 months of age [33,34]. Inaddition to assessing the behavioral effects on cognition, anxiety and depression elicited byWSAq, we also investigated the extract’s impact on Aβ plaque pathology and markers ofoxidative stress and neuroinflammation.2. Materials and Methods2.1. WSAq Preparation and Chemical AnalysisWS root (Batch number 201000162) harvested in 2019 at Oregon’s Wild Harvest (Red-mond, OR, USA) was obtained in powdered form and authenticated by genetic testingas previously described [35]. Voucher samples of the root powder are deposited at theOregon State University Herbarium (voucher number OSC-V-265405) and in our labora-tory (code number BEN-WS-8). Dried aqueous extract (WSAq) of the root was preparedas previously described [32]. Briefly, powdered root (160 g) was refluxed with boilingwater (2 L) for 90 min. The extract was filtered while still warm and the filtrate cooled,Antioxidants 2024, 13, 1164 3 of 15frozen and lyophilized to yield a dry powder. Several batches of WSAq were made usinga standardized protocol from the same starting root material (BEN-WS-8), and vouchersamples are stored in our laboratory (code numbers BEN-WSAq-16, BEN-WSAq-18 andBEN-WSAq-19). BEN-WSAq-18 and BEN-WSAq-19 were used in feeding experiments. Tar-geted analysis of withanolides using liquid chromatography coupled to multiple reactionmonitoring mass spectrometry (LC-MRM-MS) and chemical fingerprinting by untargetedanalysis using liquid-chromatography coupled to high-resolution tandem mass spectrome-try (LC-HRMS/MS) were performed on BEN-WSAq-16 using our published methods [35].Marker withanolides determined by LC-MRM-MS (µg/g extract; reported are average± S.D values derived from three technical replicates at 0.025 mg/mL plus three technicalreplicates at 0.25 mg/mL; n = 6 technical replicates): withanolide A (1157.70 ± 119. 88),withanone (832.52 ± 29.02), withaferin A (166.99 ± 14.14), withanoside IV (99.03 ± 6.02),12-deoxywithastramonolide (50.91 ± 5.84), withanoside V (29.94 ± 2.28) and withanolide B(1.49 ± 0.12).2.2. AnimalsExperiments were carried out in line with the NIH Guidelines for the care and useof laboratory animals and were given approval by the Institutional Animal Care and UseCommittee of the Veteran’s Administration Portland Health Care System (VAPORHCS;IACUC #4688-21). The 5xFAD and B6SJLF mice were purchased from Jackson Laboratory,Bar Harbor, ME, USA, and kept in a climate-controlled facility with a 12 h light/dark cycle.The 5xFAD transgenic male mice were paired with B6SJLF1 females to maintain the colony.Wild-type (WT) littermates were used as controls for each experiment.At 6 months, male and female 5xFAD mice began treatment with either 0 mg/mL,0.5 mg/mL or 2.5 mg/mL lyophilized WSAq extract administered via drinking waterad libitum for 4 weeks. Mice were not individually housed and, therefore, individualconsumption could not be measured based on average consumption per cage adjusted forthe number of mice in each cage and the average weight of all mice the doses correspondto approximately 75 mg/kg/day and 375 mg/kg/day. Age-matched WT littermates weretreated with water containing 0 mg/mL for the same duration. Water was changed twiceweekly to maintain water and additive quality. In the final week of treatment, miceunderwent behavioral testing which included open field, then object location memoryand lastly the forced swim test (Figure 1). Behavioral testing occurred only at the end oftreatment (and not prior to and following treatment) in order to avoid the confoundingeffects of learning on performance in the task.Antioxidants 2024, 13, x FOR PEER REVIEW 3 of 16 previously described[35]. Voucher samples of the root powder are deposited at the Ore-gon State University Herbarium (voucher number OSC-V-265405) and in our laboratory (code number BEN-WS-8). Dried aqueous extract (WSAq) of the root was prepared as previously described [32]. Briefly, powdered root (160 g) was refluxed with boiling water (2 L) for 90 min. The extract was filtered while still warm and the filtrate cooled, frozen and lyophilized to yield a dry powder. Several batches of WSAq were made using a stand-ardized protocol from the same starting root material (BEN-WS-8), and voucher samples are stored in our laboratory (code numbers BEN-WSAq-16, BEN-WSAq-18 and BEN-WSAq-19). BEN-WSAq-18 and BEN-WSAq-19 were used in feeding experiments. Tar-geted analysis of withanolides using liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM-MS) and chemical fingerprinting by untargeted analysis using liquid-chromatography coupled to high-resolution tandem mass spectrom-etry (LC-HRMS/MS) were performed on BEN-WSAq-16 using our published meth-ods[35]. Marker withanolides determined by LC-MRM-MS (µg/g extract; reported are av-erage ± S.D values derived from three technical replicates at 0.025 g/mL plus three tech-nical replicates a 0.25 mg/mL; n = 6 technical replicates): withanolide A (1157.70 ± 119. 88), withanone (832.52 ± 29.02), withaferin A (166.99 ± 14.14), withanoside IV (99.03 ± 6.02), 12-deoxywithastramonolide (50.91 ± 5.84), withanoside V (29.94 ± 2.28) and withanolide B (1.49 ± 0.12). 