Extra-virgin Avocado (Persea americana Mill.,Laucaceae) Oil Improves Cognitive Impairment in D-galactose-induced Alzheimer’s Disease Model on Ovariectomized Wistar Rat

Extra-virgin Avocado (Persea americana Mill.,Laucaceae) Oil Improves Cognitive Impairment in D-galactose-induced Alzheimer’s Disease Model on Ovariectomized Wistar Rat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Extra-virgin Avocado (Persea americana Mill.,Laucaceae) Oil Improves Cognitive Impairment in D-galactose-induced Alzheimer’s Disease Model on Ovariectomized Wistar Rat Christelle Massop Wamba Ndé, Sefirin Djiogue, Charline Florence Awounfack, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2036318/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Avocado is a tree’s fruit ( Persea Americana Mill.) of the Laucaceae family. It was reported that consumption of avocado improved cognitive performance. No study has yet been carried out regarding the properties of avocado oil supplementation on the occurrence of Alzheimer's disease. The objective of the present study was to evaluate the effects of extra-virgin avocado oil on a model of D-galactose-induced Alzheimer's disease in ovariectomized Wistar rats. Methods: To accomplish this, 54 female rats were used, of which 42 were ovariectomized (OVX) and 12 underwent white surgery (SHAM). Fourteen days after surgery, the animals were divided into 9 groups of 6 animals each: SHAM+Veh and OVX+Veh groups receiving the vehicle; SHAM + D-gal and OVX+D-gal groups receiving D-galactose and vehicle; OVX+D-gal+E2V and OVX+D-gal+DNPZ groups receiving D-galactose and reference drugs (estradiol valerate and donepezil respectively) and 3 test groups (OVX+D-gal+AO1; OVX+D-gal+AO2 and OVX+D-gal+AO3) receiving D-galactose each and extra-virgin avocado oil at the doses of 0.25, 0.5 and 1 mL/kg respectively. The treatment was carried out during 70 days during which memory disorders were evaluated using the Object Recognition , Y-Maze and MWM tests. Some biochemical parameters regarding memory function were evaluated on hippocampus homogenate 10%. Isolated brain was fixed in 10% formalin for histological analysis. Results: As results, compared to SHAM+Veh group, deterioration of both non-spatial and spatial memory (short- and long-term) was observed in OVX animals threated with D-galactose. In addition, a significant decrease in relative hippocampal weight (p < 0.001), Ach (p < 0.001), Glu (p < 0.001), GSH (p < 0.001), CAT (p < 0.05), and SOD (p < 0.001) activities, and a significant (p < 0.001) increase in Methylglyoxal, MDA, and NO2- . was noted in OVX+D-gal group. Compared to OVX+D-gal group, the treatment with extra-virgin avocado oil at all tested doses reversed or prevented the negative effects induced by ovariectomy and/or by D-galactose on biochemical and oxidative stress biomarkers. The analysis of hippocampus microarchitecture shows that the extra-virgin avocado oil induced a significant decrease (p < 0.05; p < 0.01; p < 0.001) of neuronal loss in CA1 and CA3 hippocampal region. Conclusions: Taken together, these results suggest that avocado oil possesses neuroprotective properties and can be consumed or supplemented to prevent the onset of Alzheimer's disease. Avocado oil Persea americana memory ovariectomy D-galactose Alzheimer's disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Alzheimer’s disease (AD) is the main cause of dementia worldwide with 60–70% of cases ( 1 ). With the increase of the aged population, the global prevalence of dementia is rising sharply. It was estimated about 50 million cases of dementia in 2019 and it is expected to increase by 152 million in 2050 if nothing is done ( 1 ). Alzheimer's disease is a chronic and irreversible neurodegenerative dementia that affects daily life through progressive memory loss, cognitive dysfunction and reduced learning abilities (2; 3; 4). Women are 2 to 3 times more likely to develop AD than men ( 5 ). This difference in the incidence of AD by sex has been attributed to the increase in life expectancy which is considerably higher in women than in men ( 6 ), as well as biological and hormonal differences, which results in a possible neuroprotective effects of estrogen that falls at menopause in women ( 7 ). In experiment animals, ovariectomy has been used by several authors to reflect postmenopausal status (8;9;10). In addition, ovariectomy has been shown to lead to a gradual decline in cognitive function and locomotor activity in rats ( 10 ). Several studies have shown that administration of D-galactose in rodents mimics the natural aging process by inducing neurodegeneration similar to that observed in AD (11; 12; 13; 14). In addition, it has been reported that ovariectomy associated to chronic administration of D-galactose act synergistically to accelerate the pathophysiology of Alzheimer's disease ( 15 ). In modern medicine, the molecules used to manage Alzheimer's disease, act essentially in alleviating the symptoms and do not prevent the progression of the disease. Moreover, these molecules have many side effects and have a high cost ( 16 ). Hence the search for an alternative treatment is increasing. In recent years, dietary approaches to prevent AD with natural ingredients of plants origin (such as the "Mediterranean diet») attract great attention. The "Mediterranean diet" is rich in monounsaturated fatty acids and many reports revealed that such diet is beneficial for the prevention of neurodegenerative diseases during aging, and the improvement of cognitive performances (17; 18; 19). The avocado is a tree’s fruit ( Persea americana Mill.) of the Laucaceae family. Its cultivation is widespread in all tropical regions of the world. More than 200 varieties are known. The pulp of this fruit has high oil content (5–30% depending on the variety) ( 20 ). Its consumption is increasing significantly worldwide and it provides a lipid profile almost identical to olive oil ( 21 ). Cheng et al . ( 22 ) reported that American seniors who consumed avocado had improved cognitive performance than non-consumers. Similarly, some authors have shown that consuming an avocado fruit for 12 weeks or 6 months improves cognitive performance in the elderly (23; 24). In the literature, few studies have been published on the properties of avocado oil targeting cognitive functions ( 25 , 26 ). These studies are limited on reflex maturation, postnatal somatic development and memory acquisition in newborn rats, during the adolescent phase and in adults ( 25 ) as well as on mitochondrial brain function in diabetic rats ( 26 ). However, no study has yet been conducted on the properties of avocado oil on Alzheimer's disease occurrence. The aim of the present study was therefore to evaluate the effects of extra-virgin avocado oil on a model of D-galactose-induced Alzheimer's disease in ovariectomized Wistar rats. 2. Material And Methods 2.1. Material 2.1.1. Plant material Different parts of Persea americana (stem bark, leaves, flowers and fruits) were collected in the orchard of the Institute of Agricultural Research for Development (IRAD) of Foumbot (Department of Noun, West Region, Cameroun). These botanical samples were identified at the National Herbarium of Cameroon (HNC) by comparison to the existing specimens under the voucher number 57756 HNC. The avocado fruits, Fuerte variety, were provided by Mrs. KEMAYOU MBIEJI Flavie Christelle, researcher at IRAD. After harvesting they were immediately cleaned and stored in a cool and dried place until they ripened. 2.1.2. Experimental animals In this study, animals used were adult female Wistar rats,12–14 weeks old, weighing between 150 and 200 g. Animals were housed in the animal facility of the Laboratory of Animal Physiology, University of Yaoundé 1 (temperature, room temperature; humidity 50–80%; 12 hlight–dark cycle). They had free access to a standard soy-free rat diet (SSniff GmbH, Soest, Germany) and were provided tap water ad libitum. The research was conducted in accordance with the guidelines of the institutional Ethic Committee of Cameroon’s Ministry of Scientific Research and Technological Innovation, which has adopted the guidelines established by the European Union on Animal Care (CEE Council 86/609; Reg.no.FWA-IRD0001954). 2.1.3. Chemicals Donepezil (donepezil hydrochloride 10 mg, ARROW Generics, 69007 LYON, France) and Estradiol valerate (Progynova® 2mg, Delpharm laboratory, France) were used as reference drugs. D-galactose (BDH chemical Ltd pools, Great Britain) was used to induce neurodegeneration in rodents similar to that observed in Alzheimer's disease. 2.2. Methods 2.2.1. Oil extraction and determination of doses After washing, the avocado fruits were ripped, and the pulp withdrawn and manually mixed in the laboratory mortar. The mixture was dried at 60 ° C ( 27 ) for 5 hours in an electric oven brand (PHILIPS) to remove traces of water. The dried pulp was pressed mechanically using a manual press and the resulting oil was filtered before being stored in the dark vials at -20°C. With 200 g of avocado fruit (Fuerte variety), our extraction process led to10 mL of extra-virgin avocado oil. The doses of administration were prepared based on the report of general consumption of avocado fruit ( 24 ). The equivalent doses in each rat were extrapolated from the human dose (0.14 mL/kg BW) to afford 0.89 mL/kg BW (~ 1 mL/kg BW). To obtain a dose response curve of the extra-virgin avocado oil,2 other doses (0.25, 0.5 mL/kg BW) were generated. 2.2.2. Behavioral Assessments Object Recognition test The "Object Recognition" (OR) test is a suitable test for the evaluation of hippocampo-dependent memory processes in rodents ( 28 ). The principle of this test is based on the natural affinity of rodents with the new object ( 29 ). In this study the OR test was carried out in an Open Field (OF) device, using 4 objects: 2 objects (A1 and A2) identical to each other, and two objects (B and C) different from each other (shape, color and texture) and different from the first two. The OR protocol used was adapted from that described by Djiogue et al . ( 10 ). The test was performed on days 59 to 61 of the experiment. During this test, the following parameters were evaluated: the time spent exploring identical objects during the familiarization phase, the time spent exploring one of the old and new objects 3 hours after the familiarization stage (A and B) and the time spent exploring one of the old and new objects 24 hours after the familiarization stage (A and C) during the test phases. The exploration time was recorded when the rats touched the object, or approached within 2 cm of the object in question ( 30 ). Parameters were recorded via a video recording system using a camera placed above the pool and connected to a computer. To avoid animal’s perturbation due to urine and faeces, between two tests, the device was cleaned with 70% ethanol solution and dry cloth. The data were reported on parameter sheets provided for this purpose. The data obtained were expressed in proportion to the time the animal spent on the new object (IR recognition index). Y-Maze test The Y-Maze test is a recognition memory test used to assess short-term spatial working memory. This test is based on the animal's natural tendency to explore novelty. The Y-labyrinth test was carried out in a wooden device with three identical arms (11 x 50 x 32 cm each) separated by an angle of 120̊ placed in a room with the visual cues on the blackberries. The protocol used was adapted to the one described by Fernanda et al. (31). The test was performed on day 63 of the experiment. During this test, the number of entries and the time spent in each arm were recorded via a video recording system using a camera placed above the pool and connected to a computer. The percentage of the number of entries into the new arm was determined in relation to the number of entries into the familiar arms; and the percentage of time spent in the new arm was determined in relation to the total time spent in the device (sum of time in two familiar arms, and new arm as well as central area). To avoid animal’s perturbation due to urine and faeces, the device was cleaned with 70% ethanol solution and dry cloth between two tests. Morris Water Maze test The Morris water Maze (MWM) test allows the accurate and reproducible measurement of spatial memory and is a very sensitive tool for assessing damage to the hippocampus ( 32 ). In the present study, the MWM test was conducted in a black circular tank (diameter 120 cm × height 50 cm) half-filled with water located in a lighted room. An 8-cm diameter black drainage platform (color-matched to the device to make it invisible) was placed in a fixed position (south quadrant of the device), submerged 1.0 cm under the water surface. The protocol for this test was adapted from Mandeep and Yash ( 33 ). The test was performed on days 64 to 68 of the experiment. The test consisted of an acquisition phase of 4 days and a retention phase on day 5. Acquisition phase: three trials were conducted per day, with a break time of 15 minutes between trials. At the end of each test the animals were cleaned properly and returned to their original cages. The principle of MWM was that when the rats escaped from the water by climbing on the platform, they learned the spatial location of the platform from any starting position in the pool. Retention phase: during this phase, to evaluate the spatial memorization, the platform was removed, each rat was released into water in one of the fixed target facing the target quadrant and had 60 seconds of swimming. The latency time to reach the exact position of the platform, the time spent in the target quadrant and the number of entries in the target quadrant of the platform were recorded via a video recording system using a camera placed above the pool and connected to a computer. 