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Background: Several findings suggest neuroinflammation as a contributing factor for the onset of psychiatric disor ‑ ders such as Alzheimer’s disease, depression, and anxiety. There is increasing evidence pointing out that the Medi‑ terranean diet influences brain and behavior. Mediterranean herbs and spices have been shown to be within those components of the Mediterranean diet involved in cognitive enhancement. Thus, we investigated the influence of Mediterranean natural extracts (MNE), Rosemary extract (RE) and Glycyrrhiza glabra root extract (GGRE), on cognitive behavior. Results: Adult zebrafish were exposed to RE or GGRE (100 and 250 mg/L) treatments. Both MNE improved memory retention during the T‑maze test, although no improvements were observed during the novel object preference. Similarly, chronic administration of RE (150 mg/Kg) and GGRE (150 mg/Kg) improved, respectively, spatial and retention memory, as assessed by the Morris Water Maze (MWM), and the Elevated Plus Maze (EPM) in healthy male rats. However, no improvements were observed during the novel object recognition. Finally, male, and female rats were chronically treated with lipopolysaccharide [(LPS) 300 ug/kg] and orally administered with RE. Interestingly, RE reversed LPS‑induced cognitive deficit during the MWM and EPM in female rats. Conclusions: We found that MNE improved cognition in both zebrafish and rats. Moreover, MNE rescued LPS‑ induced cognitive impairment in a gender‑specific manner. Therefore, our study supports the view that zebrafish represent a valuable preclinical model for drug discovery in neuroscience. These findings contribute to an excit ‑ ing and growing body of research suggesting that MNE may play an important role in the prevention of cognitive impairment. Keywords: Cognition, Memory, Learning, Mediterranean Natural extracts, Neuroinflammation Background Chronic inflammation is now considered to be central to the pathogenesis of psychiatric disorders including Alz- heimer’s disease, depression, and anxiety. Recent find - *Correspondence: firstname.lastname@example.org; ings have shown that dysregulation of the inflammatory email@example.com; firstname.lastname@example.org system may lead to memory dysfunction and cognitive Eurecat, Centre Tecnològic de Catalunya, Unitat de Nutrició I Salut, Reus, Spain impairment both at preclinical  and clinical  level. Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pusceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 2 of 13 Accumulating translational evidence has proposed that cognitive impairment commonly used to study the effects the quality of diet is a crucial and common determinant of neuroinflammation in the development of neuropsy - for mental health [3, 4]. Healthful eating patterns such as chiatric diseases. the Mediterranean diet, have been shown to offer protec - u Th s, the aim of this study was to investigate the effect tive effects on brain function, such as memory and cogni - of two Mediterranean natural extracts (MNE) in mem- tive processes [5, 6]. A growing number of data indicates ory and cognition, using a screening system based on that native Mediterranean herbs and spices are within zebrafish and testing the most prominent extracts in a those components of the Mediterranean diet involved in murine model of cognitive impairment. memory and cognitive enhancement [7–12]. Liquorice is a root extract from the Glycyrrhiza glabra Materials and methods plant that is widely distributed from southern Europe Husbandry to the northwestern part of China. A number of studies All procedures and protocols involving the care and use have shown that chronic Glycyrrhiza glabra roots extract of laboratory animals were reviewed and approved by (GGRE) improves cognition in rodents as assessed by the the Animal Ethics Committee of the Technological Unit passive avoidance test, the elevated plus maze (EPM) and of Nutrition and Health of EURECAT (Reus, Spain) and the Morris Water Maze (MWM) [9, 13]. GGRE has also the Generalitat de Catalunya (DAAM 10,026). All sec- been shown to ameliorate cognitive impairment induced tions of this report adhere to the ARRIVE Guidelines for by diazepam- scopolamine- and ethanol-induced amne- reporting animal research. sic effects [9, 14]. Rosmarinus officinalis is a perennial herb native to the Zebrafish Mediterranean region. One study showed that chronic A total of 80 adult (6–8 months old; 3–4 cm long; wild- administration of rosemary extract (RE) improved cogni- type) male and female short-finned Danio rerio zebrafish tion in senescence Accelerated Mouse-Prone 8 (SAMP8) (0.4–1 g) of heterogeneous genetic