2.2. Anim ls Experiments were carried out in line with the NIH Guidelines for the care and use of laboratory animals and were given approval by the Institutional Animal Care and Use Committee of the Veteran’s Administration Portland Health Care System (VAPORHCS; IACUC #4688-21). The 5xFAD and B6SJLF mice were purchased from Jackson Laboratory, Bar Harbor, ME, USA, and kept in a climate-controlled facility with a 12 h light/dark cycle. The 5xFAD transgenic male mice were paired with B6SJLF1 females to maintain the col-ony. Wild-type (WT) littermates were used as controls f r e h experiment. At 6 months, male and female 5xFAD mice began treatment with eith r 0 mg/mL, 0.5 mg/mL or 2.5 mg/mL lyophilized WSAq extract administered via drinking water ad libi-tum for 4 weeks. Mice were not individually housed and, therefore, individual consump-tion could not be measured base on average consumption per cage adjusted for the num-ber of mice in each cage and the average weight of ll mice the doses correspond to ap-proxi ately 75 mg/kg/day and 375 mg/kg/day. Age-matched WT littermates were treated with water containing 0 mg/mL for the same duration. Water was changed twice weekly to maintain water and additive quality. In the final week of treatment, mice underwent behavioral tes ing which included open field, then object l cation memory and lastly the forced swim test (Figure 1). Be avioral testi g occurred only at the end f treatment (and not prior to and following treatment) in order to avoid the confounding effects of learning on performance in the task. Figure 1. Treatment timeline for experiments. At the conclusion of behavioral testing, the mice were euthanized according to VAPORHCS guidelines and tissue was harvested. Experimental groups ranged from 13 to Figure 1. Treatment timeline for experiments.At the conclusion of behavioral testing, the mice were euthanized according to VA-PORHCS guideli es and tissue was harvested. Experimental groups ranged from 13 to15 animals per condition with slight variations between groups owing to litters that werenot evenly divided between 5xFAD and WT (Table 1). All treatment groups included bothmale and female mice (Supplementary Table S1).Antioxidants 2024, 13, 1164 4 of 15Table 1. Number of animals per experimental group.0 mg/mL 0.5 mg/mL 2.5 mg/mLWT 14 -- --5xFAD 15 13 132.3. Object Location Memory Test (OLM)The OLM test for spatial working memory was carried out in a square apparatus(39 cm × 39 cm × 39 cm). In the preliminary portion of the test, mice were habituatedto the apparatus for 5 min per day for two days without any objects present in the field.On day 3, mice were introduced to the arena with two identical objects in fixed locationsand were allowed to explore for three 10 min “training” intervals. Two hours and 24 hafter the final training session, testing began, wherein mice were placed in the apparatus,but one of the two objects was moved from its original location to a new location in thefield. The novel location was changed between the 2 h and the 24 h tests. Mice were thenallowed to explore both objects for 5 min during each interval. The time spent exploringthe novel location relative to the total time exploring both locations was scored manuallyby a blinded investigator using AnyMaze software version 6.13 and expressed as a percent.The objects used were of similar height and width. No preference was recorded in thisstudy for any object. Increased time with the object in the novel location reflects improvedspatial memory.2.4. Open Field Test (OF)The OF test is used to assess anxiety in mice. Animals are placed in a square arena(39 cm × 39 cm × 39 cm) and allowed to explore for 5 min. Time spent in different locationsin the arena was quantified automatically using AnyMaze software. Increased time in thecenter of the arena indicates lower anxiety, while increased time spent in the periphery ofthe arena indicates higher anxiety [36].2.5. Forced Swim Test (FST)The FST evaluates depressive-like behavior. Mice are placed into a cylindrical container(45 cm in height, 20 cm in diameter) containing lukewarm water for 6 min and their timeimmobile is automatically scored using a camera and AnyMaze software. Greater timeimmobile is indicative of increased depressive-like behavior [37].2.6. ImmunohistochemistryRight brain hemispheres were incubated in 4% paraformaldehyde for 24 h at room temper-ature and then transferred to phosphate-buffered saline (PBS), 15% and 30% sucrose solutionsfor 24 h each before being stored at −80 ◦C for sectioning. Coronal sections (40 µm) wereobtained by slicing right hemisphere samples held at−20 ◦C in Optimal Cutting Temperature(O.C.T.) Compound (Sakura Finetek, Torrance, CA, USA) on a freezing microtome.Sections were then stored in a sectioning solution (15% glycerol, 10% Tris-HCl bufferedsaline (TBS), diluted in diH2O) before further processing. During immunostaining, sectionswere placed in a quenching solution (30% methanol, 10% hydrogen peroxide, 10% TBS)for endogenous catalase activity and then blocked (2% bovine serum albumin, 10% horseserum, 2% triton x, 10% 10× TBS, diluted in diH2O). Sections were then incubated with oneof the three following primary antibodies diluted 1:1000 in PBS: anti-Aβ polyclonal antibody(Thermo Scientific, Waltham, MA, USA); GFAP (Glial fibrillary acidic protein; Invitrogen,Waltham, MA, USA); IBA1 (Ionized calcium-binding adaptor molecule 