2.2.3. Experimental design To accomplish this, 54 female rats were used. 42 of them were suggested to the bilateral ovariectomy (OVX) using the dorsal approach (34; 10). The 12others were used as SHAM. Fourteen days after surgery, the animals were divided into 9 groups of 6 animals each: Group 1 (SHAM + Veh): SHAM rats were given distilled water (Veh, vehicle) per os ; Group 2 (OVX + Veh): OVX rats were given distilled water per os ; Group 3 (SHAM + D-gal): SHAM rats were given D-galactose (D-gal, 150 mg/kg, i.p.) and distilled water per os simultaneously; Group 4 (OVX + D-gal): OVX rats were given D-galactose (150 mg/kg, i.p.) and distilled water per os simultaneously; Group 5 (OVX + D-gal + E2V): OVX rats were given D-galactose (150 mg/kg, i.p.) and estradiol valerate (E2V, 1 mg/kg, p.o.) simultaneously; Group 6 (OVX + D-gal + DNPZ): OVX rats were given D-galactose (150 mg/kg, i.p.) and donepezil (DNPZ, 1mg/kg, p.o) simultaneously; Groups 7, 8 and 9 (OVX + D-gal + AO1; OVX + D-gal + AO2 and OVX + D-gal + AO3 respectively, Test groups): these OVX rats were given D-galactose each (150 mg/kg, i.p.) and the extra-virgin avocado oil per os at the dose of 0.25, 0.5 and 1 mL/kg respectively. The treatment was carried out for 70 days during which the memory disorders were evaluated using the Object Recognition, Y-Maze and MWM tests (as shows on the diagram). At the end of the experiment, the animals were euthanized and brain isolate was immediately weighed. the hippocampus was isolated in the right hemisphere of each animal on a block of NaCl ice, weighed and used to prepare homogenate 10% on Tris buffer solution (HCl 50 mM; KCl 150 mM; pH 7.4). The homogenate was used for biochemical analysisas well as evaluation of some oxidative stress biomarkers. The left hemisphere of each animal was fixed in formaldehyde 10% for histological analysis. 2.2.4. BiochemicalAssay Some markers of memory function (Acetylcholine, Glutamate) and Methylglyoxal was measured in 10% of hippocampus homogenate using commercial Kits. The Acetylcholine content was measured using the quantitative ELISA analysis technique according to the instructions described on BioVision's Acetylcholine ELISA Kit. The Glutamate content was measured by the colorimetric method according to the manufacturer instructions described on the BioVision Glutamate Assay Kit.The determination of Methylglyoxal (MG) was performed by the fluorometric method according to the instructions described on the BioVision Methylglyoxal (fluorometric) assay kit. All kits were provided by BioVion Incorporated, CA 95035 USA. 2.2.5. Oxidative stress biomarkers evaluation Hippocampal levels of malondialdehyde (MDA), nitrites(NO2 − .), catalase activity (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) were determined using methods described respectively by Wilbur et al. , ( 35 ), Fermor et al . ( 36 ), Sinha ( 37 ), Misra and Fridovish ( 38 ) and Ellman ( 39 ). 2.2.6. Histological analysis Sections of 5-µm thickness were obtained from brain tissues embedded in paraffin wax using a standard microtome. Brain tissues section were processed for Haematoxylin and Eosin staining based on the method described by Smith and Bruton ( 40 ). The mounted slides were allowed to dry and prepared ready for microscopy. Slides observation was done under the Scientico STM-50 optical microscope equipped with a Celestron 44421 brand digital camera connected to a computer for microphotographs. The J image software (version 1.48 for Window) was used for quantification of viable neurons in the Cornu Ammonis 1 (CA1) and 3 (CA3) of the hippocampus. 2.2.7. Statistical Analysis The data are expressed as mean ± SEM and analysed with one-way ANOVA followed by the Dunnett post-test (GraphPad Prism ® Software, version 8.0.1.244, San Diego, CA, USA). A p value < 0.05 was considered significant. 3. Results And Discussion 3.1. Results 3.1.1. Effects of extra-virgin avocado oil on brain and hippocampus relative weight Figure 1 shows the effects of extra-virgin avocado oil on brain and hippocampus relative weight after 70 days of D-galactose treatment on ovariectomized rats. Compared to SHAM + Veh group, the association of ovariectomy and D-galactose (OVX + D-gal) induced a non-significant decrease of brain relative weight (Fig. 1 A). all the treated ovariectomized rats, receiving D-galactose + estradiol valerate (1 mg/kg), D-galactose + donepezil (1 mg/kg), D-galactose + extra-virgin avocado oil (0.25, 0.5 and 1 mL/Kg),showed non-significant increase of relative brain weight compare to OVX + D-gal group (Fig. 1 A). The analysis of Fig. 1 B shows that the association of ovariectomy and D-galactose (OVX + D-gal) induced a significant ( p < 0.001) decrease of hippocampus relative weight in comparison to SHAM + Veh group. Compared to OVX + D-gal group, the treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at all tested doses induced a significant (p < 0.05; p < 0.01; p < 0.001) increase of relative hippocampus weight, as well as estradiol valerate (1 mg/kg) (Fig. 1 B). 3.1.2. Effects of extra-virgin avocado oil on non-spatial memory evaluated by the Object Recognition test The evaluation of short-term memory by Object Recognition (OR) test shows that compared to SHAM + Veh, the association of ovariectomy and D-galactose (OVX + D-gal) induced a non-significant variation of the percentage of time spent on the novel object (Fig. 2 A). The treatment of ovariectomized ratcombined to D-galactose exposure with extra-virgin avocado oil and the group treated with donepezil (1 mg/kg) + D-galactose induced an increase of this parameter. This effect was significant (p < 0.05; p < 0.001) at the dose 0.25 ml/kg of extra-virgin avocado oil and donepezil (1 mg/kg) (Fig. 2 A). As shown on Fig. 2 B, the evaluation of long-term memory by OR test shows that the association of ovariectomy and D-galactose (OVX + D-gal) induced a significant (p < 0.01) decrease of the percentage of time spent on the novel object when compared to SHAM + Veh (Fig. 2 A). The treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at all tested doses as well as E2V (1 mg/kg) induced a significant (p < 0.05; p < 0.001) increase of the percentage of time spent on the novel object(Fig. 2 B) in comparison to OVX + D-gal group. 3.1.3. Effects of extra-virgin avocado oil on short-term spatial memory evaluated by the Y- Maze test Figure 3 represents the effects of avocado extra-virgin oil on the percentage of time spent and the number of entries in the new arm of the Y- Maze. Analysis of the results shows that the association of ovariectomy and D-galactose (OVX + D-gal) significantly (p < 0.001) decrease the time spent (Fig. 3 A) and the number of entries (Fig. 3 B) in the new arm compared to the non-ovariectomized group treated with vehicle (SHAM + veh). The treatment of ovariectomized animal combined to D-galactose exposure with avocado extra-virgin oil as well as the reference drug induced an increase of these parameters in comparison to OVX + D-gal group. The increase induced by avocado extra-virgin oil on the time spent in the new arm was significant (p < 0.001) at the doses of 0.5 and 1 mL/kg (Fig. 3 A). All tested doses as well as reference drugs induced a significant (p < 0.01; p < 0.001) increase of the number of entries in the new arm (Fig. 3 B). 3.1.4. Effects of extra-virgin avocado oil on long-term spatial memory evaluated by the Morris water Maze test Analysis of the results presented on Fig. 4 A shows a significant (p < 0.001) increase of the latency time of entries in target quadrant in ovariectomized animals combined to D-galactose exposure (OVX + D-gal) compared with non-ovariectomized animals receiving vehicle (SHAM + veh). When compared to OVX + D-gal group, extra-virgin avocado oil at all tested doses as well as E2V and DNPZ, induced a significant (p < 0.001) decrease of the latency time of entries in target quadrant (Fig. 4 A). Results presented in Fig. 4 B and C shows that compared to SHAM + veh group, the OVX + D-gal group shows a decrease in the number of entries (B) and the percentage of time spent (C) in the target quadrant. These effect of D-galactose in ovariectomized animals was significant (p < 0.001) regarding the percentage of time spent (Fig. 4 C). The treatment of ovariectomized animals combined to D-galactose exposure with extra-virgin avocado oil as well as E2V and DNPZ, induced an increase of the number of entries (B) and the percentage of time spent (C) in the target quadrant in comparison to OVX + D-gal group. The effect of extra-virgin avocado oil was significant (p < 0.001) at all tested doses on the percentage of time spent (Fig. 4 C). 3.1.5. Effects of extra-virgin avocado oil on some markers of cognitive function As shows in Fig. 5 A and B, the association of ovariectomy and D-galactose induced a significant (p < 0.001) decrease of Acetylcholine (A) and Glutamate (B) levels in the hippocampus, when compared to SHAM + veh group. The treatment of ovariectomized animals combined to D-galactose exposure with extra-virgin avocado oil (at all tested doses) as well as E2V and DNPZ, induced a significant (p < 0.001) increase of these parameters when compared to OVX + D-gal group (Fig. 5 A and B). Analysis of the results presented on Fig. 5 C shows a significant (p < 0.001) increase of Methylglyoxal level in the hippocampus of ovariectomized animals combined to D-galactose exposure (OVX + D-gal) compared to non-ovariectomized animals receiving vehicle (SHAM + veh). When compared to OVX + D-gal group, the groups treated with extra-virgin avocado oil at all tested doses as well as E2V, induced a significant (p < 0.01; p < 0.001) decrease of Methylglyoxal level (Fig. 5 C). 3.1.6. Effects of extra-virgin avocado oil on some markers of oxidative stress The evaluation of oxidative stress markers shows that compared to SHAM + Veh group, the association of ovariectomy and D-galactose (OVX + D-gal) induce a significant (p < 0.001) increase of MDA and Nitrite level (NO 2 −. ) in the hippocampus in comparison to SHAM + Veh (Fig. 6 A and B respectively). The treatment of ovariectomized rat combined to D-galactose exposure with extra-virgin avocado oil as well as E2V and DNPZ induced a significant (p < 0.001) decrease of these parameters when compared to OVX + D-gal group (Fig. 6 A and B). The evaluation of oxidative stress markers also shows that the treatment of ovariectomized (OVX + D-gal) rats with D-galactose induced a significant (p < 0.05; p < 0.001) decrease of GSH level, Catalase and SOD activitiesin the hippocampus in comparison to SHAM + Veh (Fig. 6 C, D and E respectively). The treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at all tested doses as well as E2V and DNPZ induced a significant (p < 0.05; p < 0.01; p < 0.001) increase of GSH level, Catalase and SOD activitiesin the hippocampus in comparison to OVX + D-gal group (Fig. 6 C, D and E respectively). 3.1.7. Effects of extra-virgin avocado oil on microarchitecture and the number of viable neurons in CA1 and CA3 regions of the hippocampus Figure 7 and 8 shows the effects of avocado extra-virgin oil on the number of viable neurons in CA1 and CA3 regions and hippocampus microarchitectures respectively. The analysis of the results presented indicates an intact architecture of the different layers of the hippocampus with intact neurons, the nucleus large, round or oval, and the nucleoli clearly observed, indicating the presence of viable neurons in the normal control group (SHAM + veh) (Figs. 7 and 8 ). In contrast, neuronal disorganization (Nds) with larger intercellular spaces, neuronal degeneration (Nd) revealed by the presence of neurons with hyperchromatic nuclei (Hn) and shrunken cytoplasm (Fig. 8 ) and a significant (p < 0.001) decrease in the number of viable neurons in the CA1 and CA3 (Fig. 7 ) were observed in animals of the ovariectomized group which received D-gal (OVX + D-gal) when compared to SHAM + veh group. the treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at doses of 0.25, 0.5 and 1ml/kg PC, as well as donepezil and estradiol valerate, prevented the neurodegeneration induced by ovariectomy and D-galactose (Figs. 7 and 8 ). The treatment induced a pronounced decrease in the number of neurons with hyperchromatic and shrunken nuclei (Fig. 8 ) and a significant (p < 0,05; p < 0,01; p < 0,001) increase in the number of viable neurons (Fig. 7 ) compared to ovariectomized animals that received D-gal injection (OVX + D-gal). 