background were mice. Specifically, RE improved cognition as assessed by obtained from ZF Biolabs (Madrid, Spain). Fish were kept the T-maze test and the novel object recognition (NOR) at 28.5 ± 0.5 °C on a 14:10-h light/dark cycles  in a . Similarly, RE improved spatial memory during the standalone aquatic flow-through system (Dohse Aqua - MWM in repetitive mild Traumatic brain injury (rmTBI) ristik GmbH & Co. KG, Grafschaft-Gelsdorf, Germany), rats . provided with constant filtration and aeration, at a den - Rodents are an excellent tool for advances towards sity of 2 fish per liter. Water used in the system consisted research in the central nervous system (CNS). However, of reverse osmosis water supplemented with 0.8 g/L sea the high cost of animal housing and the length of the salts (OSMO FIT, Hobby, Germany). Adult fish were fed experimental studies , critically slow down CNS drug daily with commercially available flake fish food (Ocean discovery and the identification of novel mechanisms Nutrition, Newark, U.S.A.) and brine shrimps. Fish were of brain function and dysfunction. Among vertebrates, acclimated to the aquatic system for 2 weeks before zebrafish (Danio rerio) has revealed as a complementary experiment begins. Behavioral tests took place during the model to experimental studies in rodents for research light phase between 08:00 and 18:00 h. All efforts were in neuroscience . Zebrafish show multiple advan - made to minimize the number of animals used and their tages, including high physiological and genetic homol- discomfort. After experiments, zebrafish were eutha - ogy to mammals, rapid development, ease of genetic nized by hypothermia. and experimental manipulations, sensitivity to cogni- tive tests as well as cost- and space-effectiveness . Rats Although zebrafish are newcomers to studies of learn - Male and female (3 weeks old; 80 g) Sprague–Dawley rats ing and memory, they are capable of performing well in (Envigo RMS Spain, Barcelona, Spain) were maintained a range of learning tasks such as, avoidance learning , in the local animal unit. Food and water were available alternation spatial memory task , associative learning ad libitum and rats were maintained on a 12:12-h light/ task , object recognition , and automated learning dark cycles with temperature at 22 ± 1 °C. Male and paradigm . female rats were housed in separate animal rooms to Bacterial lipopolysaccharide (LPS) is a potent activa- avoid hormonal effects on the cognitive tests . A total tor of the immune system. Several studies have shown of 80 rats were single housed in plastic cages with saw- that chronic administration of LPS in rodents alters the dust bedding in an enriched environment with shredded signaling between the immune system and the brain paper and a cardboard roll. Rats were left undisturbed affecting emotional mood and cognitive behavior [1, 24]. except for routine cage cleaning, twice per week, and Therefore, LPS represents an excellent murine model of body weight were measured weekly until treatments. P usceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 3 of 13 After experiments, adult rats were fastened for 5 h (from Behavioral testing 09.00 to 14.00 h) without restriction of water and killed Zebrafish by decapitation. Zebrafish underwent a behavior test battery spread over 5 days: T-maze on days one and two; the novel object preference (NOP) on day five. All behavioral Treatments tests were performed in the morning between 9.00 and MNE 12.00 h. GGRE (6.2% glycyrrhizic acid) and RE (6% rosmarinic acid) were obtained by Ebro Regaliz (Barcelona, Spain) and Nutrafur (Murcia, Spain), respectively. T‑maze T-maze was used to measure spatial memory as Zebrafish described previously . The apparatus consisted of a Combined males and females of equal proportion were white plexiglass maze with a T shape, filled with 10 cm randomly selected for exposure to GGRE, or RE at a con- of tank water. The long arm (50 × 10 cm) was inter- centration of either 100 or 250 mg/L of water (N = 10/ sected at one of its ends by a short arm (50 × 10 cm). group). Treatments were administered by immersion into One of the ends of the short arm opens to a larger res- a novel water tank for 30 min to minimize stress . ervoir (30 cm square) 5 cm deeper than the rest of the After treatment, fish were transferred to another water maze. The reservoir was enriched with artificial plants tank system for 5 min before behavioral tests. To mini- and color stones to offer a favorable habitat to the fish. mize the number of animals used, each fish performed By nature zebrafish would spend more time in the a behavior test battery. Each fish was exposed to the reservoir than