1; Proteintech,Rosemont, IL, USA); NRF2 (Abcam, Waltham, MA, USA) and visualized using biotinylatedsecondary antibodies.ImageJ software (Rasband, W.S., ImageJ, version 1.54k) was used to perform thequantification of antibody staining. Images were converted to greyscale, and a tracing toolwas used to outline the area of the cortex or hippocampus and the area outlined was noted.Antioxidants 2024, 13, 1164 5 of 15Contrast thresholding was adjusted according to background staining to highlight onlyintense staining. Staining was quantified for three coronal sections at different sectioningdepths from each right hemisphere sample. Finally, the extent of staining was expressedas a percentage of area stained against the total area of the region in question, and meanvalues for each sample were calculated from the three sections analyzed.2.7. Gene Expression AnalysisLeft brain hemispheres were sub-dissected by brain region and frozen at −80 ◦C. RNAwas extracted from one half of the cortex from the left hemisphere of each mouse brain usingTRI reagent solution per the manufacturer’s protocol (Invitrogen). Reverse transcriptionwas performed on the RNA product using a SuperScript™ III RT cDNA synthesis kit(Invitrogen) also per the manufacturer’s protocol. Quantitative PCR (qPCR) was performedusing a QuantStudio™ 12K Flex Real-time PCR System (Applied Biosystems, Rosemont, IL,USA) using the following Taqman primers from Thermo: NRF2 (nuclear factor erythroid-derived 2-like 2, also called NFE2L2); HMOX1 (heme oxygenase 1); NQO1 (NAD(P)Hquinone dehydrogenase 1); GCLC (Glutamate-Cysteine Ligase Catalytic Subunit); SYP(synaptophysin); PSD95 (post synaptic density protein 95, also called DLG4); GAPDH(glyceraldehyde-3-phosphate dehydrogenase). Relative expression was quantified usingthe delta delta CT method normalized to GAPDH expression.2.8. Statistical AnalysisAll bar graphs show error bars that reflect standard error of the mean. Statisticalsignificance for graphed data was calculated using ANOVAs with Sidak pairwise posthoc testing. No interactions were found between sex and any outcomes measured and soresults are presented with male and female mice together. All analyses were performedusing GraphPad Prism version 10 software (GraphPad Software, Inc., La Jolla, CA, USA).3. Results3.1. WSAq Improves Spatial Memory in 5xFAD MiceIn the OLM test, spatial memory retention was tested at 2 h (Figure 2A) and 24 h(Figure 2B) after the final training session. The 5xFAD mice receiving no WSAq spentsignificantly less time exploring the object in the novel location relative to their WT coun-terparts at both testing time points (Figure 2A,B). At the 2 h test, performance in the 5xFADmice was significantly improved by 2.5 mg/mL WSAq treatment. There was a similartrend in the 0.5 mg/mL treated group, but it did not reach significance (Figure 2A). In the24 h test, treatment with both concentrations of WSAq attenuated the deficit seen in thevehicle-treated 5xFAD mice (Figure 2B). At both time points, neither WSAq-treated groupwas significantly different from the WT mice.Antioxidants 2024, 13, x FOR PEER REVIEW 6 of 16 Figure 2. WSAq improves OLM performance in 5xFAD mice. WSAq treatment attenuated deficits in OLM performance at both 2 hours (A) and 24 hours (B). n = 9–12 per treatment group * p < 0.05. 3.2. Anxiety-Related and Depressive-like Behavior Is Reduced by WSAq in 5xFAD Mice The 5xFAD mice showed increased anxiety compared to the WT mice, as measured by the reduced time in the center of the OF (Figure 3). WSAq treatment attenuated this decrease in time in the center to levels comparable to the WT mice. A significant increase in time in the center was observed in the 0.5 mg/mL group compared to the vehicle-treated 5xFAD mice and a similar but non-significant trend was seen with the 2.5 mg/mL-treated mice (Figure 3). Figure 3. WSAq improves performance on the OF in 5xFAD mice. WSAq improved performance on the OF at the 0.5 g/L dose and approached significant improvement at the 2.5 g/L dose. * p < 0.05, ** p < 0.01. n = 8–12 per treatment group. During the FST, the 5xFAD control group spent significantly more time immobile than the WT group (Figure 4), indicating greater depressive-like behavior. Both concen-trations of WSAq reduced time immobile to a similar extent in the 5xFAD mice (Figure 4). Figure 2. Cont.Antioxidants 2024, 13, 1164 6 of 15Antioxidants 2024, 13, x FOR PEER REVIEW 6 of 16 Figure 2. WSAq improves OLM performance in 5xFAD mice. WSAq treatment attenuated deficits in OLM performance at both 2 hours (A) and 24 hours (B). n = 9–12 per treatment group * p < 0.05. 