3.2. DISCUSSION Neurogenesis Decrease in the hippocampus is indicative of age-related memory impairment in rats and humans ( 41 ). It has been shown that ovariectomy and chronic D-galactose exposure act synergistically to accelerate the pathophysiology of Alzheimer's Disease ( 15 ). The objective of the present study was to evaluate the effects of extra-virgin avocado oil on a D-galactose-induced model of Alzheimer' s Disease (AD) in ovariectomized Wistar rats. D-galactose is a sugar capable of causing cognitive impairment by inducing accelerated aging through oxidative stress and inflammation ( 14 ). Knowing that the hippocampus is a cortical structure that deals with the process of memorization, formation, storage of new memories and the connection of some information with these memories ( 42 ), it has been reported that hippocampal atrophy is strongly correlated with dementia and can be considered as a predictor of AD ( 43 ). In this study, it was found that compared to SHAM + Veh group, the administration of D-galactose to ovariectomized (OVX + D-gal) rats induced a non-significant decrease of brain relative weight and a significant decrease in the hippocampus relative weight. This would be attributed to a decrease in neurogenesis in the hippocampus (due to the lack of estrogen) and the deleterious effects of D-galactose. In fact, estrogens act as a regulator of cellular processes in the hippocampus; they promote neurogenesis ( 44 ) and D-galactose resulted in a decrease in hippocampal relative weight ( 45 ). Oral administration of extra-virgin avocado oil (at doses of 0.25, 0.5 and 1 ml/kg) significantly increased hippocampus relative weight of ovariectomized animals combined to D-galactose exposure. These effects of extra-virgin avocado oil could be attributed to phytonutrients found in avocado such as carotenoids (lutein and zeaxanthin) ( 46 ) which are able to improve cognitive function ( 47; 24). In this study, memory function was evaluated by a series of behavioral tests. As results compared to SHAM + Veh group, OVX group received D-galactose (OVX + D-gal) showed an impairment of non-spatial memory (evaluated by OR test) as well as spatial memory (evaluated by Y-maze and MWM tests respectively). This memory impairment was revealed in the OR test by a decrease in the time spent on the novel object (recognition index) in the short and long term. However, the treatment with extra-virgin avocado oil as well as reference drugs significantly increased the time spent on the new object in the short- and long-term suggesting an improvement of the non-spatial memory. Indeed, rodents are naturally curious and in the presence of a novel and old object, they remember the old object and therefore spend more time exploring the novel object ( 48 ). In the Y-maze test, animals from OVX + D-gal group presented a significant decrease in the time spent and the number of entries in the new arm of Y-maze tasks in comparison to SHAM + veh group. Fernanda et al. ( 31 ) reported that in the Y-maze tasks, alterations in short-term spatial memory were noticed by a decrease in the time spent and number of entries in the new arm. However, as well as to estradiol valerate and donepezil, the treatment with avocado extra-virgin oil at all tested doses significantly increased these parameters, suggesting an improvement in short-term spatial memory. In the MWM test used to assess long-term spatial memory, alterations in memory were confirmed by a significant increase of the latency time of entries in the target quadrant and a significant decrease in the time spent in the same quadrant. The treatment of animals with avocado extra-virgin oil (at the tested doses) as well as the reference drugs significantly reduced the latency time of entries in the target quadrant and significantly increased the time spent in the target quadrant. It is well known that estrogen deprivation following ovariectomy impairs learning and memory capacity in female rats ( 49 ) and ovariectomy is associated with progressive loss of non-spatial and spatial memory ( 10 ). The learning and memory deficits observed in ovariectomized animals combined to D-galactose exposure corroborate those of Weam et al . ( 15 ) who showed that estrogen depletion and intraperitoneal administration of D-galactose act synergistically to accelerate the pathophysiology of Alzheimer's disease. The alteration of non-spatial and spatial memory (short and long term) induced by ovariectomy combined to D-galactose administration were improved by the treatment with extra-virgin avocado oil (0.25, 0.5 and 1 ml/kg). This suggests that avocado oil possesses neuroprotective properties although these effects seem to be specific for each type of memory. These observations are in accordance with statements that avocado may be beneficial for cognition throughout the life span, potentially influencing different cognitive domains at different stages of life ( 22 ). The effects of avocado on working memory have been correlated to its richness in carotenoids specifically lutein (22; 50). In AD, dysfunction of Acetylcholine and glutamate is strongly correlated with cognitive decline ( 51 ). There is strong evidence that a decrease in brain glutamate and acetylcholine levels correlates with the severity of Alzheimer's disease (52; 53). In this study, D-galactose administration combined with ovariectomy (OVX + D-gal) resulted in a significant decrease in acetylcholine and glutamate levels in these animals in comparison to SHAM + veh group. This suggests that estrogen depletion and D-gal ingestion act synergistically to accelerate cholinergic and glutaminergic neurodegeneration, thereby disrupting the metabolism of these neurotransmitters. Indeed, it is known that chronic administration of D-galactose leads to oxidative stress and neuroinflammation resulting in losses of cholinergic and glutaminergic neurons ( 13 ). Compared to OVX + D-gal group, the treatment with extra-virgin avocado oil at all tested doses induced an increase of these parameters. These results indicate that extra-virgin avocado oil could protect cholinergic and glutaminergic neurons against alterations induced by estrogen deficiency combined with D-gal injection. This could explain the improved cognitive performance observed in animals treated with extra-virgin avocado oil in the OR, Y-maze and MWM tests. The neuroprotective effects of extra-virgin avocado oil were similar to those of estradiol valerate and donepezil (an acetylcholine esterase inhibitor), suggesting that the compounds present in the extra-virgin oil could interfere with acetylcholine metabolism, probably by inhibiting of acetylcholine esterase activity or acting as phytoestrogens (mechanisms to be explored) by binding to estrogens located in dendritic spines, dendrites, axons and nuclei of hippocampal pyramidal neurons ( 54 ). The D-galactose administration is known to induce brain senescence through Methylglyoxal (MG) accumulation ( 55 ). Methylglyoxal is a highly reactive carbonyl compound that propagates glycation reactions and thus capable of generating advance glycation end products (AGEs) ( 56 ). It has been reported that AGEs accumulation on hippocampus cause dysfunction and death of neurons, thus contributing to the pathophysiology of AD by promoting amyloid-beta accumulation and hyperphosphorylation of tau protein (57; 58). The results obtained in this study shows that compared to the SHAM group, OVX + D-gal group presented a significant increase in the level of MG in the hippocampus. As describe by Li et al . ( 55 ), administration of D-gal in rodents for 10 weeks strongly increased MG levels. This could justify the memory and learning deficits observed in the OR, Y-maze and MWM tests ( 42 ). Compared to OVX + D-gal group, the administration of extra-virgin avocado oil (0.25, 0.5 and 1 ml/kg) to ovariectomized animals receiving D-galactose induced a significant decrease of MG levels in the hippocampus. These effects could be attributed to the presence of secondary metabolites in this fruit that could interact with MG metabolism and reduce his accumulation in the hippocampus. Oxidative stress is known to play a crucial role in the pathophysiology of AD ( 59 ), which includes the activation of NADPH oxidase in astrocytes ( 60 ), mitochondrial dysfunction ( 61 ), increased amyloid-beta neurotoxicity ( 62 ), and synaptic dysfunction responsible for neuronal apoptosis ( 63 ). In the present study, compared to non-ovariectomized animals that received the vehicle, ovariectomy combined with D-galactose administration resulted in a significant increase of MDA and NO 2 −. levels; and a significant decrease of GSH level, CAT and SOD activities in the hippocampus. This suggests that ovariectomy and D-galactose act synergistically to accelerate oxidative damage. It was reported that in the hippocampus, D-gal inhibits the activity of triosephosphate isomerase (TPI) and glyoxalase1 (GLO1) leading to the accumulation of MG which reacts with long-lived proteins to cause irreversible cross-linking of proteins into AGEs ( 64 ). Advanced glycation end products, by binding to their RAGE receptors, activate intracellular inflammatory signalling pathways and produce reactive oxygen species (ROS) while decreasing antioxidant enzymes activity ( 58 ). Furthermore, MDA is an important marker of lipid peroxidation ( 65 ), NO 2 −. is considered to mediate amyloid beta-induced cerebral vascular dysfunction ( 66 ); high concentrations of MDA and NO 2 − induce cell death ( 67 ). The treatment of ovariectomized animals receiving D-galactose with extra virgin avocado oil (0.25, 0.5 and 1 ml/kg) induced a significant decrease of the levels of MDA and NO 2 −. levels as well as a significant increase of the levels of GSH level, SOD and CAT activities. As antioxidant enzymes, SOD is the first line of cellular defence against oxidative damage ( 68 ) and it converts ROS to hydrogen peroxide (H 2 O 2 ) ( 65 ); Glutathione (GSH) destroys the H 2 O 2 produced as well as toxic organic peroxides ( 65 ) and Catalase (CAT) converts hydrogen peroxide into water and molecular oxygen and prevents the formation of the highly reactive and dangerous hydroxyl radical ( 69 ). These antioxidative effects of extra virgin avocado oil could be attributed to some active metabolites present in the avocado such as phenolics, flavonoids and carotenoids capable of scavenging free radical and increasing the activity of endogenous antioxidant enzymes ( 70 ). Avocado is rich in vitamin such as vitamins A, B, C, E, K1 and betaine (Dreher and Davenport, 2013). It is well known that vitamin C (ascorbate) and vitamin E (or α-tocopherol) are considered the main antioxidant molecules (provided by the diet) used by cells ( 71 ). In addition, Gao et al . ( 72 ) reported that DHA present in avocado is able to protect the hippocampus from oxidative stress and neuronal apoptosis ( 72 ). The analysis of brain microarchitecture shows that compared to SHAM + veh group, neuronal disorganization with larger intercellular spaces; neuronal degeneration revealed by the presence of neurons with hyperchromatic nuclei and shrunken cytoplasm; and a significant decrease in the number of viable neurons in the CA1 and CA3 region were observed in animals of the ovariectomized group that received D-galactose only. It is well known that estrogen depletion is associated with neurodegeneration which induced a reduction in the density of dendritic spines of pyramidal neurons in the CA1 region of the hippocampus (73; 9). In addition, D-galactose has the ability to induce a decrease of the growth and reproduction of nerve cells, damage of hippocampal neurons an attenuate the expression of neurotrophic growth factors by stimulating of oxidative stress in the brain ( 74 ). The result obtained in this study supported the hypothesis that estrogen depletion combined with D-galactose act synergistically to accelerate the pathophysiology of AD ( 15 ). In contrast, the treatment with extra-virgin avocado oil at all tested doses significantly attenuated neurodegenerative changes, as shown by a pronounced decrease in the number of neurons with hyperchromatic and shrunken nuclei and a significant increase in the number of viable neurons compared to animals in the ovariectomized group that received D-galactose. These neuroprotective effects of extra-virgin avocado oil could be due to the presence of active metabolic compounds such as phenolics, flavonoids, and carotenoids present in this fruit ( 70 ) that would protect neurons through their antioxidant capacity or by interacting with various signalling pathways that regulate neuron tracking and differentiation. 