in the rest of the maze . The other assigned treatment before each behavioral test (twice per end of the long arm was connected to a starting zone fish) (Fig. 1). Control fish were kept under the same con - (30 × 10 cm) by a white removable door (Fig. 2). The ditions used for the treated fish, but without exposure to protocol consists in three trials [T-Maze T1 (TMT1), any treatment. TMT2 and TMT3]. During each trial, fish were indi - vidually placed in the starting zone for 1 min with the door closed. Then, the start box was raised and low - Rats ered after the fish exited. During the TMT1, fish were Two sets of animals were used for two parallel experi- given a maximum of 4 min to fully explore the maze ments (Fig. 1). and encounter the reservoir where they were left for at least 20 s before the end of the trial. Each fish repeated (1) Healthy rats. Adult male rats were randomly allo- the trial 3 h (TMT2) and 24 h (TMT3) later. After each cated to the following experimental groups: control, trial, fish were returned to their home tank. All trials GGRE (150 mg/Kg) or RE (150 mg/Kg), (n = 10/ were video-recorded, and the time needed to find the group). reservoir [transfer latency time (TLT)] was quantified (2) LPS-treated rats. Adult male and female rats were using a digital tracking system (ANY-Maze, version randomly allocated to the following experimental 4.82). groups: control, LPS, LPS + RE (n = 10/ group). Once animals were four weeks old underwent oral NOP administration of either GGRE (150 mg/Kg) or RE NOP was performed as previously described . (150 mg/Kg) for six weeks. Treatments were dissolved Briefly, zebrafish were subjected to 5 min of acclima - in low-fat condensed milk:water (1:10 v/v) and adminis- tion to the novel tank (29 × 14 × 18 cm tank filled with tered by micropipette. Controls were treated with low-fat 14 L of tank water) followed by 10 min of habituation condensed milk:water (1:10 v/v), only. As for the LPS- to two identical objects (training phase). After a recov- treated rats’ study, LPS (300 ug/kg, i.p.) was administered ery period of 10 min, one of the two original objects for seven days, during the fourth week of the study, 2 h was randomly replaced with a novel object and inter- after RE or vehicle treatments, when animals were seven actions were monitored for an additional 10 min (test- weeks old. Controls for the LPS group were i.p. injected ing phase). To avoid thigmotaxis influence, the objects with 0.9% sterile saline water for the same period. All were placed one in front of the other in an equal dis- treatments were administered every day, between 09.00 tance between the center of the tank and the walls. and 10.00 h. Animals underwent behavioral testing An 8.4 cm × 8.4 cm box was superimposed over each immediately after LPS treatment (Fig. 1). GGRE and RE object and the time the fish spent within the boxes was treatments were based on previous studies where similar recorded and scored automatically (ANY-Maze, version doses were applied [41, 42]. 4.82). Pusceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 4 of 13 Zebrafish 3 days break RE, GGRE (♂ ,♀ zebrafish) T-maze NOP Healthy Rats RE, GGRE (♂ SD rats) LPS Rats RE, LPS (♂,♀ SD rats) Fig. 1 Study design. Experimental timeline showing A treatment and animal behavior in zebrafish; B treatment and animal behavior in healthy adult male rats; C treatment and animal behavior in LPS‑treated adult male and female rats Rats for 30 min. All experiments were carried out between Behavioral tests were carried out during the fifth [EPM, 9:00 am and 2:00 pm. NOR and open field (OF)] and sixth (MWM) week of MNE administration, when the animals were eight and EPM nine weeks of age, respectively (Fig. 1). Before each EPM was performed to assess retention memory as behavioral test, rats were habituated to the test room described previously  with some modification. The P usceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 5 of 13 T-maze Control Control RE 100mg/L GGRE100 mg/L RE 250mg/L GGRE250 mg/L 100 100 75 75 ** 50 50 ** ** # 25 25 0h 3h 24h 0h 3h 24h Water 100mg/L 250mg/L ** Ctrl RE GGRE NOP Water 125 Water 100mg/L 100mg/L 250mg/L 250mg/L 75 75 Ctrl RE GGRE Ctrl RE GGRE Fig. 2 Mediterranean natural extracts improved memory retention in zebrafish. A Time curve representing TLT during TMT1, TMT2 and TMT3 of the T‑maze test; B AUC representing TLT during the T ‑maze test; C Time and interaction scores during the NOP task expressed as exploration ratio (%). Data are presented as the mean ± SEM. (∗ P < 0.05; ∗ ∗ P < 0.01 compared to control; # P < 0.05 compared to 250 mg/L) On the training phase, rats were placed individually at maze consisted of two