Kaurenoic Acid from Annona senegalensisAttenuates Hippocampal NMDA Receptor Overexpression and Enhances Cognitive Function in a Rat Model of PTZ-Induced Seizures

Kaurenoic Acid from Annona senegalensisAttenuates Hippocampal NMDA Receptor Overexpression and Enhances Cognitive Function in a Rat Model of PTZ-Induced Seizures | 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 Kaurenoic Acid from Annona senegalensis Attenuates Hippocampal NMDA Receptor Overexpression and Enhances Cognitive Function in a Rat Model of PTZ-Induced Seizures Samuel Sunday Dare, Folarin O. Royhaan, Bamidele P. Fakunle, Olukayode O. Odubela, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6829238/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: Overactivation of N-methyl-D-aspartate receptors (NMDARs) is implicated in seizure-induced excitotoxicity and neurodegeneration. Kaurenoic acid, a diterpenoid from Annona senegalensis , has shown anticonvulsant potential, but its modulatory effect on NMDARs in epilepsy remains unclear. This study investigates the neuroprotective and cognitive effects of kaurenoic acid and its impact on hippocampal NMDAR expression in a PTZ-induced seizure model in rats. Methods: Kaurenoic acid (KNA) was isolated from the ethyl acetate fraction of A. senegalensis leaves. Thirty male Wistar rats were divided into five groups (n=6): control, PTZ only, KNA (400 or 800 mg/kg, p.o) + PTZ, and phenobarbital (PB, 10 mg/kg, i.p) + PTZ. Seizure activity was scored using Racine’s scale. Cognitive performance was assessed via the Open Field and Novel Object Recognition tests. Hippocampal NMDAR levels were measured using ELISA. Data were analyzed with ANOVA and Tukey’s post hoc test. Results: KNA significantly increased seizure thresholds in myoclonic (173±33 s) and tonic-clonic seizures (444±41 s), comparable to PB (233±43 s and 534±42 s), p<0.0001. KNA improved thigmotaxis (98%) and preserved cognitive function with a positive discrimination index (44±17%), whereas PB impaired cognition (−61±18%). Locomotor activity decreased with both KNA and PB. PTZ elevated NMDAR expression (27±1 µmol) compared to control (22.8±0.2 µmol), while both KNA and PB reduced NMDAR levels (17±3 and 23±1 µmol, respectively). Conclusion: Kaurenoic acid demonstrated strong anticonvulsant and neuroprotective effects, effectively lowering hippocampal NMDAR expression and preserving cognitive function better than phenobarbital. These findings support its therapeutic potential as a safer alternative for seizure management. Molecular Biology Cellular & Molecular Neuroscience Neurobiology of Disease Animal Behavior NMDA receptor epileptic seizures kaurenoic acid hippocampus cognition neuroprotection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION N-methyl-D-aspartate receptors (NMDARs) overexcitation has been linked to neurological conditions that induce neuronal death, including epilepsy, stroke, Alzheimer's disease (AD), and Parkinson's disease, according to related research (Chen et al., 2022 ; Essiz et al., 2021 ). The receptor is composed of 4 subunits derived from 3 gene families: GluN1-3. Each receptor is a tetramer composed of 2 GluN1 subunits and either 2 GluN2 or 2 GluN3 subunits. The NMDAR's pharmacological regulation is determined by the different subunit binding site combinations. The NR2 subunit contains the glutamate binding site, while the NR1 subunit has the glycine binding site. Different neuroanatomic manifestations are seen for these binding sites (Kapur, 2018 ; Sivakumar et al., 2022 ). In epilepsy, NMDAR regulation has drawn a lot of attention due to its role in the brain's neuroexcitatory and functional plasticity. NMDARs' functions and mechanisms in epilepsy are yet unknown, but they have been shown to contribute to seizures. During seizures in temporal lobe epilepsy (TLE), glutamate levels in the extracellular fluid increase which can directly activate NMDARs and result in neuroexcitatory injury. Cognitive impairment may be exacerbated by impaired NMDAR function. It should come as no surprise that NMDARs are a very desirable therapeutic target. Autoantibodies against NMDARs cause a distinctive condition that highlights the vital role these receptors play in behavior, cognition, motor, respiratory, and autonomic regulation (Benarroch, 2011 ). Using a range of techniques, including immunoblotting, binding affinities, and evaluation of subunit gene expression, some researchers have assessed the