4. Conclusion The aim of the present study was to evaluate the effects of extra-virgin avocado oil on a model of D-galactose-induced Alzheimer's disease in ovariectomized Wistar rats. Extra-virgin avocado oil treatment of ovariectomized rats receiving D-galactose resulted in the improvement of both non-spatial and spatial memory as well as anti-oxidative activities. The Extra-virgin avocado oil also prevented the neurodegeneration. These observed effects suggested that this fruit possesses neuroprotective properties and could justify its traditional use. The avocado oil can be consumed or supplemented to prevent the onset of Alzheimer's disease. Abbreviations AD: Alzheimer's disease; D-gal: D-galactose; Ach: Acethylcholine; Glu: Glutamate; MG: Methylglyoxal; NO 2 - . : Nitrites; MDA: Malondialdehyde; SOD: superoxide dismutase, CAT: catalase; GSH: Glutathione reduct; ANOVA: Analysis of variance; i.p.: Intraperitoneally; p.o: Per os ; SEM: Standard error of the mean; AO: avocado oil;OVX: ovariectomized; SHAM: sham operated; E2V: Oestradiol valerate; OR: Object Recognition; OF:Open Field; MWM: Morris water Maze. Declarations Ethics approval All experiments were carried out in accordance with the Cameroon National Ethic Committee for animal experiments, which adopted all procedures recommended by the European Union on the protection of animals used for scientific purposes (CEE Council 86/ 609; Reg. no. FWA-IRD 0001954). Consent for Publication Not applicable Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing financial and non-financial interests. Funding We received no funding support for the present study. Authors’ contributions SD and DN conceived the study, supervised the data collection and analysis as well as the manuscript preparation and proofreading. CMW, CFA, FMT, RNDT performed the experiments and data collection, participated to the first draft manuscript writing. FZG participated to the manuscript writing and data analysis. ACKNOWLEDGMENTS The authors are thankful to Ms. KEMAYOU MBIEJI Flavie Christelle, researcher at the Institute of Agricultural Research for Development (IRAD) of Foumbot (Department of Noun, West Region of Cameroon), for her technical assistance during the identification and harvesting of the fruits of the avocado of the Fuerte variety. References Alzheimer’s Disease International (ADI). (2019). Alzheimer's World Report 2019 : Attitudes towards dementia. London : Alzheimer’s Disease International. Heckl S , Pipkom R, Nagele T, Vogel U, Kuker W and Voight K. (2004). Molecular imaging: Bridging the gap between neuroradiology and neurohistology. Histology and Histopathology . 19:651-668. Baulac S, LaVoie MJ, Kimberly WT, Strahle J, Wolfe MS, Selkoe D.J and Weiming X (2003). Functional g-secretase complex assembly in Golgi/trans- Golgi network: interactions among presenilin, nicastrin, Aph1, Pen-2, and g-secretase substrates. Neurobiology of Disease . 14: 194–204. 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(2006). Ovariectomized rats show decreased recognition memory and spine density in the hippocampus and prefrontal cortex. Brain Research . 1126: 176–182. Hua X, Lei M, Zhang Y, Ding J , Han Q, Hu G and Xiao M. (2007). Long-term D-galactose injection combined with ovariectomy serves as a new rodent model for Alzheimer’s disease. Life Sciences . 80 (20): 1897–1905. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2036318","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":134718817,"identity":"fd38433a-a58f-45c8-a461-47312b7e5fa6","order_by":0,"name":"Christelle Massop Wamba Ndé","email":"","orcid":"","institution":"Université de Yaoundé I","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christelle","middleName":"Massop Wamba","lastName":"Ndé","suffix":""},{"id":134718818,"identity":"7deb8c44-e303-482f-9c33-06c0dc3e5ed5","order_by":1,"name":"Sefirin Djiogue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYFACxgcHGAyA9IGDDSCuHIg48ACvFmYDFC3GYC0JBLRA6AMQKhGsEZ8W+fZmxsMVBXcY+A4ebnz4tc0mfX7Y4YdAW+zkdBuwazE4c5jh4BmDZwySBw42G8u2peVuvJ1mANSSbGx2AIcWiXygJwwOMxgcONgmLdl2OHfj7ASQlgOJ23BokZ//mAFZy/90w9npH/BqYbjBjNAi+bHtQIK8dA5+WwzOJIO18ID9wnAu2XCDdE7BgQQD3H6Rbz/M/LHhz2E5vhvHHz78UWYnLz87ffOHDxV2cri0wAAPg8QBBmYekL1glQb4lUMAfwMD4w+QvQ3EqB4Fo2AUjIKRBADhW24fUkFZnAAAAABJRU5ErkJggg==","orcid":"","institution":"Université de Yaoundé I","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sefirin","middleName":"","lastName":"Djiogue","suffix":""},{"id":134718819,"identity":"50647686-2b34-44e1-b88b-0fea34f9b727","order_by":2,"name":"Charline Florence Awounfack","email":"","orcid":"","institution":"Université de Yaoundé I","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Charline","middleName":"Florence","lastName":"Awounfack","suffix":""},{"id":134718820,"identity":"df34b348-8f74-42a6-ba81-38d37e79708e","order_by":3,"name":"Franklin Zemo Gamo","email":"","orcid":"","institution":"Université de Yaoundé I","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Franklin","middleName":"Zemo","lastName":"Gamo","suffix":""},{"id":134718821,"identity":"ddbcf84f-cc35-4642-9001-ed2e4b30de61","order_by":4,"name":"Florette Motoum Tedjo","email":"","orcid":"","institution":"Université de Yaoundé I","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Florette","middleName":"Motoum","lastName":"Tedjo","suffix":""},{"id":134718822,"identity":"668f23ad-826e-42c2-8414-313e6ca66e2c","order_by":5,"name":"Rudig Nikanor Djikem Tadah","email":"","orcid":"","institution":"Université de Yaoundé I","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rudig","middleName":"Nikanor Djikem","lastName":"Tadah","suffix":""},{"id":134718823,"identity":"9bf305d3-f12c-4925-9c80-a257b777a762","order_by":6,"name":"Dieudonné Njamen","email":"","orcid":"","institution":"Université de Yaoundé I","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dieudonné","middleName":"","lastName":"Njamen","suffix":""}],"badges":[],"createdAt":"2022-09-06 06:59:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2036318/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2036318/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26356654,"identity":"28a409ba-c9e7-493b-a105-78e1542869fc","added_by":"auto","created_at":"2022-09-12 17:59:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66823,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocado oil on the brain (A) and hippocampus (B) relative weight.\u003c/p\u003e\n\u003cp\u003eEach bar represents the mean ± SEM, n = 6. #p\u0026lt; 0.05, ###p\u0026lt; 0.001 compared to SHAM+Veh;p\u0026lt; 0.001; * p\u0026lt; 0.05, ** p \u0026lt;0.01, *** p\u0026lt; 0.001 compared to OVX+D-gal.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/f229b8e39926b369afffdf41.png"},{"id":26356656,"identity":"f401dcdd-ad14-4600-852a-7b24f7e768e4","added_by":"auto","created_at":"2022-09-12 17:59:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73520,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocado oil on the percentage of time spent on a novel object in non-spatial short-term memory (A) and long-term memory (B).\u003c/p\u003e\n\u003cp\u003eEach bar represents the mean ± SEM, n = 6. ## p\u0026lt; 0.01 compare to SHAM+Veh;* p\u0026lt; 0.05, ** p \u0026lt; 0.01, *** p\u0026lt; 0.001 compared to OVX+D-gal.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/37fb39dec929eb5d27a770bf.png"},{"id":26356657,"identity":"27c895ce-9ba6-4541-8af1-147753de14db","added_by":"auto","created_at":"2022-09-12 17:59:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72357,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocado oil on the percentage of time spentin the new arm (A) and percentage of number of entries in the new arm (B).\u003c/p\u003e\n\u003cp\u003eEach bar represents the mean ± SEM, n = 6. ###p\u0026lt; 0.001 compared to SHAM+Veh;** p \u0026lt; 0.01, *** p\u0026lt; 0.001 compared to OVX+D-gal.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/6c28862fac930ba23d6f383f.png"},{"id":26356898,"identity":"eb0a4c58-0448-41e3-b916-ee91c812c2bb","added_by":"auto","created_at":"2022-09-12 18:04:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71725,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocado oil on latency time of entries(A), the number of entries (B) and the percentage of time spent (C) in target quadrant.\u003c/p\u003e\n\u003cp\u003eEach bar represents the mean ± SEM, n = 6. \u0026nbsp;### p\u0026lt; 0.001 compared to SHAM+Veh;*** p\u0026lt; 0.001 compared to OVX+D-gal.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/8f93dd794d88353f47b7a620.png"},{"id":26356659,"identity":"a9ba4200-d7d5-45a2-9ffc-042fbc597068","added_by":"auto","created_at":"2022-09-12 17:59:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88008,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocadooil on Acetylcholine (A), Glutamate (B) and Methylglyoxal(C) levelsin the hippocampus.\u003c/p\u003e\n\u003cp\u003eEach bar represents the mean ± SEM, n = 6. # p\u0026lt; 0.05, ### p\u0026lt; 0.001 compared to SHAM+Veh;** p\u0026lt; 0.01, *** p\u0026lt; 0.001 compared to OVX+D-gal.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/7d7fbe78c98ee1244416861b.png"},{"id":26356660,"identity":"6031eeff-d9c1-41be-a983-e4f5f88dced1","added_by":"auto","created_at":"2022-09-12 17:59:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105781,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocado oil on MDA (A), Nitrite (B) and GSH (C) levels, and Catalase(D) and SOD (E) activitiesin the hippocampus.\u003c/p\u003e\n\u003cp\u003eEach bar represents the mean ± SEM, n = 6. # p\u0026lt; 0.05, ### p\u0026lt; 0.001 compared to SHAM+Veh;* p\u0026lt; 0.05, ** p\u0026lt; 0.01, *** p\u0026lt; 0.001 compared to OVX+D-gal.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/d1359ef5a727932b56addc34.png"},{"id":26356661,"identity":"c1e2c930-4f66-49ff-a86b-f7aa1ab8b944","added_by":"auto","created_at":"2022-09-12 17:59:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":70526,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocado oil on the number of viable neurons in CA1 (A) and CA3 (B) regions of the hippocampus.\u003c/p\u003e\n\u003cp\u003eEach bar represents the mean ± SEM, n = 6. ### p\u0026lt; 0.001 compare to SHAM+Veh;* p\u0026lt; 0.05, ** p\u0026lt; 0.01, *** p\u0026lt; 0.001 compared to OVX+D-gal.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/cc4ce99c0a8c45da1a1f317e.png"},{"id":26356899,"identity":"e16524d4-4150-488f-b351-835b1f4173ad","added_by":"auto","created_at":"2022-09-12 18:04:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":665557,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of extra-virgin avocado oil on microarchitecture of the hippocampus.\u003c/p\u003e\n\u003cp\u003eHematoxylin-eosin staining, ×200. CA1 and 3: Cornu Ammonis 1 and 3, Nn = normal neuron, Nds = neuronal disorganization; Vn: viable neuron; Hn: hyperchromatic nucleus; Nd: neuronal degeneration.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/d7c0eb01849e428d2c4c00f1.png"},{"id":26356900,"identity":"b64c7d38-4e5c-4517-b85e-95b33c8079e2","added_by":"auto","created_at":"2022-09-12 18:04:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2635506,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2036318/v1/7b5753e6-3571-4209-8e00-8cc3568cfd9a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extra-virgin Avocado (Persea americana Mill.,Laucaceae) Oil Improves Cognitive Impairment in D-galactose-induced Alzheimer’s Disease Model on Ovariectomized Wistar Rat","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is the main cause of dementia worldwide with 60\u0026ndash;70% of cases (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). With the increase of the aged population, the global prevalence of dementia is rising sharply. It was estimated about 50\u0026nbsp;million cases of dementia in 2019 and it is expected to increase by 152\u0026nbsp;million in 2050 if nothing is done (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Alzheimer's disease is a chronic and irreversible neurodegenerative dementia that affects daily life through progressive memory loss, cognitive dysfunction and reduced learning abilities (2; 3; 4). Women are 2 to 3 times more likely to develop AD than men (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). This difference in the incidence of AD by sex has been attributed to the increase in life expectancy which is considerably higher in women than in men (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), as well as biological and hormonal differences, which results in a possible neuroprotective effects of estrogen that falls at menopause in women (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn experiment animals, ovariectomy has been used by several authors to reflect postmenopausal status (8;9;10). In addition, ovariectomy has been shown to lead to a gradual decline in cognitive function and locomotor activity in rats (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Several studies have shown that administration of D-galactose in rodents mimics the natural aging process by inducing neurodegeneration similar to that observed in AD (11; 12; 13; 14). In addition, it has been reported that ovariectomy associated to chronic administration of D-galactose act synergistically to accelerate the pathophysiology of Alzheimer's disease (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn modern medicine, the molecules used to manage Alzheimer's disease, act essentially in alleviating the symptoms and do not prevent the progression of the disease. Moreover, these molecules have many side effects and have a high cost (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Hence the search for an alternative treatment is increasing. In recent years, dietary approaches to prevent AD with natural ingredients of plants origin (such as the \"Mediterranean diet\u0026raquo;) attract great attention. The \"Mediterranean diet\" is rich in monounsaturated fatty acids and many reports revealed that such diet is beneficial for the prevention of neurodegenerative diseases during aging, and the improvement of cognitive performances (17; 18; 19).