3.2. Anxiety-Related and Depressive-like Behavior Is Reduced by WSAq in 5xFAD Mice The 5xFAD mice showed increased anxiety compared to the WT mice, as measured by the reduced time in the center of the OF (Figure 3). WSAq treatment attenuated this decrease in time in the center to levels comparable to the WT mice. A significant increase in time in the center was observed in the 0.5 mg/mL group compared to the vehicle-treated 5xFAD mice and a similar but non-significant trend was seen with the 2.5 mg/mL-treated mice (Figure 3). Figure 3. WSAq improves performance on the OF in 5xFAD mice. WSAq improved performance on the OF at the 0.5 g/L dose and approached significant improvement at the 2.5 g/L dose. * p < 0.05, ** p < 0.01. n = 8–12 per treatment group. During the FST, the 5xFAD control group spent significantly more time immobile than the WT group (Figure 4), indicating greater depressive-like behavior. Both concen-trations of WSAq reduced time immobile to a similar extent in the 5xFAD mice (Figure 4). Figure 2. WSAq improves i ice. SAq treatment attenuated deficits inOLM performance at both 2 ours (A) and 24 hours (B). n = 9–12 per treatment group * p < 0.05.3.2. Anxiety-Related and Depressive-like Behavior Is Reduced by WSAq in 5xFAD MiceThe 5xFAD mice showed increased anxiety compared to the WT mice, as measuredby the reduced time in the center of the OF (Figure 3). WSAq treatment attenuated thisdecrease in time in the center to levels comparable to the WT mice. A significant increase intime in the center was observed in the 0.5 mg/mL group compared to the vehicle-treated5xFAD mice and a similar but non-significant trend was seen with the 2.5 mg/mL-treatedmice (Figure 3).Antioxidants 2024, 13, x FOR PEER REVIEW 6 of 16 Figure 2. WSAq improves OLM performance in 5xFAD mice. WSAq treatment attenuated deficits in OLM performance at both 2 hours (A) and 24 hours (B). n = 9–12 per treatment group * p < 0.05. 3.2. Anxiety-Related and Depressive-like Behavior Is Reduced by WSAq in 5xFAD Mice The 5xFAD mice showed increased anxiety compared to th WT mice, as measured by the reduced time in the center of the OF (Figure 3). WSAq treatment attenuated this decrease in time in the center to levels comparable to the WT mice. A significant increase i time in the c nt r was observed in th 0.5 mg/mL group compared to the vehicle-treated 5xFAD mice and a similar but non-significant trend was seen with the 2.5 mg/mL-treated ice (Figure 3). Figure 3. WSAq improves performance on the OF in 5xFAD mice. WSAq improved performance on the OF at the 0.5 g/L dose and approached significant improvement at the 2.5 g/L dose. * p < 0.05, ** p < 0.01. n = 8–12 per treatment group. During the FST, the 5xFAD control group spent significantly more time immobile than the WT group (Figure 4), indicating greater depressive-like behavior. Both concen-trations of WSAq reduced time immobile to a similar extent in the 5xFAD mice (Figure 4). Figure 3. WSAq improves performance on the OF in 5xFAD ice. WSAq i proved performance on theOF at the 0.5 g/L dose and approached significant improvement at the 2.5 g/L dose. * p < 0.05, ** p < 0.01.n = 8–12 per treatment group.Durin the ST, the 5xFAD control group spent significantly mor time immobile thanthe WT group (Figure 4), indicating greater depressive-like behavior. Both concentrationsof WSAq reduced time immobile to a similar extent in the 5xFAD mice (Figure 4).Antioxidants 2024, 13, x FOR PEER REVIEW 7 of 16 Figure 4. WSAq improves performance on the FST in 5xFAD mice. WSAq improved performance on the FST at both the 0.5 g/L and 2.5 g/L doses. WSAq treated groups were not significantly differ-ent from the WT group. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 9–12 per treatment group. 3.3. Aβ Plaque Burden Is Reduced in WSAq-Treated 5xFAD Mice WSAq reduced the Aβ plaque burden in the 5xFAD mice (Figure 5A). In both the cortex (Figure 5B) and hippocampus (Figure 5C), 2.5 mg/mL WSAq reduced the plaque burden significantly compared to the 5xFAD mice that received 0 mg/mL WSAq. A simi-lar, non-significant trend was also observed in the cortex of the WSAq 0.5 mg/mL-treated mice. Figure 5. Aβ accumulation is attenuated following WSAq treatment. (A) Representative images from each 5xFAD treatment condition. In both the (B) cortex and (C) hippocampus a significant reduction of pan-Aβ staining at the 2.5 g/L dose was observed, * p < 0.05, ** p < 0.01. n = 13–15 per treatment group. 3.4. WSAq Reduces Astrocytic and Microglial Activation in 5xFAD Mice Astrocytic activation was quantified via GFAP expression. The 5xFAD vehicle-treated mice had significantly higher GFAP expression than their WT littermates (Figure 6A). The 5xFAD 0 mg/mL 5xFAD 0.5 mg/mL 5xFAD 2.5 mg/mL Figure 4. WSAq improves performance on the FST in 5xFAD mice. WSAq improved performance on theFST at both the 0.5 g/L and 2.5 g/L doses. WSAq tr at d groups were not significantly different from theWT group. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 9–12 per treatment group.Antioxidants 2024, 13, 1164 7 of 153.3. Aβ Plaque Burden Is Reduced in WSAq-Treated 5xFAD MiceWSAq reduced the Aβ plaque burden in the 5xFAD mice (Figure 5A). In both thecortex (Figure 5B) and hippocampus (Figure 5C), 2.5 mg/mL WSAq reduced the plaqueburden significantly compared to the 5xFAD mice that received 0 mg/mL WSAq. A similar,non-significant trend was also observed in the cortex of the WSAq 0.5 mg/mL-treated mice.Antioxidants 2024, 13, x FOR PEER REVIEW 7 of 16 Figure 4. WSAq improves performance on the FST in 5xFAD mice. WSAq improved performance on the FST at both the 0.5 g/L and 2.5 g/L doses. WSAq treated groups were not significantly differ-ent from the WT group. * p < 0.05, ** p < 0.01, *** p < 0.001. n = 9–12 per treatment group. 