potential changes of NMDARs in epilepsy, given that NMDAR activity is crucial for neuronal excitation in the central nervous system. The primary excitatory input to the hippocampus is glutamatergic impulses from the entorhinal cortex, and a change in glutamate-mediated excitability may play a role in the pathophysiology of epileptic discharges (Carter et al., 2011 ). Furthermore, as NMDAR antagonists offer defense against such damage, NMDARs may be in charge of the selective excitotoxic cell death of certain hippocampus neuronal populations brought on by seizures (Meldrum, 1993 ). Research has demonstrated that certain AEDs influence glutamatergic synapses via NMDA receptors, which are typically excitatory amino acid (glutamate) receptors. By blocking these excitatory receptors, these AEDs lessen impulse conduction (Das et al., 2002 ; Howard et al., 2011 ). Numerous studies have examined the role of NMDARs in epileptic seizures and kaurenoic acid, a deterpenoids isolated from Annona senegalensis root back extract has shown an anticonvulsant potential and phenobarbital with notable effects (Chen et al., 2022 ; Okoye et al, 2013 ); however the relationship between kaurenoic acid activities and NMDARs with respect to epileptic seizure is unclear. This study is aimed to investigate the prophylactic potential of kaurenoic acid isolated from Annona senegalensis pers leaves on the level of expression of NMDARs in the hippocampus of PTZ-induced seizure in Wistar rats. MATERIALS AND METHODS Plant Material, Extraction and Isolation of Kaurenoic Acid Annona senegalensis leaves were collected from the “Ishaka-Bushenyi” Municipality along Mbarara-Kasese Road, Western Uganda. Geographic coordinates are 0.5424° S and 30.1965° E. The Plant was identified harvested and processed to obtain 100g dry powder of the aqueous crude according to Dare et al, 2024 . To isolate kaurenoic acid, solvent–solvent fractionation by separation funnel method with methanol, ethyl acetate, and n-hexane in the increasing polarity order was employed to obtain the respective fractions. To carry out this process, the dried crude aqueous extract was transferred to a separation funnel, equal volume of n-hexane added and the funnel was shaken vigorously to partition non-polar compounds into the n-hexane layer. The funnel was allowed to settle then the upper (n-hexane) layer was separated from the lower (aqueous) layer. For ethyl acetate extraction, ethyl acetate was added to the remaining aqueous extract in the separation funnel, shaken vigorously to separate the semi-polar compounds including kaurenoic acid, which partitioned into the ethyl acetate phase and allowed to settle. The layer of ethyl acetate was gathered and allowed to evaporate at a lower pressure. A silica gel column was used for purification by column chromatography, with n-hexane serving as the initial elution solvent. By dissolving the concentrated ethyl acetate extract in a tiny amount of solvent (n-hexane acetate, 90:10), the sample was put onto the column. To obtain a sub-fraction (80:20), the ethyl acetate fraction was further eluted using gradient mixes of n-hexane and ethyl acetate (90:10 to 20:80). Based on the yield and potency, this sub-fraction was further identified as kaurenoic acid (Okoye et al., 2013 ; Mtunzi et al., 2017 ). HPLC Characterization of Kaurenoic Acid Methanol, ethyl acetate, and n-hexane extract of Annona senegalensis were subjected to HPLC analysis using a JASCO LC-4000 Series, Japan, HPLC system. The system contained employed an asymmetric twin-piston solvent delivery system SSQD (Slow Suction, Quick Delivery) which provides significantly better flow and pressure profiles and UV-Visible Detectors (UV-4070/4075). Reverse phase column chromatographic separation was performed using Octadecylsilane, Size: 4.6mm X 150–250mm column with ethyl acetate mobile phase. For sample preparation, filtration was carried out to remove any particulates that could clog the column and the sample was diluted to an appropriate concentration suitable for analysis (50µm). For solvent selection, HPLC-graded solvent which are free from impurities (water, 99.8% methanol, 99.8% acetonitrile, 99.8% isopropanol, 99.8% ethanol and 99.9% ethyl acetate were used. Samples were stored in sterile samples and refrigerated to maintain stability. The standard concentrations used for analytical applications ranged from 10–70 µg/mL, 1–2% of the total column volume and 1 µg/µL concentration