\u003c/p\u003e \u003cp\u003eThe avocado is a tree\u0026rsquo;s fruit (\u003cem\u003ePersea americana\u003c/em\u003eMill.) of the Laucaceae family. Its cultivation is widespread in all tropical regions of the world. More than 200 varieties are known. The pulp of this fruit has high oil content (5\u0026ndash;30% depending on the variety) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Its consumption is increasing significantly worldwide and it provides a lipid profile almost identical to olive oil (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Cheng \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) reported that American seniors who consumed avocado had improved cognitive performance than non-consumers. Similarly, some authors have shown that consuming an avocado fruit for 12 weeks or 6 months improves cognitive performance in the elderly (23; 24). In the literature, few studies have been published on the properties of avocado oil targeting cognitive functions (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). These studies are limited on reflex maturation, postnatal somatic development and memory acquisition in newborn rats, during the adolescent phase and in adults (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) as well as on mitochondrial brain function in diabetic rats (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, no study has yet been conducted on the properties of avocado oil on Alzheimer's disease occurrence. The aim of the present study was therefore to evaluate the effects of extra-virgin avocado oil on a model of D-galactose-induced Alzheimer's disease in ovariectomized Wistar rats.\u003c/p\u003e"},{"header":"2. Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Material\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Plant material\u003c/h2\u003e \u003cp\u003eDifferent parts of \u003cem\u003ePersea americana\u003c/em\u003e (stem bark, leaves, flowers and fruits) were collected in the orchard of the Institute of Agricultural Research for Development (IRAD) of Foumbot (Department of Noun, West Region, Cameroun). These botanical samples were identified at the National Herbarium of Cameroon (HNC) by comparison to the existing specimens under the voucher number 57756 HNC. The avocado fruits, Fuerte variety, were provided by Mrs. KEMAYOU MBIEJI Flavie Christelle, researcher at IRAD. After harvesting they were immediately cleaned and stored in a cool and dried place until they ripened.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Experimental animals\u003c/h2\u003e \u003cp\u003eIn this study, animals used were adult female Wistar rats,12\u0026ndash;14 weeks old, weighing between 150 and 200 g. Animals were housed in the animal facility of the Laboratory of Animal Physiology, University of Yaound\u0026eacute; 1 (temperature, room temperature; humidity 50\u0026ndash;80%; 12 hlight\u0026ndash;dark cycle). They had free access to a standard soy-free rat diet (SSniff GmbH, Soest, Germany) and were provided tap water ad libitum. The research was conducted in accordance with the guidelines of the institutional Ethic Committee of Cameroon\u0026rsquo;s Ministry of Scientific Research and Technological Innovation, which has adopted the guidelines established by the European Union on Animal Care (CEE Council 86/609; Reg.no.FWA-IRD0001954).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Chemicals\u003c/h2\u003e \u003cp\u003eDonepezil (donepezil hydrochloride 10 mg, ARROW Generics, 69007 LYON, France) and Estradiol valerate (Progynova\u0026reg; 2mg, Delpharm laboratory, France) were used as reference drugs. D-galactose (BDH chemical Ltd pools, Great Britain) was used to induce neurodegeneration in rodents similar to that observed in Alzheimer's disease.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Methods\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Oil extraction and determination of doses\u003c/h2\u003e \u003cp\u003eAfter washing, the avocado fruits were ripped, and the pulp withdrawn and manually mixed in the laboratory mortar. The mixture was dried at 60 \u0026deg; C (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) for 5 hours in an electric oven brand (PHILIPS) to remove traces of water. The dried pulp was pressed mechanically using a manual press and the resulting oil was filtered before being stored in the dark vials at -20\u0026deg;C. With 200 g of avocado fruit (Fuerte variety), our extraction process led to10 mL of extra-virgin avocado oil. The doses of administration were prepared based on the report of general consumption of avocado fruit (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The equivalent doses in each rat were extrapolated from the human dose (0.14 mL/kg BW) to afford 0.89 mL/kg BW (~\u0026thinsp;1 mL/kg BW). To obtain a dose response curve of the extra-virgin avocado oil,2 other doses (0.25, 0.5 mL/kg BW) were generated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Behavioral Assessments\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eObject Recognition test\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe \"Object Recognition\" (OR) test is a suitable test for the evaluation of hippocampo-dependent memory processes in rodents (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The principle of this test is based on the natural affinity of rodents with the new object (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In this study the OR test was carried out in an Open Field (OF) device, using 4 objects: 2 objects (A1 and A2) identical to each other, and two objects (B and C) different from each other (shape, color and texture) and different from the first two. The OR protocol used was adapted from that described by Djiogue \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The test was performed on days 59 to 61 of the experiment. During this test, the following parameters were evaluated: the time spent exploring identical objects during the familiarization phase, the time spent exploring one of the old and new objects 3 hours after the familiarization stage (A and B) and the time spent exploring one of the old and new objects 24 hours after the familiarization stage (A and C) during the test phases. The exploration time was recorded when the rats touched the object, or approached within 2 cm of the object in question (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Parameters were recorded via a video recording system using a camera placed above the pool and connected to a computer. To avoid animal\u0026rsquo;s perturbation due to urine and faeces, between two tests, the device was cleaned with 70% ethanol solution and dry cloth. The data were reported on parameter sheets provided for this purpose. The data obtained were expressed in proportion to the time the animal spent on the new object (IR recognition index).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eY-Maze test\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe Y-Maze test is a recognition memory test used to assess short-term spatial working memory. This test is based on the animal's natural tendency to explore novelty. The Y-labyrinth test was carried out in a wooden device with three identical arms (11 x 50 x 32 cm each) separated by an angle of 120̊ placed in a room with the visual cues on the blackberries. The protocol used was adapted to the one described by Fernanda \u003cem\u003eet al.\u003c/em\u003e (31). The test was performed on day 63 of the experiment. During this test, the number of entries and the time spent in each arm were recorded via a video recording system using a camera placed above the pool and connected to a computer. The percentage of the number of entries into the new arm was determined in relation to the number of entries into the familiar arms; and the percentage of time spent in the new arm was determined in relation to the total time spent in the device (sum of time in two familiar arms, and new arm as well as central area). To avoid animal\u0026rsquo;s perturbation due to urine and faeces, the device was cleaned with 70% ethanol solution and dry cloth between two tests.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eMorris Water Maze test\u003c/span\u003e \u003c/p\u003e \u003cp\u003eThe Morris water Maze (MWM) test allows the accurate and reproducible measurement of spatial memory and is a very sensitive tool for assessing damage to the hippocampus (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In the present study, the MWM test was conducted in a black circular tank (diameter 120 cm \u0026times; height 50 cm) half-filled with water located in a lighted room. An 8-cm diameter black drainage platform (color-matched to the device to make it invisible) was placed in a fixed position (south quadrant of the device), submerged 1.0 cm under the water surface. The protocol for this test was adapted from Mandeep and Yash (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The test was performed on days 64 to 68 of the experiment. The test consisted of an acquisition phase of 4 days and a retention phase on day 5. Acquisition phase: three trials were conducted per day, with a break time of 15 minutes between trials. At the end of each test the animals were cleaned properly and returned to their original cages. The principle of MWM was that when the rats escaped from the water by climbing on the platform, they learned the spatial location of the platform from any starting position in the pool. Retention phase: during this phase, to evaluate the spatial memorization, the platform was removed, each rat was released into water in one of the fixed target facing the target quadrant and had 60 seconds of swimming. The latency time to reach the exact position of the platform, the time spent in the target quadrant and the number of entries in the target quadrant of the platform were recorded via a video recording system using a camera placed above the pool and connected to a computer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Experimental design\u003c/h2\u003e \u003cp\u003eTo accomplish this, 54 female rats were used. 42 of them were suggested to the bilateral ovariectomy (OVX) using the dorsal approach (34; 10). The 12others were used as SHAM. Fourteen days after surgery, the animals were divided into 9 groups of 6 animals each:\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroup 1\u003c/b\u003e(SHAM\u0026thinsp;+\u0026thinsp;Veh): SHAM rats were given distilled water (Veh, vehicle) \u003cem\u003eper os\u003c/em\u003e;\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroup 2\u003c/b\u003e(OVX\u0026thinsp;+\u0026thinsp;Veh): OVX rats were given distilled water \u003cem\u003eper os\u003c/em\u003e;\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroup 3\u003c/b\u003e(SHAM\u0026thinsp;+\u0026thinsp;D-gal): SHAM rats were given D-galactose (D-gal, 150 mg/kg, i.p.) and distilled water \u003cem\u003eper os\u003c/em\u003e simultaneously;\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroup 4\u003c/b\u003e(OVX\u0026thinsp;+\u0026thinsp;D-gal): OVX rats were given D-galactose (150 mg/kg, i.p.) and distilled water \u003cem\u003eper os\u003c/em\u003e simultaneously;\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroup 5\u003c/b\u003e(OVX\u0026thinsp;+\u0026thinsp;D-gal\u0026thinsp;+\u0026thinsp;E2V): OVX rats were given D-galactose (150 mg/kg, i.p.) and estradiol valerate (E2V, 1 mg/kg, p.o.) simultaneously;\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroup 6\u003c/b\u003e(OVX\u0026thinsp;+\u0026thinsp;D-gal\u0026thinsp;+\u0026thinsp;DNPZ): OVX rats were given D-galactose (150 mg/kg, i.p.) and donepezil (DNPZ, 1mg/kg, p.o) simultaneously;\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroups 7, 8 and 9\u003c/b\u003e (OVX\u0026thinsp;+\u0026thinsp;D-gal\u0026thinsp;+\u0026thinsp;AO1; OVX\u0026thinsp;+\u0026thinsp;D-gal\u0026thinsp;+\u0026thinsp;AO2 and OVX\u0026thinsp;+\u0026thinsp;D-gal\u0026thinsp;+\u0026thinsp;AO3 respectively, Test groups): these OVX rats were given D-galactose each (150 mg/kg, i.p.) and the extra-virgin avocado oil \u003cem\u003eper os\u003c/em\u003e at the dose of 0.25, 0.5 and 1 mL/kg respectively.