3.3. Aβ Plaque Burden Is Reduced in WSAq-Treated 5xFAD Mice WSAq reduced the Aβ plaque burden in the 5xFAD mice (Figure 5A). In both the cortex (Figure 5B) and hippocampus (Fig e 5C), 2.5 mg/mL WSAq reduced the plaque burden significantly compared to the 5xFAD mice that received 0 mg/mL WSAq. A simi-lar, non-significant trend was also bserved in the cortex of the WSAq 0.5 g/mL-treated mice. Figure 5. Aβ accumulation is attenuated following WSAq treatment. (A) Representative images from each 5xFAD treatment condition. In both the (B) cortex and (C) hippocampus a significant reduction of pan-Aβ staining at the 2.5 g/L dose was observed, * p < 0.05, ** p < 0.01. n = 13–15 per treatment group. 3.4. WSAq Reduces Astrocytic and Microglial Activation in 5xFAD Mice Astrocytic activation was quantified via GFAP expression. The 5xFAD vehicle-treated mice had significantly higher GFAP expression than their WT littermates (Figure 6A). The 5xFAD 0 mg/mL 5xFAD 0.5 mg/mL 5xFAD 2.5 mg/mL Figure 5. Aβ accumulation is attenuated following WSAq treatment. (A) Representative images fromeach 5xFAD treatment condition. In both the (B) cortex and (C) hippocampus a significant reduction ofpan-Aβ staining at the 2.5 g/L dose was observed, * p < 0.05, ** p < 0.01. n = 13–15 per treatment group.3.4. WSAq Reduces Astrocytic and Microglial Activation in 5xFAD MiceAstrocytic activation was quantified via GFAP expression. The 5xFAD vehicle-treatedmice had significantly higher GFAP expression than their WT littermates (Figure 6A).The 5xFAD mice treated with a 0.5 mg/mL dose of WSAq had a significant reductionin activated astrocytes in both the cortex (Figure 6B) and hippocampus (Figure 6C) ascompared to the vehicle-treated 5xFAD mice. Interestingly, the same reduction was notseen in the 5xFAD mice treated with 2.5 mg/mL WSAq (Figure 6B,C).Antioxidants 2024, 13, x FOR PEER REVIEW 8 of 16 5xFAD mice treated with a 0.5 mg/mL dose of WSAq had a significant reduction in acti-vated astrocytes in both the cortex (Figure 6B) and hippocampus (Figure 6C) as compared to the vehicle-treated 5xFAD mice. Interestingly, the same reduction was not seen in the 5xFAD mice treated with 2.5 mg/mL WSAq (Figure 6B,C). Figure 6. Astrocytic activation is reduced following WSAq treatment. Representative images of GFAP staining given by (A). A significant reduction in GFAP staining was observed at the 0.5 g/L dose but was not observed in the 2.5 g/L group in both the cortex (B) and hippocampus (C). **** p < 0.0001. n = 7–15 per treatment group. The 5xFAD control mice also had significantly higher levels of microglial activation, as seen in IBA1 expression, than their WT counterparts (Figure 6A). The 2.5 mg/mL dose of WS reduced IBA1 staining significantly in the hippocampus of the 5xFAD mice (Figure 6C), and a similar trend was observed in the cortex (Figure 7B). Figure 7. WSAq reduces microglial activation in 5xFAD mice. Representative images of Iba1 staining given by (A). Treatment with 2.5 g/L WSAq reduced activated microglia in the hippocampus (C) and approached significant reduction of IBA1 in the cortex (B). Activation following WSAq 0.5 g/L Figure 6. Astrocytic activation is reduced following WSAq treatment. Representative images of GFAPstaining given by (A). A significant reduction in GFAP staining was observed at the 0.5 g/L dose butwas not observed in the 2.5 g/L group in both the cortex (B) and hippocampus (C). **** p < 0.0001.n = 7–15 per treatment group.Antioxidants 2024, 13, 1164 8 of 15The 5xFAD control mice also had significantly higher levels of microglial activation, asseen in IBA1 expression, than their WT counterparts (Figure 6A). The 2.5 mg/mL dose ofWS reduced IBA1 staining significantly in the hippocampus of the 5xFAD mice (Figure 6C),and a similar trend was observed in the cortex (Figure 7B).Antioxidants 2024, 13, x FOR PEER REVIEW 8 of 16 5xFAD mice treated with a 0.5 mg/mL dose of WSAq had a significant reduction in acti-vated astrocytes in both the cortex (Figure 6B) and hippocampus (Figure 6C) as compared to the vehicle-treated 5xFAD mice. Interestingly, the same reduction was not seen in the 5xFAD mice treated with 2.5 mg/mL WSAq (Figure 6B,C). Figure 6. Astrocytic activation is reduced following WSAq treatment. Representative images of GFAP staining given by (A). A significant reduction in GFAP staining was observed at the 0.5 g/L dose but was not observed in the 2.5 g/L group in both the cortex (B) and hippocampus (C). **** p < 0.0001. n = 7–15 per treatment group. The 5xFAD control mice also had significantly higher levels of microglial activation, as seen in IBA1 expression, than their WT counterparts (Figure 6A). The 2.5 mg/mL dose of WS reduced IBA1 staining significantly in the hippocampus of the 5xFAD mice (Figure 6C), and a similar trend was observed in the cortex (Figure 7B). Figure 7. WSAq reduces microglial activation in 5xFAD mice. Representative images of Iba1 staining given by (A). Treatment with 2.5 g/L WSAq reduced activated microglia in the hippocampus (C) and approached significant reduction of IBA1 in the cortex (B). Activation following WSAq 0.5 g/L Figure 7. WSAq reduces microglial activation in 5xFAD mice. Representative images of Iba1 staininggiven by (A). Treatment with 2.5 g/L WSAq reduced activated microglia in the hippocampus (C) andapproached significant reduction of IBA1 in the cortex (B). Activation following WSAq 0.5 g/L was notsignificantly different from the 5xFAD controls. * p < 0.05, **** p < 