of the extract. Data collection and processing were done using ChromNAV Chromatography Data System (CDS) software and the detection wavelength was between 200 and 400 nm. The injection volume of samples and reference solutions was 3–5µL, and the flow rate was 3.0 mL/min. By monitoring retention time and examining UV spectra, the peaks were found at 20 and 400nm Retension Time (5min) a little quantity of the reference standards. Experimental Animals and Design For this investigation, thirty (30) adult male wistar rats were acquired and kept in the animal house in hygienic cages with a 12-hour light and 12-hour dark cycle at an average ambient temperature between 18 0 C and 26 0 C, fed with pelletized rat diet and given unlimited water. Two weeks to the start of the experiment, the animals were weighed (150–250 grams) and grouped into five (5) groups at random (n = 6) with identification marks and allowed to become familiar with their new environment. Group 1 (received distilled water as negative control) and Group 2 (received PTZ as positive control) while Group 3 (received low dose of KNA), Group 4 (received high dose of KNA) and Group 5 (received phenobarbital) before PTZ. Seizure Activity and Behavioural Study PTZ (Sigma–Aldrich, P6500, Lot No. MKCM4261) was used to induce seizures and seizure-behaviour of individual animal was recorded with a PC Logitech B905 digital Webcam connected to a laptop in a square open-field standard box (100 x 100 x 50 cm) for 10 minutes. The seizure behavioural activities of the animals were scored according to Racine scale 3 and 6 characterized by myoclonic neck jerks with sharp spikes followed by spike-wave discharges EEG correlates and clonic, tonic-clonic seizures, and wild jumping with high amplitude, spike-wave discharges EEG correlate respectively. To investigate the prophylactic potential of KNA and PB against PTZ-induced seizure, 400mg/kg and 800mg/kg of KNA were administered orally to group 3 and group 4 respectively while group 5 received 10mg/kg of phenobarbital (PB) thirty minutes before administration of PTZ (40mg/kg), and these were compared with group 1 and group 2 which received 1m/kg orally and 40mg/kg PTZ intraperitoneally. Thigmotaxis, Locomotor Behaviour and Discriminative Index To determine the thigmotaxtic index, a metric used to assess anxiogenic and anxiolytic behaviour, the percentage of the 10-minute test duration that the individual stays next to the maze's outer wall, a sign of anxiety-like behavior was measured (Zhang et al., 2023 ) while the total ambulatory distance covered during the open field test (OFT), measured as an indicator of locomotor activity. To ascertain the non-spatial or recognition, novel object recognition test (NORT) was employed. The test was conducted 20 to 24 hours after the medicine was administered in a 100 × 100 cm square open-field standard box with walls 50 cm high. A Logitech B905 2MP Portable Webcam was mounted on top of the box and linked to a laptop to capture the animals' exploratory activities. The test process was divided into three stages: recognition, familiarization, and habituation. During the habituation phase, each animal was carefully put in the object-free box and given five minutes to explore. The initial exploration session of the habituation phase was followed by the familiarization phase 24 hours later. Each animal was given ten minutes to investigate after two identical plastic items (A and B) were positioned in the box's neighboring corners, about ten centimeters from the walls. After fifteen minutes, item B was used to start the recognition phase. It was then swapped out for a new object C that was different in color and form but had a comparable size, texture, and weight. For ten minutes, each animal was left alone in the box, and the amount of time it spent exploring the objects was noted (Fig. 1) The videos recorded were analyzed using ANY-maze 7.3 software to track the object exploration time which was used to calculate the percentage novel object preference index [TC / (TC + TA) x 100] and discrimination ratio [(TC – TA) / (TC + TA)], where TA and TC signify time spent exploring object A and C respectively. The object exploration time was calculated by making two virtual zones around each object (A and C) using the software and the time spent in each zone was calculated (Antunes and Biala, 2012 Mazumder et al., 2017 ). Level of Expression of NMDA Receptor Using ELISA Rat