open arms (50 × 10 com) and two the end of the open arm, facing it away from the central enclosed arms (50 × 10 × 40 cm) that all extended from platform. The time that the animal took to move from a common central platform (10 × 10 cm). The apparatus the open arm to one of the two enclosed arms (4 paws was raised 60 cm above the floor on a central pedestal. Eplorationratio- AUC time (%) Transferlatency time (s) Eploration ratio - interactions(%) Transferlatencytime(s) Pusceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 6 of 13 in) was recorded. The same procedure was repeated (NW), Southeast (SE) and Southwest (SW), and four 60 min (acquisition) and 24 h (retention) after training. starting positions, North (N), East (E), West (W), South To become acquainted with the EPM, if the rat failed to (S), were located at equal distances along the pool rim. enter the enclosed arm within 120 s, it was guided to one A circular white platform (10 cm diameter) was placed of them and let it to explore the maze for further 60 s in the center of the NW quadrant and submerged 1.5 cm before to be returned to its home cage (training phase below the water surface. Water was white colored with a only). Animal behavior was recorded and TLT (acquisi- non-toxic white paint to hide the platform. Distal cues tion and retention phases) was analyzed using a track- were arranged around the maze to provide landmarks ing system (ANY-Maze, version 4.82). The apparatus was that the animals could use to navigate to the platform. wiped clean with 70% ethanol before testing each animal. Acquisition training NOR Rats underwent five days of training that consisted of four NOR was used to assess recognition memory as trials/day. At the beginning of each trial rats were indi- described previously  with some modifications. vidually placed in one of the starting positions (N, E, S, or Briefly, rats were placed in a grey plywood rectangu - W) facing the wall of the tank and allowed to explore the lar box (70 × 45 × 45 cm) for 5 min for habituation to maze for 60 s. A different starting position was used for the novel arena. The day after, rats were habituated to each of the 4 trials on a given day arranged in a semi-ran- two identical objects [(A), acquisition phase)] placed in dom pattern. Once rats reached the platform, they were the back left and right corners of the experimental box held there for 20 s. If the platform was not found within for 3 min. After a delay of 1 h, one of the two familiar the given time, rats were gently assisted to the platform objects was replaced with a novel object (B) and rats were by the experimenter and detained there for 20 s. replaced in the middle of the box at the mid-point of the wall opposite the sample objects for a total time of 3 min Removal phase (retention phase). Box and objects were cleaned with On day six (removal) the platform was removed, and the alcohol 70% to avoid any cue smell between each trial. rats were allowed to freely explore the maze for 60 s. Rats Direct contacts with the objects, include any contact with were individually placed into the quadrant diagonally mouth, nose or paw or minimal defined distance (< 2 cm), opposite to the one originally hosting the platform. The were recorded and scored automatically (ANY-Maze, amount of time spent in the quadrant where the platform version 4.82). Memory discrimination index (MDI), [(B was previously hidden was video recorded and automati- exploration time – A exploration time)/ (B exploration cally analyzed using a tracking system (ANY-Maze, ver- time + A exploration time)], was used to express recogni- sion 4.82). tion memory in rats . Statistical analysis OF All the data are expressed as mean ± standard error of To assess anxiety-like behavior rats were individu- the mean (SEM). Repeated measure (RM)-ANOVA was ally placed in a wooden grey box (70 × 45 × 45 cm) for performed to analyze differences over time among treat - 5 min. A central area (20 × 40 cm) was drawn in the floor ments, whereas ANOVAs analysis followed by LSD’s of the apparatus to score time and number of entries in post-hoc test was performed to analyze single-time points the inner zone. Distance moved, velocity, percentage of differences between treatment groups. Student’s t-test time spent in inner zone and frequency of inner zone was used for single statistical comparisons. The level of entries were recorded and analyzed using a tracking sys- statistical significance was set at bilateral 5% (p < 0.05). All tem (ANY-Maze, version 4.82). The apparatus was wiped statistical analysis was performed with the SPSS Statistics clean with 70% ethanol before testing each animal. 