0.0001. n = 11–15 per treatment group.3.5. Antioxidant Response Is Upregulated in 5xFAD Mice Following WSAq TreatmentAn increase in the cortical expression of the antioxidant regulatory factor NRF2 wasseen in the 5xFAD mice treated with both 0.5 mg/mL and 2.5 mg/mL WSAq (Figure 8A).There was also a significant increase in the cortical expression of the NRF2-regulatedantioxidant enzyme NQO1 in the 5xFAD mice treated with 2.5 mg/mL WSAq. A similartrend towards increased expression of the NRF2-regulated antioxidant enzymes GCLCand HMOX1 was also seen in low and high WSAq-treated 5xFAD mice (Figure 8A). Theprotein expression of NRF2 was also increased in the cortex of the 5xFAD mice treated with2.5 mg/mL WSAq (Figure 8B).Antioxidants 2024, 13, x FOR PEER REVIEW 9 of 16 was not significantly different from the 5xFAD controls. * p < 0.05, **** p < 0.0001. n = 11–15 per treatment group. 3.5. Antioxidant Response Is Upregulated in 5xFAD ice Following SAq Treatment An increase in the cortical expression of the antioxidant regulatory factor RF2 as seen in the 5xF ice treated ith both 0.5 mg/ L and 2.5 g/ L S q (Figure 8 ). There as also a significant increase in the cortical expression of the NRF2-regulated an-tioxidant enzyme NQO1 in the 5xFAD mice treated it 2.5 g/ S . si ilar tre t r s i cre se e ressi f t e -re l te ti i t e es l i l i -tr t i ( i r ). tei expre sion of NRF2 was also increased in the cortex of the 5xFAD mice treated with 2.5 mg/mL WSAq (Figure 8B). Figure 8. WSAq upregulates antioxidant response in the cortex of 5xFAD mice. (A) Gene expression of antioxidant response genes is increased in the cortex of WSAq treated 5xFAD mice. (B) NRF2 protein expression is likewise increased in the cortex of 5xFAD mice treated with 2.5 g/L WSAq. * p < 0.05. n = 9–12 per treatment condition. Figure 8. Cont.Antioxidants 2024, 13, 1164 9 of 15Antioxidants 2024, 13, x FOR PEER REVIEW 9 of 16 was not significantly different from the 5xFAD controls. * p < 0.05, **** p < 0.0001. n = 11–15 per treatment group. 3.5. Antioxidant Response Is Upregulated in 5xFAD Mice Following WSAq Treatment An increase in the cortical expression of the antioxidant regulatory factor NRF2 was seen in the 5xFAD mice treated with both 0.5 mg/mL and 2.5 mg/mL WSAq (Figure 8A). There was also a significant increase in the cortical expression of the NRF2-regulated an-tioxidant enzyme NQO1 in the 5xFAD mice treated with 2.5 mg/mL WSAq. A similar trend towards increased expression of the NRF2-regulated antioxidant enzymes GCLC and HMOX1 was also seen in low and high WSAq-treated 5xFAD mice (Figure 8A). The protein expression of NRF2 was also increased in the cortex of the 5xFAD mice treated with 2.5 mg/mL WSAq (Figure 8B). Figure 8. WSAq upregulates antioxidant response in the cortex of 5xFAD mice. (A) Gene expression of antioxidant response genes is increased in the cortex of WSAq treated 5xFAD mice. (B) NRF2 protein expression is likewise increased in the cortex of 5xFAD mice treated with 2.5 g/L WSAq. * p < 0.05. n = 9–12 per treatment condition. Figure 8. S q upregulates antioxidant response in the cortex of 5xF ice. ( ) ene expressionf a ti i r s is i crease i the cortex of SAq treated 5xFAD mice. ( )r t i i i creased in the cortex of 5xFAD mice treated with 2.5 g/L WSAq.* p < 0.05. n = 9–12 per treatment condition.4. DiscussionThis study investigated the effects of an aqueous extract of ashwagandha root (WSAq)in the 5xFAD mouse model of Aβ accumulation. Four weeks of oral treatment with WSAqresulted in improved spatial memory as well as reduced anxiety-related and depressive-like behavior. These changes were accompanied by a reduction in Aβ plaque burden andmarkers of neuroinflammation and increased antioxidant response.The behavioral results from this study are in line with previous reports of the effectsof WS extracts. Anxiolytic and anti-depressive effects of WS were demonstrated in bothrodent and human models. In mice, WS root and leaf extracts increased mobility in the FSTin Swiss albino mice [38] and oral treatment with a modified WS root extract ameliorateddepressive and anxious behavior in a model of foot-shock-induced stress [30]. In humans,8 weeks of daily administration of the product Sensoril®, containing both WS leaf and rootextract, reduced stress, anxiety and depression in adults aged between 18 and 60 yearsold [39]. Similarly, 60 days of treatment with WS root extract standardized for 2.5% ofthe withanolide compounds found in WS improved stress and anxiety metrics in healthyadults exhibiting mild to moderate symptoms of those endpoints [40]. The same durationof treatment with the product Shoden®, containing a WS root and leaf extract standardizedto 35% withanosides, also improved scores for anxiety and stress in healthy adults [28].Similar results were seen in stressed adults as well. Improvements in anxiety and stressscores were also seen in stressed adults given Shoden® [28] and in adults reporting a historyof chronic stress, WS extracts were reported to significantly reduce stress assessment andanxiety scores [41,42].Preliminary studies using WS in human trials have also provided evidence for itscognition-enhancing capabilities. A systematic review of five studies exploring the effectof WS on cognition found evidence for improved