N-Methyl-d-aspartate (NMDA) Receptor 2 ELISA Kit, Bioassay Technology Laboratory, Zhejiang, China (Dhamad and Abdal Rhida, 2020; Tajima et al., 2023 ) was used to investigate the level of the expression of NMDA Receptor 2 in the hippocampus of the animals. Following the bahavioural tests, the animals were anaesthetized with halothane (Piramal Pharma Limited, India), brain was extracted and the hippocampus removed by micro-dissection. The hippocampal tissue was homogenized with buffer following established techniques or the manufacturer's instructions, centrifuged and the supernatant collected. To carry out the assay, all the reagents, samples and standards were prepared brought to room temperature before use according to the user instruction. The sample and ELISA reagent were added into each well and incubate for 1 hour at 37°C. The plates were washed 5 times and the substrate solution A and B were added and incubated in the dark for 10 minutes at 37°C. Afterwards the stop solution was added and the blue color changed into yellow immediately. The optical density (OD value) of each well was determined immediately using a microplate reader set to 450 nm within 10 minutes after adding the stop solution. Statistical Analysis GraphPad (8.0.2) was used to perform an analysis of variance (ANOVA) and a Tukey post hoc test on the collected data (Geoff et al., 2007 ). The findings are shown as mean ± SEM and p-value of less than 0.05 was considered statistically significant. Ethical Consideration Ethical Clearance was obtained from OOU Research Ethics Committee (OOU-REC) with reference: OOU/SCIENG/EC/0008/240924. Animals used were properly handled and cared for, with strict adherence to the standard protocol and institutional guidelines. RESULTS Seizure Severity Assessment KNA and phenobarbital demonstrated significant anticonvulsant effects in PTZ-induced seizure models, delaying the onset of seizure activities at different stages. Both compounds delayed the initial occurrence of first neck jerks (Fig. 1) and clonic, tonic-clonic seizure (lying on the side) (Fig. 2), corresponding to Racine scale 3 and 6 characterized by sharp spikes followed by spike-wave discharges and high-amplitude polyspikes spike-wave discharges EEG correlate respectively. Assessment of Thigmotaxis, Locomotor Activities and Discriminative Index The thigmotaxis index was reduced in the PTZ group compared to the control group. This reduction was ameliorated in the groups receiving low and high doses of KNA and PB. While the control, KNA, and PB groups spent more time near the walls, indicating a preference for the periphery, there was no statistically significant difference among the groups (Fig. 3) The total ambulatory distance covered during the OFT, measured as an indicator of locomotor activity, revealed no statistically significant differences among the groups. The PTZ, KNA, and PB groups showed a reduction in the distance traveled compared to the control (Fig. 4). The discriminative index, a measures learning and memory, was used to assess the animals' cognitive ability using the novel object recognition test (NORT). Except for the PB group, which had a statistically significant negative discriminative index (p < 0.05), all the groups displayed a positive discriminative index. In contrast to the other groups, the high dosage of KNA showed a comparatively higher discriminative index, indicating enhanced cognitive function (Fig. 5) Determination of the Level of Expression of NMDA Receptor in the Hippocampus The level of NMDA receptor expression was upregulated in the PTZ group compared to the control but was relatively downregulated in the KNA and PB groups. The effect of the KNA high dose was statistically significant (p < 0.05) compared to the PTZ group (Fig. 6) DISCUSSION The prophylactic potential of KNA and PB against PTZ-induced seizure in rats investigated in this study presented a delayed onset of myoclonic and clonic, tonic-clonic seizure with KNA showing a better performance than PB. The observed anticonvulsant activity of KNA is consistent with previous findings. Okoye et al. ( 2013 ) reported that KNA, isolated from the root bark of Annona senegalensis , significantly delayed the onset of myoclonic spasms and tonic-clonic phases in PTZ-induced seizures in mice, demonstrating a dose-dependent effect. Similarly, Lima et al. (2007) found that kaurenoic acid inhibited tonic hind-limb extension in spinal seizures induced by sudden