\u003c/p\u003e \u003cp\u003eThe treatment was carried out for 70 days during which the memory disorders were evaluated using the Object Recognition, Y-Maze and MWM tests (as shows on the diagram). At the end of the experiment, the animals were euthanized and brain isolate was immediately weighed. the hippocampus was isolated in the right hemisphere of each animal on a block of NaCl ice, weighed and used to prepare homogenate 10% on Tris buffer solution (HCl 50 mM; KCl 150 mM; pH 7.4). The homogenate was used for biochemical analysisas well as evaluation of some oxidative stress biomarkers. The left hemisphere of each animal was fixed in formaldehyde 10% for histological analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. BiochemicalAssay\u003c/h2\u003e \u003cp\u003eSome markers of memory function (Acetylcholine, Glutamate) and Methylglyoxal was measured in 10% of hippocampus homogenate using commercial Kits. The Acetylcholine content was measured using the quantitative ELISA analysis technique according to the instructions described on BioVision's Acetylcholine ELISA Kit. The Glutamate content was measured by the colorimetric method according to the manufacturer instructions described on the BioVision Glutamate Assay Kit.The determination of Methylglyoxal (MG) was performed by the fluorometric method according to the instructions described on the BioVision Methylglyoxal (fluorometric) assay kit. All kits were provided by BioVion Incorporated, CA 95035 USA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Oxidative stress biomarkers evaluation\u003c/h2\u003e \u003cp\u003eHippocampal levels of malondialdehyde (MDA), nitrites(NO2\u003csup\u003e\u0026minus;\u003c/sup\u003e.), catalase activity (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) were determined using methods described respectively by Wilbur \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), Fermor \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), Sinha (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), Misra and Fridovish (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and Ellman (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6. Histological analysis\u003c/h2\u003e \u003cp\u003eSections of 5-\u0026micro;m thickness were obtained from brain tissues embedded in paraffin wax using a standard microtome. Brain tissues section were processed for Haematoxylin and Eosin staining based on the method described by Smith and Bruton (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The mounted slides were allowed to dry and prepared ready for microscopy. Slides observation was done under the Scientico STM-50 optical microscope equipped with a Celestron 44421 brand digital camera connected to a computer for microphotographs. The J image software (version 1.48 for Window) was used for quantification of viable neurons in the \u003cem\u003eCornu Ammonis\u003c/em\u003e 1 (CA1) and 3 (CA3) of the hippocampus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and analysed with one-way ANOVA followed by the Dunnett post-test (GraphPad Prism \u0026reg; Software, version 8.0.1.244, San Diego, CA, USA). A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.1. Results\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.1.1. Effects of extra-virgin avocado oil on brain and hippocampus relative weight\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the effects of extra-virgin avocado oil on brain and hippocampus relative weight after 70 days of D-galactose treatment on ovariectomized rats. Compared to SHAM\u0026thinsp;+\u0026thinsp;Veh group, the association of ovariectomy and D-galactose (OVX\u0026thinsp;+\u0026thinsp;D-gal) induced a non-significant decrease of brain relative weight (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). all the treated ovariectomized rats, receiving D-galactose\u0026thinsp;+\u0026thinsp;estradiol valerate (1 mg/kg), D-galactose\u0026thinsp;+\u0026thinsp;donepezil (1 mg/kg), D-galactose\u0026thinsp;+\u0026thinsp;extra-virgin avocado oil (0.25, 0.5 and 1 mL/Kg),showed non-significant increase of relative brain weight compare to OVX\u0026thinsp;+\u0026thinsp;D-gal group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eThe analysis of Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB shows that the association of ovariectomy and D-galactose (OVX\u0026thinsp;+\u0026thinsp;D-gal) induced a significant ( p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease of hippocampus relative weight in comparison to SHAM\u0026thinsp;+\u0026thinsp;Veh group. Compared to OVX\u0026thinsp;+\u0026thinsp;D-gal group, the treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at all tested doses induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of relative hippocampus weight, as well as estradiol valerate (1 mg/kg) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec18\"\u003e\n \u003ch2\u003e3.1.2. Effects of extra-virgin avocado oil on non-spatial memory evaluated by the Object Recognition test\u003c/h2\u003e\n \u003cp\u003eThe evaluation of short-term memory by Object Recognition (OR) test shows that compared to SHAM\u0026thinsp;+\u0026thinsp;Veh, the association of ovariectomy and D-galactose (OVX\u0026thinsp;+\u0026thinsp;D-gal) induced a non-significant variation of the percentage of time spent on the novel object (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The treatment of ovariectomized ratcombined to D-galactose exposure with extra-virgin avocado oil and the group treated with donepezil (1 mg/kg)\u0026thinsp;+\u0026thinsp;D-galactose induced an increase of this parameter. This effect was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at the dose 0.25 ml/kg of extra-virgin avocado oil and donepezil (1 mg/kg) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eAs shown on Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, the evaluation of long-term memory by OR test shows that the association of ovariectomy and D-galactose (OVX\u0026thinsp;+\u0026thinsp;D-gal) induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) decrease of the percentage of time spent on the novel object when compared to SHAM\u0026thinsp;+\u0026thinsp;Veh (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at all tested doses as well as E2V (1 mg/kg) induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of the percentage of time spent on the novel object(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) in comparison to OVX\u0026thinsp;+\u0026thinsp;D-gal group.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec19\"\u003e\n \u003ch2\u003e3.1.3. Effects of extra-virgin avocado oil on short-term spatial memory evaluated by the Y- Maze test\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e represents the effects of avocado extra-virgin oil on the percentage of time spent and the number of entries in the new arm of the Y- Maze. Analysis of the results shows that the association of ovariectomy and D-galactose (OVX\u0026thinsp;+\u0026thinsp;D-gal) significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease the time spent (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA) and the number of entries (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB) in the new arm compared to the non-ovariectomized group treated with vehicle (SHAM\u0026thinsp;+\u0026thinsp;veh). The treatment of ovariectomized animal combined to D-galactose exposure with avocado extra-virgin oil as well as the reference drug induced an increase of these parameters in comparison to OVX\u0026thinsp;+\u0026thinsp;D-gal group. The increase induced by avocado extra-virgin oil on the time spent in the new arm was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at the doses of 0.5 and 1 mL/kg (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). All tested doses as well as reference drugs induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of the number of entries in the new arm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n \u003ch2\u003e\u003cstrong\u003e3.1.4. Effects of extra-virgin avocado oil on long-term spatial memory evaluated by the Morris water Maze test\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eAnalysis of the results presented on Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA shows a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of the latency time of entries in target quadrant in ovariectomized animals combined to D-galactose exposure (OVX\u0026thinsp;+\u0026thinsp;D-gal) compared with non-ovariectomized animals receiving vehicle (SHAM\u0026thinsp;+\u0026thinsp;veh). When compared to OVX\u0026thinsp;+\u0026thinsp;D-gal group, extra-virgin avocado oil at all tested doses as well as E2V and DNPZ, induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease of the latency time of entries in target quadrant (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eResults presented in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB and C shows that compared to SHAM\u0026thinsp;+\u0026thinsp;veh group, the OVX\u0026thinsp;+\u0026thinsp;D-gal group shows a decrease in the number of entries (B) and the percentage of time spent (C) in the target quadrant. These effect of D-galactose in ovariectomized animals was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) regarding the percentage of time spent (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The treatment of ovariectomized animals combined to D-galactose exposure with extra-virgin avocado oil as well as E2V and DNPZ, induced an increase of the number of entries (B) and the percentage of time spent (C) in the target quadrant in comparison to OVX\u0026thinsp;+\u0026thinsp;D-gal group. The effect of extra-virgin avocado oil was significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at all tested doses on the percentage of time spent (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec20\"\u003e\n \u003ch2\u003e3.1.5. Effects of extra-virgin avocado oil on some markers of cognitive function\u003c/h2\u003e\n \u003cp\u003eAs shows in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and B, the association of ovariectomy and D-galactose induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease of Acetylcholine (A) and Glutamate (B) levels in the hippocampus, when compared to SHAM\u0026thinsp;+\u0026thinsp;veh group. The treatment of ovariectomized animals combined to D-galactose exposure with extra-virgin avocado oil (at all tested doses) as well as E2V and DNPZ, induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of these parameters when compared to OVX\u0026thinsp;+\u0026thinsp;D-gal group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and B).\u003c/p\u003e\n \u003cp\u003eAnalysis of the results presented on Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC shows a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of Methylglyoxal level in the hippocampus of ovariectomized animals combined to D-galactose exposure (OVX\u0026thinsp;+\u0026thinsp;D-gal) compared to non-ovariectomized animals receiving vehicle (SHAM\u0026thinsp;+\u0026thinsp;veh). When compared to OVX\u0026thinsp;+\u0026thinsp;D-gal group, the groups treated with extra-virgin avocado oil at all tested doses as well as E2V, induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease of Methylglyoxal level (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec21\"\u003e\n \u003ch2\u003e3.1.6. Effects of extra-virgin avocado oil on some markers of oxidative stress\u003c/h2\u003e\n \u003cp\u003eThe evaluation of oxidative stress markers shows that compared to SHAM\u0026thinsp;+\u0026thinsp;Veh group, the association of ovariectomy and D-galactose (OVX\u0026thinsp;+\u0026thinsp;D-gal) induce a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of MDA and Nitrite level (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;.\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003ein the hippocampus in comparison to SHAM\u0026thinsp;+\u0026thinsp;Veh (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA and B respectively). The treatment of ovariectomized rat combined to D-galactose exposure with extra-virgin avocado oil as well as E2V and DNPZ induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease of these parameters when compared to OVX\u0026thinsp;+\u0026thinsp;D-gal group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA and B).