executive function, attention, reactiontime, and cognitive task performance, but low-quality studies and heterogeneity of thestudy populations limit the impact of these findings [43]. Stronger evidence of cognitiveenhancement elicited by WS was reported in pre-clinical models. Oral treatment withvarious WS preparations was shown to improve cognitive deficits in rodent models ofscopolamine-, hypoxia- and high-fat diet-induced cognitive impairments [27,44].Despite the evidence for cognitive-enhancing, anxiolytic and anti-depressive effects ofWS, the exact molecular mechanisms through which these behavioral changes are elicitedremain poorly defined. In the present study, the behavioral improvements may be related tothe reduction in Aβ plaque burden in WSAq-treated 5xFAD mice. This finding is consistentAntioxidants 2024, 13, 1164 10 of 15with existing literature on the effects of WS on Aβ [45–47]. A similar plaque reductionwas reported in the APPswe, PSEN1 transgenic model of Aβ accumulation following a30-day treatment period with a powdered root extract suspended in ethanol [48]. Thestudy proposed a unique mechanism by which WS affects Aβ clearance from the braininto the periphery by modulating liver production of low-density lipoprotein receptor-related protein (LRP) which traffics Aβ from the brain [48]. There have also been in vitroreports of constituent compounds from WS reducing the levels of Aβ by inhibiting Aβsecretion [49,50]. Future studies are needed to determine whether the Aβ lowering effectof WSAq is the result of altered production or clearance in 5xFAD mice. The effects of WSon Aβ are interesting in light of the newer FDA approved Aβ lowering therapies [51,52].While these therapies are major breakthroughs there are still limitations including a narrowwindow to initiate treatment in a specific population of AD patients as well as somesafety concerns [53]. WSAq may prove to be an alternative that does not suffer from thesame limitations, although that remains to be seen as it continues to be developed forclinical testing.In this study, there was a potent effect of WSAq on microglial and astrocytic activationin the 5xFAD mouse brain. These findings are in line with other studies that have demon-strated the anti-inflammatory effects of WS [47,54–56]. A sustained release formulation ofWS, AshwaSR, at a dose of 100 mg and containing NLT 5% total withanolides administeredonce daily to Sprague Dawley rats inhibited the expression of pro-inflammatory cytokinesIL-1β and TNF-α, and in an in vitro portion of the study, AshwaSR showed dose-dependentinhibition of IL-1B and TNF-α production from LPS-induced THP-1 human monocytes [57].Further evidence is also seen in rodent models. In young adult female Wistar rats withdiet-induced obesity, WS dry leaf powder reduced the expression of GFAP and IBA1 in thehippocampus along with other inflammatory markers [58]. Similarly, GFAP expressionwas reduced in adult male Albino Wistar rats treated with a fine leaf powder extract ofWS [59]. Although the effects of WS on neuroinflammation in humans have not been thor-oughly investigated, there is evidence for the anti-inflammatory effects of WS in numerousother conditions including lupus erythematosus, inflammatory bowel disease, rheumatoidarthritis and coronavirus [60]. It is possible that the anti-inflammatory effects of WS maybe mediators of the behavioral effects observed in this study, as neuroinflammation hasbeen linked to affective and cognitive dysfunction [61]; however, more work is neededto confirm this mechanistic link which could include a quantitative assessment of otherinflammatory markers and more in-depth microglial and astrocytic profiling.Interestingly, the effects of WS on inflammation are not unequivocally positive. Thepro-inflammatory effects of WS preparations were also reported in the literature. IncreasedGFAP expression was seen in scopolamine-exposed Swiss albino mice treated with analcoholic extract of WS leaves (i-Extract) at 100, 200 and 300 mg/kg [62]. One possiblereason for these divergent findings could be that the concentrations used were muchhigher than the concentration of WSAq used in the present study. It is possible that themaximal anti-inflammatory effects of WS are evoked at lower concentrations and oncethose concentrations are exceeded the effects diminish or are even reversed. It is alsopossible that the pro-inflammatory compounds of WSAq only attain active concentrationsat higher doses. In fact, the GFAP data from the current study support this idea, as thereduction in expression was only observed in 5xFAD mice treated with 0.5 mg/mL and not2.5 mg/mL of WSAq. The fact that lower concentrations of WS may confer greater benefitsthan higher concentrations of WS is noteworthy especially in light of recent reports of livertoxicity following WS administration [63]. Taken together, these findings underscore theimportance of precise dosing of WS for optimal effects on inflammation.WSAq also induced the expression of antioxidant response genes and the expressionof the antioxidant regulatory protein NRF2 in the cortex of treated 5xFAD mice in thepresent study which could also have contributed to the behavioral improvements observed.The antioxidant effects of WS were also reported in other model