26 software (SPSS, Inc., Chicago, IL, United States). MWM Results The MWM is a test of spatial learning and reference Mediterranean natural extracts improved memory memory for rodents that relies on distal cues to navigate retention in zebrafish from start locations around the perimeter of an open T‑maze swimming arena to locate a submerged escape platform The T-maze was used to assess memory retention in . The swimming arena consisted of a circular metal zebrafish. For RE effects on zebrafish, RM-ANOVA pool (150 cm diameter × 58 cm tall) filled with tap water revealed an overall learning improvement over time (f (23-25ºC, 40-cm deep). The maze was divided geographi - = 6.928, p = 0.002) and a significant time by treat - (2,76) cally into four quadrants: Northeast (NE), Northwest ment interaction (f = 4.513, p = 0.003), (Fig. 2A). (4,76) P usceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 7 of 13 Interestingly, significant time by treatment interaction improved learning in RE-treated rats (1-way ANOVA: was mainly due to the treatment effect lasting for 24 h. f = 3.668, p = 0.040; LSD’s post hoc test: p = 0.012; (2,26) In contrast, in the control groups cognitive abilities Fig. 3A) compared to controls. However, no improve- returned to basal at 24 h time point. Accordingly, LSD’s ments were observed in rats treated with GGRE (Fig. 3A). multiple comparisons post hoc analysis showed that To assess spatial long-term memory, a probe trial was RE supplementation reduced TLT of 50% (100 mg/L) performed on day 6. However, no improvement induced and of 30% (250 mg/L) at 24 h time point, compared to by MNE was found (f = 1.522, p = 0.236; Fig. 3A). (2,27) controls (Fig. 2A). For GGRE effects on zebrafish, RM- ANOVA revealed an overall learning improvement over EPM time (f = 15.087, p = 0.000) and a significant time by We further tested the effect of MNE on retention mem - (4,74) treatment interaction (f = 4.385, p = 0.00), (Fig. 2A). ory by assessing the EPM test. RM-ANOVA revealed that (4,74) Similar to the RE effects, significant time by treatment all experimental groups improved TLT towards one of interaction was mainly due to the long lasting effects of the enclosed arms of the maze over time (f = 51.430, (2,46) GGRE up to 24 h. Post hoc analysis showed that TLT p = 0.000; Fig. 3B). Moreover, single statistical compari- was higher in zebrafish treated with GGRE (250 mg/L) at son analysis revealed that GGRE-treated rats improved baseline compared to both controls and GGRE 100 mg/L- TLT at 24 h’ time point, when compared to controls (stu- treated zebrafish. GGRE (100 mg/L) reduced TLT of dent’s t-test: p = 0.045; Fig. 3B). Similarly, AUC analysis 25% at 3 h time point compared to controls. Moreover, confirmed memory improvements in GGRE-treated rats GGRE, at both concentrations, reduced TLT of 57% at (student’s t-test: p = 0.027; Fig. 3B). No improvements 24 h time point compared to controls (Fig. 2A). Simi- induced by RE were observed. larly, AUC analysis confirmed the improved learning in both RE- and GGRE-treated zebrafish (1-way ANOVA: f NOR = 3.092, p = 0.021; Fig. 2B). Moreover, post hoc anal- (4,75) Next, we tested whether MNE supplementation can ysis revealed that both RE and GGRE improved memory modulate recognition memory as assessed by the NOR retention at a concentration of 100 mg/L, only (Fig. 2A). task. All experimental groups successfully discriminate the novel object from the familiar one as expressed by an NOP exploration ratio score higher than 50% (Fig. 3C). How- The NOP task was used to assess recognition memory in ever, neither RE nor GGRE improved recognition mem- zebrafish. All the experimental groups successfully per - ory compared to controls. formed the training phase showing no preference for nei- ther one of the familiar objects (data not shown). When OF zebrafish were exposed to the novel and the familiar None of the treatments altered locomotor activity as objects (test phase) all the groups were able to discrimi- assessed by the OF test (Fig. 3D). No other changes such nate the familiar object from the novel one (more than as time and number of crossings in the inner zone were 50%), (Fig. 2C). However, MNE did not induce cognitive found (data not shown). improvement during the NOP task. RE reversed LPS‑induced cognitive impairment in rats Mediterranean natural extracts improved learning in a sex dependent manner and cognition in adult rats The study on healthy rats revealed a stronger effect of To confirm the findings obtained from the zebrafish RE on learning skills and spatial memory compared to study, cognitive behavior was assessed in healthy male GGRE. In accordance, we selected RE to further inves- rats chronically treated with