\u003c/p\u003e\n \u003cp\u003eThe evaluation of oxidative stress markers also shows that the treatment of ovariectomized (OVX\u0026thinsp;+\u0026thinsp;D-gal) rats with D-galactose induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease of GSH level, Catalase and SOD activitiesin the hippocampus in comparison to SHAM\u0026thinsp;+\u0026thinsp;Veh (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D and E respectively). The treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at all tested doses as well as E2V and DNPZ induced a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase of GSH level, Catalase and SOD activitiesin the hippocampus in comparison to OVX\u0026thinsp;+\u0026thinsp;D-gal group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D and E respectively).\u003c/p\u003e\n \u003ch2\u003e\u003cstrong\u003e3.1.7. Effects of extra-virgin avocado oil on microarchitecture and the number of viable neurons in CA1 and CA3 regions of the hippocampus\u003c/strong\u003e\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e shows the effects of avocado extra-virgin oil on the number of viable neurons in CA1 and CA3 regions and hippocampus microarchitectures respectively. The analysis of the results presented indicates an intact architecture of the different layers of the hippocampus with intact neurons, the nucleus large, round or oval, and the nucleoli clearly observed, indicating the presence of viable neurons in the normal control group (SHAM\u0026thinsp;+\u0026thinsp;veh) (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). In contrast, neuronal disorganization (Nds) with larger intercellular spaces, neuronal degeneration (Nd) revealed by the presence of neurons with hyperchromatic nuclei (Hn) and shrunken cytoplasm (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) and a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease in the number of viable neurons in the CA1 and CA3 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) were observed in animals of the ovariectomized group which received D-gal (OVX\u0026thinsp;+\u0026thinsp;D-gal) when compared to SHAM\u0026thinsp;+\u0026thinsp;veh group.\u003c/p\u003e\n \u003cp\u003ethe treatment of ovariectomized rats combined to D-galactose exposure with extra-virgin avocado oil at doses of 0.25, 0.5 and 1ml/kg PC, as well as donepezil and estradiol valerate, prevented the neurodegeneration induced by ovariectomy and D-galactose (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). The treatment induced a pronounced decrease in the number of neurons with hyperchromatic and shrunken nuclei (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) and a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05; p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; p\u0026thinsp;\u0026lt;\u0026thinsp;0,001) increase in the number of viable neurons (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) compared to ovariectomized animals that received D-gal injection (OVX\u0026thinsp;+\u0026thinsp;D-gal).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec22\"\u003e\n \u003ch2\u003e3.2. DISCUSSION\u003c/h2\u003e\n \u003cp\u003eNeurogenesis Decrease in the hippocampus is indicative of age-related memory impairment in rats and humans (\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e). It has been shown that ovariectomy and chronic D-galactose exposure act synergistically to accelerate the pathophysiology of Alzheimer\u0026apos;s Disease (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). The objective of the present study was to evaluate the effects of extra-virgin avocado oil on a D-galactose-induced model of Alzheimer\u0026apos; s Disease (AD) in ovariectomized Wistar rats. D-galactose is a sugar capable of causing cognitive impairment by inducing accelerated aging through oxidative stress and inflammation (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e). Knowing that the hippocampus is a cortical structure that deals with the process of memorization, formation, storage of new memories and the connection of some information with these memories (\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e), it has been reported that hippocampal atrophy is strongly correlated with dementia and can be considered as a predictor of AD (\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e). In this study, it was found that compared to SHAM\u0026thinsp;+\u0026thinsp;Veh group, the administration of D-galactose to ovariectomized (OVX\u0026thinsp;+\u0026thinsp;D-gal) rats induced a non-significant decrease of brain relative weight and a significant decrease in the hippocampus relative weight. This would be attributed to a decrease in neurogenesis in the hippocampus (due to the lack of estrogen) and the deleterious effects of D-galactose. In fact, estrogens act as a regulator of cellular processes in the hippocampus; they promote neurogenesis (\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e) and D-galactose resulted in a decrease in hippocampal relative weight (\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e). Oral administration of extra-virgin avocado oil (at doses of 0.25, 0.5 and 1 ml/kg) significantly increased hippocampus relative weight of ovariectomized animals combined to D-galactose exposure. These effects of extra-virgin avocado oil could be attributed to phytonutrients found in avocado such as carotenoids (lutein and zeaxanthin) (\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e) which are able to improve cognitive function \u003cspan class=\"Underline\" name=\"Emphasis\" type=\"Underline\"\u003e(\u003c/span\u003e47; 24).\u003c/p\u003e\n \u003cp\u003eIn this study, memory function was evaluated by a series of behavioral tests. As results compared to SHAM\u0026thinsp;+\u0026thinsp;Veh group, OVX group received D-galactose (OVX\u0026thinsp;+\u0026thinsp;D-gal) showed an impairment of non-spatial memory (evaluated by OR test) as well as spatial memory (evaluated by Y-maze and MWM tests respectively). This memory impairment was revealed in the OR test by a decrease in the time spent on the novel object (recognition index) in the short and long term. However, the treatment with extra-virgin avocado oil as well as reference drugs significantly increased the time spent on the new object in the short- and long-term suggesting an improvement of the non-spatial memory. Indeed, rodents are naturally curious and in the presence of a novel and old object, they remember the old object and therefore spend more time exploring the novel object (\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e). In the Y-maze test, animals from OVX\u0026thinsp;+\u0026thinsp;D-gal group presented a significant decrease in the time spent and the number of entries in the new arm of Y-maze tasks in comparison to SHAM\u0026thinsp;+\u0026thinsp;veh group. Fernanda \u003cem\u003eet al.\u003c/em\u003e (\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e) reported that in the Y-maze tasks, alterations in short-term spatial memory were noticed by a decrease in the time spent and number of entries in the new arm. However, as well as to estradiol valerate and donepezil, the treatment with avocado extra-virgin oil at all tested doses significantly increased these parameters, suggesting an improvement in short-term spatial memory. In the MWM test used to assess long-term spatial memory, alterations in memory were confirmed by a significant increase of the latency time of entries in the target quadrant and a significant decrease in the time spent in the same quadrant. The treatment of animals with avocado extra-virgin oil (at the tested doses) as well as the reference drugs significantly reduced the latency time of entries in the target quadrant and significantly increased the time spent in the target quadrant.\u003c/p\u003e\n \u003cp\u003eIt is well known that estrogen deprivation following ovariectomy impairs learning and memory capacity in female rats (\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e) and ovariectomy is associated with progressive loss of non-spatial and spatial memory (\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e). The learning and memory deficits observed in ovariectomized animals combined to D-galactose exposure corroborate those of Weam \u003cem\u003eet al\u003c/em\u003e. (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e) who showed that estrogen depletion and intraperitoneal administration of D-galactose act synergistically to accelerate the pathophysiology of Alzheimer\u0026apos;s disease. The alteration of non-spatial and spatial memory (short and long term) induced by ovariectomy combined to D-galactose administration were improved by the treatment with extra-virgin avocado oil (0.25, 0.5 and 1 ml/kg). This suggests that avocado oil possesses neuroprotective properties although these effects seem to be specific for each type of memory. These observations are in accordance with statements that avocado may be beneficial for cognition throughout the life span, potentially influencing different cognitive domains at different stages of life (\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e). The effects of avocado on working memory have been correlated to its richness in carotenoids specifically lutein (22; 50).\u003c/p\u003e\n \u003cp\u003eIn AD, dysfunction of Acetylcholine and glutamate is strongly correlated with cognitive decline (\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e). There is strong evidence that a decrease in brain glutamate and acetylcholine levels correlates with the severity of Alzheimer\u0026apos;s disease (52; 53). In this study, D-galactose administration combined with ovariectomy (OVX\u0026thinsp;+\u0026thinsp;D-gal) resulted in a significant decrease in acetylcholine and glutamate levels in these animals in comparison to SHAM\u0026thinsp;+\u0026thinsp;veh group. This suggests that estrogen depletion and D-gal ingestion act synergistically to accelerate cholinergic and glutaminergic neurodegeneration, thereby disrupting the metabolism of these neurotransmitters. Indeed, it is known that chronic administration of D-galactose leads to oxidative stress and neuroinflammation resulting in losses of cholinergic and glutaminergic neurons (\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e). Compared to OVX\u0026thinsp;+\u0026thinsp;D-gal group, the treatment with extra-virgin avocado oil at all tested doses induced an increase of these parameters. These results indicate that extra-virgin avocado oil could protect cholinergic and glutaminergic neurons against alterations induced by estrogen deficiency combined with D-gal injection. This could explain the improved cognitive performance observed in animals treated with extra-virgin avocado oil in the OR, Y-maze and MWM tests. The neuroprotective effects of extra-virgin avocado oil were similar to those of estradiol valerate and donepezil (an acetylcholine esterase inhibitor), suggesting that the compounds present in the extra-virgin oil could interfere with acetylcholine metabolism, probably by inhibiting of acetylcholine esterase activity or acting as phytoestrogens (mechanisms to be explored) by binding to estrogens located in dendritic spines, dendrites, axons and nuclei of hippocampal pyramidal neurons (\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe D-galactose administration is known to induce brain senescence through Methylglyoxal (MG) accumulation (\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e). Methylglyoxal is a highly reactive carbonyl compound that propagates glycation reactions and thus capable of generating advance glycation end products (AGEs) (\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e). It has been reported that AGEs accumulation on hippocampus cause dysfunction and death of neurons, thus contributing to the pathophysiology of AD by promoting amyloid-beta accumulation and hyperphosphorylation of \u003cem\u003etau\u003c/em\u003e protein (57; 58). The results obtained in this study shows that compared to the SHAM group, OVX\u0026thinsp;+\u0026thinsp;D-gal group presented a significant increase in the level of MG in the hippocampus. As describe by Li \u003cem\u003eet al\u003c/em\u003e. (\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e), administration of D-gal in rodents for 10 weeks strongly increased MG levels. This could justify the memory and learning deficits observed in the OR, Y-maze and MWM tests (\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e). Compared to OVX\u0026thinsp;+\u0026thinsp;D-gal group, the administration of extra-virgin avocado oil (0.25, 0.5 and 1 ml/kg) to ovariectomized animals receiving D-galactose induced a significant decrease of MG levels in the hippocampus. These effects could be attributed to the presence of secondary metabolites in this fruit that could interact with MG metabolism and reduce his accumulation in the hippocampus.