cooling, with an effective dose (ED50) of 2.5 mg/kg. In PTZ-induced seizures, KNA increased the latency to seizure onset and provided 45% and 65% protection at doses of 0.625 and 1.25 mg/kg, respectively. The thigmotaxis index was reduced insignificantly in the PTZ group compared to the control group which was ameliorated in the treatment groups receiving low and high doses of KNA and PB. The control, KNA, and PB groups spent more time near the walls, indicating a preference for the periphery and positive thigmotaxis while the PTZ group had a negative thigmotaxis showing less anxiety-related behaviour. A high thigmotaxis index indicates high axiety-like behaviour while low thigmotaxis index indicates a low anxiety-like behaviour (Kraeuter et al., 2019 , Leon, 2023 ). In this study study, PTZ demonstrated an anxiolytic effect which is agreement with the report of Kajita and Mushiake ( 2024 ) stating that anxiety-like behaviors decreased compared with those of the control group in the open field, light/dark transition, and elevated plus-maze tests after 10 PTZ injections. As for the locomotive behaviour, the total ambulatory distance covered by the animals during exploration period, a measurement of voluntary movement have been shown to be sensitive to drug treatment and disease progression (Gibbs and Crosbie-Watson, 2017 ). The decreased in the total ambulatory distance covered observed in the PTZ group compared with the control which was improved by KNA and PB suggests sedative and/or motor impairment effects of PTZ on the animals. In this study, although PTZ did not significantly raise the amount of NMDA receptor expression in the PTZ group as compared to the control group statistically, nevertheless, the upregulation observed in this finding is in line with other research showing that PTZ raises NMDA receptor subunit expression (Rahimi-Madiseh et al., 2022 ). Compared to PTZ, kaurenoic acid and phenobarbital decrease the amount of NMDA receptor subunit expression, which this study demonstrated to be statistically significant in the prophylactic trial with a greater dosage of KNA. This agrees with previous studies by (Kapur, 2018 ; Prasad et al., 2002 ) which reported that NMDA receptor antagonist could play a crucial role in seizure reduction and/or termination when combined with benzodiazepines and neuroprotection against status epilepticus-induced cell loss in the hippocampus (Mazarati and Wasterlain, 1999 ; Niquet et al., 2017 ; Kapur, 2018 ). In conclusion, kaurenoic acid outperformed phenobarbital in its ability to preserve cognitive function and successfully reduce hippocampus NMDAR expression. It also showed excellent anticonvulsant and neuroprotective effects. These results lend credence to its potential as a safer treatment option for seizure control. Declarations Data Availability statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Antunes M, Biala G (2012) The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 13:93–110 Benarroch EE (2011) NMDA receptors: recent insights and clinical correlations. Neurology 76(20):1750–1757 Carter DS, Deshpande LS, Rafiq A, Sombati S, DeLorenzo RJ (2011) Characterization of spontaneous recurrent epileptiform discharges in hippocampal–entorhinal cortical slices prepared from chronic epileptic animals. Seizure 20(3):218–224 Chen S, Xu D, Fan L, Fang Z, Wang X, Li M (2022) Roles of N-Methyl-D-Aspartate Receptors (NMDARs) in Epilepsy. Front Mol Neurosci 14:797253 Dare SS, Folarin RO, Fakunle PB, Odubela OO, Oluwatunase GO, Ekanem PE, Shallie PD (2024) Evaluating the anticonvulsant potential of kaurenoic acid isolated from annona senegalensis leaves in pentylenetetrazole-induced seizures: behavioral, cognitive, and cellular insights in Wistar rats. J Experimental Clin Anat 21(2):235–242 Das CP, Prabhakar S, Lal V, Kharbanda PS (2002) Scleroderma, stroke, optic neuropathy: a rare association. Neurol India 50(4):504 Dhamad AE, Rhida MAA (2020) COVID-19: molecular and serological detection methods. PeerJ 8:e10180 Essiz S, Gencel M, Aktolun M, Demir A, Carpenter T, Servili B (2021) Correlated conformational dynamics of the human GluN1-GluN2A type N-methyl-D-aspartate (NMDA) receptor. J Mol Model 27:162 Geoff C, Fidler F, Vaux DL (2007) Error bars in experimental biology. J Cell Biol 177(1):7–11 Gibbs EM, Crosbie-Watson RH (2017) A simple and low-cost assay for measuring ambulation in mouse models of muscular dystrophy. J visualized experiments: JoVE, (130), 