systems as well. Cul-tured BV-2 microglial cells treated with the WS constituent compounds withaferin A andAntioxidants 2024, 13, 1164 11 of 15withanolide A showed reduced LPS-induced NO production and activation of the NRF2antioxidant response pathway [64]. Similar effects were observed in vivo. WS powderedroot extract attenuated MPTP-induced deficits in superoxide dismutase (SOD) and catalase(CAT) in albino mice [65] and normalized malondialdehyde (MDA), SOD and glutathione(GSH) activity in 5xFAD mice [66]. WS was also found to exert an antioxidative effect inhuman trials. In a study of healthy adults treated with dried aqueous root extract for 6months, a significant increase in SOD was observed [67]. Other markers of antioxidantresponse and oxidative stress have similarly been reported to be altered by WS in humans,including decreased levels of MDA and nitric oxide and increased expression of GSH, SOD,and CAT enzymes [68]. The antioxidant effects of WSAq could be confirmed in futurestudies by evaluating the protein expression of the antioxidant enzymes and markers ofoxidative damage.It is important to note that many WS preparations discussed in the literature arecomplex mixtures of phytochemical compounds as is WSAq. While some preparationsare standardized to specific levels of constituent compounds, it remains to be seen whichchemical constituents mediate the beneficial effects of the extracts in the context of neu-rodegeneration. Withaferin A was shown to improve cognitive function in a mouse modelof frontotemporal lobar degeneration [69] and sominone, a metabolite of withanoside IV,enhanced location memory in healthy young mice [70]. Continued research into the anti-depressive and anxiolytic properties of constituent compounds of WS and the molecularmechanisms underpinning the observed behavioral effects will be valuable in understand-ing how WS is able to confer its beneficial effects.It is notable there was no difference in response to WSAq observed between male andfemale 5xFAD mice. This study was designed to evaluate the effects of WSAq in animalswith robust Aβ pathology. It would be interesting in future studies to explore the effects ofan earlier intervention to determine if WSAq treatment could prevent or delay the onsetof pathology. It will be important in such a study to again take into account the effectsof sex in light of the differential risk based on sex of developing AD that exists in thehuman population.5. ConclusionsThe findings from this study support the therapeutic potential for WSAq to improvecognition and reduce anxiety and depressive symptoms in the context of AD. Future studiesare needed to more fully understand the cellular mechanism by which WSAq exerts thesebeneficial effects and to identify optimal dosing for behavioral improvements. Becauseneuroinflammation and oxidative stress accompany cognitive impairment, anxiety anddepression in other conditions beyond AD, the results presented here suggest the potentialfor a broader application of WSAq to other neurodegenerative conditions as well as tosupport healthy aging.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox13101164/s1. Table S1: Number of male and female micein each treatment condition.Author Contributions: Conceptualization, N.E.G. and A.S.; formal analysis, N.E.G.; investigation,N.G.-K., O.M., M.B., J.C., A.A.M. and L.Y.; resources, N.E.G., A.S. and C.S.M.; data curation, N.G.-K.;writing—original draft preparation, N.G.-K.; writing—review and editing, N.E.G., A.S. and C.S.M.;visualization, N.E.G.; supervision, N.E.G. funding acquisition, N.E.G., A.S. and C.S.M. All authorshave read and agreed to the published version of the manuscript.Funding: This research was funded by a grant from the Oregon Partnership for Alzheimer’s Research,NIH NCCIH grant U19AT010829, NIH grant S10RR027878 and NIH grant S10OD026922.Institutional Review Board Statement: The animal work in this study was conducted in accordancewith the NIH Guidelines for the Care and Use of Laboratory Animals and was approved by theInstitutional Animal Care and Use Committee of the Portland VA Healthcare System, (VAPORHCS;IACUC #4688-21).Antioxidants 2024, 13, 1164 12 of 15Informed Consent Statement: Not applicable.Data Availability Statement: The datasets used and/or analyzed in the current study are availablefrom the corresponding author upon reasonable request.Acknowledgments: We would like to thank Samantha Guerra-Nuñez, Wyatt Hack and ChristopherHarris for their assistance in harvesting and processing tissue for this study, the Medicinal ChemistryCore at OHSU for the use of their lyophilizer, and the OSU mass spectrometry center for the use oftheir equipment.Conflicts of Interest: The authors declare no conflicts of interest.References1. 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Multidisciplinary Digital Publishing Institute
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Faculty
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Alzheimer’s disease
memory
depression
anxiety
ashwagandha
neuroinflammation
oxidative stress
beta-amyloid
5xFAD mice
Withania somnifera (Ashwagandha) Improves Spatial Memory, Anxiety and Depressive-like Behavior in the 5xFAD Mouse Model of Alzheimer’s Disease
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