either RE or GGRE. tigate whether MNE exert any protective effect in a rat model of cognitive impairment induced by neuroinflam - MWM mation. To this aim, cognitive deficit was induced by To assess whether MNE improved spatial learning and chronic injection of LPS (300 mg/kg) for 7 consecutively memory, rats were trained over five consecutive days days to both male and female rats before animal behavior to find a hidden platform in the MWM. RM-ANOVA commenced. revealed a significant effect of time (f = 53.013, (4,92) p = 0.000; Fig. 3A), only. LSD’s multiple comparisons MWM post hoc analysis showed that RE supplementation The effects of chronic administration of RE against improved learning at days 2 (p = 0.036), 3 (p = 0.018) and LPS-induced cognitive impairment during the MWM 5 (p = 0.030) of the training sessions compared to con- are shown in Fig. 4A. For RE effects on female rats, trols (Fig. 3A). Similarly, AUC analysis confirmed the RM-ANOVA revealed a significant effect of time (f Pusceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 8 of 13 MWM 60 175 35 Control RE 150 30 GGRE 125 25 100 20 75 15 50 10 25 5 0 0 0 ControlREGGRE ControlREGGRE 12 34 5 Days EPM Control RE GGRE 0h 1h 24h ControlREGGRE C D NOR OF 80 40 70 35 60 30 50 25 40 20 30 15 20 10 10 5 0 0 ControlREGGRE ControlREGGRE Fig. 3 Mediterranean natural extracts improved learning and cognition in adult rats. A MWM represented by latency time to reach the platform over 5 training days, AUC and removal phase 24 h after the last training day; B EPM represented by the time to reach the reservoir at 0 h (training), 1 h (acquisition) and 24 h (retention) as well as AUC; C NOR represented by the time spent with the objects expressed as exploration ratio (%); D distance travelled during the OF test. Data are presented as the mean ± SEM. (∗ P < 0.05 compared to control; t P < 0.05 compared to control, unpaired student’s t‑test) = 56.788, p = 0.000), a significant time by RE inter - days 2 (p = 0.000) and 3 (p = 0.049) of the training phase (4,96) action (f = 4.078, p = 0.004) and a significant time (Fig. 4A). Interestingly, LPS + RE female rats were able (4,96) by LPS interaction (f = 2.772, p = 0.031), (Fig. 4A). to find the hidden platform faster than the LPS-treated (4,96) LSD’s multiple comparisons post hoc analysis showed group both at days 2 (p = 0.049) and 5 (p = 0.049), escape latency time to be higher in the LPS group at (Fig. 4A). To assess spatial long-term memory, a probe Escape latency (s) Transfer latencytime(s) Explorationratio -Time(%) Distancetravelled (m) AUC AUC Time intargetquadrant (s) P usceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 9 of 13 MWM_Female 60 200 Control ** *** * LPS ### 50 LPS+RE # ## 125 * 30 100 # 5 0 0 0 12 34 5 Days MWM_Male Control LPS 50 LPS+RE 50 10 0 0 12 34 5 Days EPM_Female 80 2000 Control LPS LPS+RE *** 60 1500 *** ** 40 1000 ## ** 20 500 ## 0 0 0h 1h 24h EPM_Male Control LPS LPS+RE 0h 1h 24h Fig. 4 RE reversed LPS‑induced cognitive impairment in rats in a sex dependent manner. A MWM represented by latency time to reach the platform over 5 training days, AUC and removal phase 24 h after the last training day; B EPM score represented by the time to reach the reservoir at 0 h (training), 1 h (acquisition) and 24 h (retention) as well as AUC. Data are presented as the mean ± SEM. (∗ P < 0.05; ∗ ∗ P < 0.01; ∗ ∗ ∗ P < 0.001 compared to control, # P < 0.05 ## P < 0.01 compared to LPS) Control LPS LPS+RE Control LPS LPS+RE Control LPS LPS+RE Control LPS LPS+RE Control LPS LPS+RE Control LPS LPS+RE Transferlatencytime(s) Transfer latencytime(s) Escapelatency (s) Escapelatency(s) AUC AUC AUC AUC Time in target quadrant (s) Time in targetquadrant (s) Pusceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 10 of 13 trial was performed on day 6. One-way ANOVA revealed compounds to current therapeutic interventions in the a significant effect of group on time spent in the tar - treatment of cognitive impairment and suggests that get quadrant (f = 11.348, p = 0.000; Fig. 4A). LSD’s MNE may be more efficacious in females with a neuroin - (2,27) multiple comparisons post hoc analysis showed spa- flammatory profile. tial long-term memory deficit in LPS treated rats when Rodents have been traditionally used to advance our compared to controls (p = 0.026; Fig. 4A). Interestingly, knowledge on human behavior, such as learning and RE treatment reversed LPS-induced cognitive impair- cognition. However, expanding the range of experi- ment (p = 0.000; Fig. 4A). Moreover, RE + LPS treated mental domains with the use of additional models, is rats spent more time in the target quadrant compared to now considered as an important strategy