\u003c/p\u003e\n \u003cp\u003eOxidative stress is known to play a crucial role in the pathophysiology of AD (\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e), which includes the activation of NADPH oxidase in astrocytes (\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e), mitochondrial dysfunction (\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e), increased amyloid-beta neurotoxicity (\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e), and synaptic dysfunction responsible for neuronal apoptosis (\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e). In the present study, compared to non-ovariectomized animals that received the vehicle, ovariectomy combined with D-galactose administration resulted in a significant increase of MDA and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;.\u003c/sup\u003e levels; and a significant decrease of GSH level, CAT and SOD activities in the hippocampus. This suggests that ovariectomy and D-galactose act synergistically to accelerate oxidative damage. It was reported that in the hippocampus, D-gal inhibits the activity of triosephosphate isomerase (TPI) and glyoxalase1 (GLO1) leading to the accumulation of MG which reacts with long-lived proteins to cause irreversible cross-linking of proteins into AGEs (\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e). Advanced glycation end products, by binding to their RAGE receptors, activate intracellular inflammatory signalling pathways and produce reactive oxygen species (ROS) while decreasing antioxidant enzymes activity (\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e). Furthermore, MDA is an important marker of lipid peroxidation (\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e), NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;.\u003c/sup\u003e is considered to mediate amyloid beta-induced cerebral vascular dysfunction (\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e); high concentrations of MDA and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003einduce cell death (\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e). The treatment of ovariectomized animals receiving D-galactose with extra virgin avocado oil (0.25, 0.5 and 1 ml/kg) induced a significant decrease of the levels of MDA and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;.\u003c/sup\u003e levels as well as a significant increase of the levels of GSH level, SOD and CAT activities. As antioxidant enzymes, SOD is the first line of cellular defence against oxidative damage (\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e) and it converts ROS to hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e); Glutathione (GSH) destroys the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e produced as well as toxic organic peroxides (\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e) and Catalase (CAT) converts hydrogen peroxide into water and molecular oxygen and prevents the formation of the highly reactive and dangerous hydroxyl radical (\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e). These antioxidative effects of extra virgin avocado oil could be attributed to some active metabolites present in the avocado such as phenolics, flavonoids and carotenoids capable of scavenging free radical and increasing the activity of endogenous antioxidant enzymes (\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e). Avocado is rich in vitamin such as vitamins A, B, C, E, K1 and betaine (Dreher and Davenport, 2013). It is well known that vitamin C (ascorbate) and vitamin E (or \u0026alpha;-tocopherol) are considered the main antioxidant molecules (provided by the diet) used by cells (\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e). In addition, Gao \u003cem\u003eet al\u003c/em\u003e. (\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e) reported that DHA present in avocado is able to protect the hippocampus from oxidative stress and neuronal apoptosis (\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe analysis of brain microarchitecture shows that compared to SHAM\u0026thinsp;+\u0026thinsp;veh group, neuronal disorganization with larger intercellular spaces; neuronal degeneration revealed by the presence of neurons with hyperchromatic nuclei and shrunken cytoplasm; and a significant decrease in the number of viable neurons in the CA1 and CA3 region were observed in animals of the ovariectomized group that received D-galactose only.\u003c/p\u003e\n \u003cp\u003eIt is well known that estrogen depletion is associated with neurodegeneration which induced a reduction in the density of dendritic spines of pyramidal neurons in the CA1 region of the hippocampus (73; 9). In addition, D-galactose has the ability to induce a decrease of the growth and reproduction of nerve cells, damage of hippocampal neurons an attenuate the expression of neurotrophic growth factors by stimulating of oxidative stress in the brain (\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e). The result obtained in this study supported the hypothesis that estrogen depletion combined with D-galactose act synergistically to accelerate the pathophysiology of AD (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). In contrast, the treatment with extra-virgin avocado oil at all tested doses significantly attenuated neurodegenerative changes, as shown by a pronounced decrease in the number of neurons with hyperchromatic and shrunken nuclei and a significant increase in the number of viable neurons compared to animals in the ovariectomized group that received D-galactose. These neuroprotective effects of extra-virgin avocado oil could be due to the presence of active metabolic compounds such as phenolics, flavonoids, and carotenoids present in this fruit (\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e) that would protect neurons through their antioxidant capacity or by interacting with various signalling pathways that regulate neuron tracking and differentiation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe aim of the present study was to evaluate the effects of extra-virgin avocado oil on a model of D-galactose-induced Alzheimer's disease in ovariectomized Wistar rats. Extra-virgin avocado oil treatment of ovariectomized rats receiving D-galactose resulted in the improvement of both non-spatial and spatial memory as well as anti-oxidative activities. The Extra-virgin avocado oil also prevented the neurodegeneration. These observed effects suggested that this fruit possesses neuroprotective properties and could justify its traditional use. The avocado oil can be consumed or supplemented to prevent the onset of Alzheimer's disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD: Alzheimer\u0026apos;s disease; D-gal: D-galactose; Ach: Acethylcholine; Glu: Glutamate; MG: Methylglyoxal; NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003cstrong\u003e.\u003c/strong\u003e\u003c/sup\u003e: Nitrites; MDA: Malondialdehyde; SOD: superoxide dismutase, CAT: catalase; GSH: Glutathione reduct; ANOVA: Analysis of variance; i.p.: Intraperitoneally; p.o: \u003cem\u003ePer os\u003c/em\u003e; SEM: Standard error of the mean; AO: avocado oil;OVX: ovariectomized; SHAM: sham operated; E2V: Oestradiol valerate; OR: Object Recognition; OF:Open Field; MWM: Morris water Maze.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Ethics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were carried out in accordance with the Cameroon National Ethic Committee for animal experiments, which adopted all procedures recommended by the European Union on the protection of animals used for scientific purposes (CEE Council 86/ 609; Reg. no. FWA-IRD 0001954).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Consent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Availability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial and non-financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe received no funding support for the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Authors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSD and DN conceived the study, supervised the data collection and analysis as well as the manuscript preparation and proofreading. CMW, CFA, FMT, RNDT performed the experiments and data collection, participated to the first draft manuscript writing. FZG participated to the manuscript writing and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Ms. KEMAYOU MBIEJI Flavie Christelle, researcher at the Institute of Agricultural Research for Development (IRAD) of Foumbot (Department of Noun, West Region of Cameroon), for her technical assistance during the identification and harvesting of the fruits of the avocado of the Fuerte variety.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eAlzheimer\u0026rsquo;s Disease International (ADI). (2019).\u003c/strong\u003e Alzheimer\u0026apos;s World Report 2019 : Attitudes towards dementia. 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(2006).\u003c/strong\u003e Ovariectomized rats show decreased recognition memory and spine density in the hippocampus and prefrontal cortex. \u003cem\u003eBrain Research\u003c/em\u003e. 1126: 176\u0026ndash;182.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHua X, Lei M, Zhang Y, Ding J\u003c/strong\u003e\u003cstrong\u003e, Han Q, Hu G and Xiao M. (2007).\u003c/strong\u003e Long-term D-galactose injection combined with ovariectomy serves as a new rodent model for Alzheimer\u0026rsquo;s disease. \u003cem\u003eLife Sciences\u003c/em\u003e. 80 (20): 1897\u0026ndash;1905.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Avocado oil, Persea americana, memory, ovariectomy, D-galactose, Alzheimer's disease","lastPublishedDoi":"10.21203/rs.3.rs-2036318/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2036318/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAvocado is a tree’s fruit (\u003cem\u003ePersea Americana\u003c/em\u003eMill.) of the Laucaceae family. It was reported that consumption of avocado improved cognitive performance. No study has yet been carried out regarding the properties of avocado oil supplementation on the occurrence of Alzheimer's disease. The objective of the present study was to evaluate the effects of extra-virgin avocado oil on a model of D-galactose-induced Alzheimer's disease in ovariectomized Wistar rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eTo accomplish this, 54 female rats were used, of which 42 were ovariectomized (OVX) and 12 underwent white surgery (SHAM). Fourteen days after surgery, the animals were divided into 9 groups of 6 animals each: SHAM+Veh and OVX+Veh groups receiving the vehicle; SHAM + D-gal and OVX+D-gal groups receiving D-galactose and vehicle; OVX+D-gal+E2V and OVX+D-gal+DNPZ groups receiving D-galactose and reference drugs (estradiol valerate and donepezil respectively) and 3 test groups (OVX+D-gal+AO1; OVX+D-gal+AO2 and OVX+D-gal+AO3) receiving D-galactose each and extra-virgin avocado oil at the doses of 0.25, 0.5 and 1 mL/kg respectively. The treatment was carried out during 70 days during which memory disorders were evaluated using the Object Recognition\u003cstrong\u003e,\u003c/strong\u003e Y-Maze and MWM tests. Some biochemical parameters regarding memory function were evaluated on hippocampus homogenate 10%. Isolated brain was fixed in 10% formalin for histological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAs results, compared to SHAM+Veh group, deterioration of both non-spatial and spatial memory (short- and long-term) was observed in OVX animals threated with D-galactose. In addition, a significant decrease in relative hippocampal weight (p \u0026lt; 0.001), Ach (p \u0026lt; 0.001), Glu (p \u0026lt; 0.001), GSH (p \u0026lt; 0.001), CAT (p \u0026lt; 0.05), and SOD (p \u0026lt; 0.001) activities, and a significant (p \u0026lt; 0.001) increase in Methylglyoxal, MDA, and NO2-\u003cstrong\u003e.\u003c/strong\u003e was noted in OVX+D-gal group. Compared to OVX+D-gal group, the treatment with extra-virgin avocado oil at all tested doses reversed or prevented the negative effects induced by ovariectomy and/or by D-galactose on biochemical and oxidative stress biomarkers. The analysis of hippocampus microarchitecture shows that the extra-virgin avocado oil induced a significant decrease (p \u0026lt; 0.05; p \u0026lt; 0.01; p \u0026lt; 0.001) of neuronal loss in CA1 and CA3 hippocampal region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eTaken together, these results suggest that avocado oil possesses neuroprotective properties and can be consumed or supplemented to prevent the onset of Alzheimer's disease.\u003c/p\u003e","manuscriptTitle":"Extra-virgin Avocado (Persea americana Mill.,Laucaceae) Oil Improves Cognitive Impairment in D-galactose-induced Alzheimer’s Disease Model on Ovariectomized Wistar Rat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-12 17:59:21","doi":"10.21203/rs.3.rs-2036318/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54c08ca8-e14b-4c31-91d0-ea2f6755990b","owner":[],"postedDate":"September 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-28T16:59:18+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-12 17:59:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2036318","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2036318","identity":"rs-2036318","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}