for transla- controls (p = 0.023; Fig. 4A). Similarly, AUC analysis con- tional neuroscience research . Recent data suggest firmed that RE reversed LPS-induced learning impair - zebrafish as an emerging successful model to study ment in female rats (f = 7.084, p = 0.004; Fig. 4A). cognitive phenotypes and CNS related drug discov- (2,26) For RE effects on male rats, RM-ANOVA revealed a sig - ery [35–37]. Accordingly, our behavioral results show nificant effect of time (f = 25.109, p = 0.000; Fig. 4A), that both GGRE and RE improved retention memory (4,56) only. Moreover, LSD’s multiple comparisons post hoc as indicated by the T-maze test. Similarly, RE has been analysis revealed learning deficit at day 5 of the train - shown to improve cognitive function in a zebrafish ing sessions induced by LPS administration to male rats model of amnesia induced by scopolamine . We (p = 0.0322; Fig. 4A). However, no further changes were also found that improvement in retention memory was observed in male rats, as referred to AUC analysis and best at the lowest concentration used (100 mg/L). This the total time in the target quadrant (Fig. 4A). Therefore, suggests that at high doses (250 mg/L) both GGRE and data indicated that RE protective effects in response to RE may have off-target activity. Ceiling effects have LPS stimulation were more pronounced in female than been previously observed with other natural extracts male rats. in rats [39, 40]. Interestingly, the results obtained in zebrafish correlated with those observed in healthy EPM male rats. Specifically, RE demonstrated improve - The effects of chronic administration of RE against LPS- ments in spatial learning as indicated by the MWM. induced cognitive impairment during the EPM are shown Moreover, a better recall memory was observed in in Fig. 4A. For RE effects on female rats, RM-ANOVA GGRE treated rats at 24 h, but not 1 h, from the train- revealed a significant effect of time (f = 69.210, ing phase of the EPM task. In a similar study, GGRE (2,54) p = 0.000), a significant time by LPS interaction (f improved memory functions in a mice model of LPS- = 8.170, p = 0.001), (Fig. 4A). LSD’s multiple com- induced neuroinflammation . Moreover, RE has (2,54) parisons post hoc analysis showed that LPS induced been shown to rescue learning and spatial memory cognitive retention impairment in female rats at both 1 deficits in a rat model of epilepsy induced by treatment (p = 0.000) and 24 h (p = 0.002), (Fig. 4B). Interestingly, with kainic acid . Interestingly, a randomized clini- TLT score was lower in LPS + RE female rats compared cal trial observed that RE treatment positively affected to the LPS-treated (p = 0.030; Fig. 4B). Moreover, RE memory performance, anxiety, depression, and sleep reversed LPS-induced memory deficit at 24 h’ time point quality in 34 Iranian university students . On the (p = 0.002; Fig. 4B). AUC analysis confirmed the protec - other hand, no improvements were found in recogni- tive effect exerted by RE against LPS-induced cognitive tion memory either in zebrafish or in rats as assessed deficit as shown during the EPM test (f = 13.679, by the NOP and the NOR tests, respectively. There is (2,26) p = 0.000; Fig. 4B). No RE effects were observed in male a wide agreement that both spatial learning and long- rats (Fig. 4B). term memory are largely dependent on the integrity of the hippocampus [44, 45], whereas its role in recogni- Discussion tion memory is controversial. A number of studies The present studies demonstrate that selective MNE suggest no effect of hippocampal lesion in recognition can induce cognitive enhancement in adult zebrafish. memory [46, 47], while other studies reported signifi - Moreover, we showed that chronic administration of cant impairments [48, 49]. Moreover, it is suggested both GGRE and RE had similar cognitive improvements that tasks of recognition memory are mainly supported in healthy rats. Additionally, we demonstrated that LPS- by cortical structures adjacent to the hippocampus and induced cognitive impairment was reversed by chronic that are less dependent to hippocampal malfunction- RE administration in female rats only. Our findings ing [50, 51]. For instance, the hippocampus receives provide additional support for MNE as potential mem- projections from the adjacent perirhinal and entorhi- ory enhancement as well as potential complementary nal cortices which have been shown to severely disrupt P usceddu et al. Behavioral and Brain Functions (2022) 18:5 Page 11 of 13 Senescence Accelerated Mouse‑Prone 8; SE: Southeast; SEM: Standard error of recognition memory in rodents . Thus, our data the mean; SW: Southwest; TLT: Transfer latency time; TMT: T‑Maze Trial; W: West. may suggest that the observed improvements in spatial learning and long-term memory induced by GGRE and Acknowledgements We thank Ms Anna Antolín, Ms Gertruda Chomiciute, Ms Cristina Egea Vilches, RE may be due to their action on neuronal circuitries Ms Iris Triguero for their technical assistance in performing the experimental specifically involved in the regulation of hippocampal- studies. dependent tasks both in zebrafish and rats. However, it Authors’ contribution should be mentioned that improvements in recognition MMP: Formal analysis, Writing—Original draft, Visualization, Conceptualization memory induced by RE and GGRE have been previ- JH‑B: Investigation FP: Project administration LA: Project Administration AC: ously reported in mice [41, 52]. The discrepancy with Conceptualization, Methodology, Editing JMDB: Conceptualization, Editing LB: Conceptualization, Methodology. All authors read and approved the final our findings may be found in the applied animal model manuscript. of cognitive impairment and the type of rodents used. Further analysis is warranted to shed more light on this Funding MMP has received funding from the European Union’s Horizon 2020 research issue. and innovation programme under the Marie Skłodowska‑ Curie grant agree‑ Growing evidence suggests that females are more ment No 712949 ( TECNIOspring PLUS) and from the Agency for Business susceptible to the development of mood and cognitive Competitiveness of the Government of Catalonia. LB has received funding from the Spanish government under the Juan de la Cierva‑Formación fel‑ deficits compared to males, leading to a higher risk for lowship. This work was financially supported by ACCIO through the project psychopathologies . Neuroinflammation has been KETRENOVIN of the RIS3CAT initiative (COMRDI 15‑1‑0028); and by the Centre indicated as one of the main factors that contribute to sex for the Development of Industrial Technology (CDTI) of the Spanish Ministry of Science and Innovation under grant agreement: TECNOMIFOOD project. differences in psychiatric diseases such as Alzheimer’s CER‑20191010. disease, depression and anxiety [54, 55]. LPS injections represent a widely used animal model of neuroinflamma - Data availability We will make our data available should it be required. tion that has been shown to disrupt memory and cogni- tion in rodents [1, 56]. We observed that, at the chosen Declarations dose of 300 ug/kg, LPS chronic administration impaired females’ learning, and cognition of a greater extent com- Ethics approval and consent to participate pared to male rats. Interestingly, RE chronic treatment All procedures and protocols involving the care and use of laboratory animals were reviewed and approved by the Animal Ethics Committee of the demonstrated protective cognitive effects in response to Technological Unit of Nutrition and Health of EURECAT (Reus, Spain) and the LPS chronic injection. However, such effect was blunted Generalitat de Catalunya (DAAM 10026). All sections of this report adhere to by LPS administration in male rats. Such discrepancy the ARRIVE Guidelines for reporting animal research. Full efforts were made to diminish the use of animals and to improve their wellbeing. may be found on the different immunological response between genders that in turn may be associated with the Consent for publication gender-specific outcomes from RE treatment. Further Not applicable. research is warranted. Competing interests In conclusion, we demonstrated that zebrafish can be The authors declare no conflict of interest. considered as a valuable preclinical model for drug dis- Author details covery in neuroscience. The results obtained in zebrafish Eurecat, Centre Tecnològic de Catalunya, Unitat de Nutrició I Salut, Reus, are, to a great extent, comparable to those observed in Spain. Departament de Bioquímica I Biotecnologia, Grup de Recerca en healthy rats. Moreover, we provided further evidence Nutrigenòmica, Universitat Rovira I Virgili, Tarragona, Spain. that neuroinflammation is involved in the regulation of Received: 26 October 2020 Accepted: 12 February 2022 learning and cognition using a rat model of LPS-induced memory impairment. Notably, the protective effects of the selected MNE against LPS-induced memory impair- ment were gender-specific. Finally, this study shed light References on the beneficial role of MNE administration in enhanc - 1. 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Behavioral and Brain Functions – Springer Journals
Published: Feb 25, 2022
Keywords: Cognition; Memory; Learning; Mediterranean Natural extracts; Neuroinflammation
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