56772 Howard P, Twycross R, Shuster J, Mihalyo M, Rémi J, Wilcock A (2011) Anti-epileptic drugs. J Pain Symptom Manag 42(5):788–804 Kajita Y, Mushiake H (2024) Dynamic changes in seizure state and anxiety-like behaviors during pentylenetetrazole kindling in rats. Epilepsy Behav 159:110019 Kapur J (2018) Role of NMDA receptors in the pathophysiology and treatment of status epilepticus. Epilepsia Open 3(S2):165–168 Kraeuter AK, Guest PC, Sarnyai Z (2019) The Open Field Test for Measuring Locomotor Activity and Anxiety-Like Behavior. Methods in Molecular Biology (Clifton, N.J.), 1916, 99–103 Leon Y (2023), November 21 Open Field Tests: Mastering Animal Behavior Research. Amuza Inc. https://www.amuzainc.com/blog/how-to-set-up-an-open-field-test-for-animal-behavior-research/ Mazarati AM, Wasterlain CG (1999) N-methyl-D-asparate receptor antagonists abolish the maintenance phase of self-sustaining status epilepticus in rat. Neurosci Lett 265(3):187–190 Mazumder AG, Sharma P, Patial V, Singh D (2017) Crocin attenuates kindling development and associated cognitive impairments in mice via inhibiting reactive oxygen species-mediated NF‐κB activation. 120(5):426–433Basic & clinical pharmacology & toxicology Meldrum BS (1993) Excitotoxicity and selective neuronal loss in epilepsy. Brain Pathol 3(4):405–412 Mtunzi FM, Ejidike IP, Ledwaba I, Ahmed A, Pakade VE, Klink MJ, Modise SJ (2017) Solvent–solvent fractionations of Combretum erythrophyllum (Burch.) leave extract: Studies of their antibacterial, antifungal, antioxidant and cytotoxicity potentials. Asian Pac J Trop Med 10(7):670–679 Niquet J, Baldwin R, Norman K, Suchomelova L, Lumley L, Wasterlain CG (2017) Simultaneous triple therapy for the treatment of status epilepticus. Neurobiol Dis 104:41–49 Okoye TC, Akah PA, Omeje EO, Okoye FBC, Nworu CS (2013) Anticonvulsant effect of kaurenoic acid isolated from the root bark of Annona senegalensis. Pharmacol Biochem Behav 109:38–43 Prasad A, Williamson JM, Bertram EH (2002) Phenobarbital and MK-801, but not phenytoin, improve the long-term outcome of status epilepticus. Ann Neurol 51(2):175–181 Rahimi-Madiseh M, Lorigooini Z, Boroujeni SN, Taji M, Amini-Khoei H (2022) The Role of the NMDA Receptor in the Anticonvulsant Effect of Ellagic Acid in Pentylenetetrazole-Induced Seizures in Male Mice. Behavioural Neurology, 2022(1), 9015842 Sivakumar S, Ghasemi M, Schachter SC (2022) Targeting NMDA receptor complex in management of epilepsy. Pharmaceuticals 15(10):1297 Tajima T, Hata K, Kusakabe J, Miyauchi H, Badshah JS, Kageyama S, Hatano E (2023) Anti-complement 5 antibody ameliorates antibody-mediated rejection after liver transplantation in rats. Front Immunol 14:1186653 Zhang XY, Diaz-delCastillo M, Kong L, Daniels N, MacIntosh-Smith W, Abdallah A, Domanski D, Sofrenovic D, Yeung TP, Skel), Valiente D, Vollert J, Sena E, Rice AS, Soliman N (2023) A systematic review and meta-analysis of thigmotactic behaviour in the open field test in rodent models associated with persistent pain. PLoS ONE, 18(9), e0290382 Additional Declarations The authors declare no competing interests. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6829238","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467210084,"identity":"5590d14c-e732-4b95-ad97-506372c60d65","order_by":0,"name":"Samuel Sunday Dare","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYFCCBCCuSGAwIFHLGZK1MLaRokW+PfnZh4/z0uTNGZgffmBsqyWsxeDMM+OZM7flGO5sYDOWYGw7ToQWiQRjZt5tFYwbDjCYAV14jAiHzUj/zPx3ToX9hgPs34jTwnAjx5iZsSEnccMBHpAtNUQ47MybYsaeY2nJO5t5iiUSzh0gwmHt6ZsZftQk225nb9/44UNZHREOgwNmBlAcHSZFCwSQZMsoGAWjYBSMEAAAfLQ5MgQCu6EAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-7022-9818","institution":"Kabale University, Uganda","correspondingAuthor":true,"prefix":"","firstName":"Samuel","middleName":"Sunday","lastName":"Dare","suffix":""},{"id":467210085,"identity":"f173b1f3-d801-49c5-8db3-f0a2632bf9e9","order_by":1,"name":"Folarin O. Royhaan","email":"","orcid":"","institution":"Olabisi Onabanjo University, Ago-Iwoye, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Folarin","middleName":"O.","lastName":"Royhaan","suffix":""},{"id":467210086,"identity":"0b6aa77b-0309-44ea-81d6-a9851a894d78","order_by":2,"name":"Bamidele P. Fakunle","email":"","orcid":"","institution":"Olabisi Onabanjo University, Ago-Iwoye, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Bamidele","middleName":"P.","lastName":"Fakunle","suffix":""},{"id":467210087,"identity":"a9a29f36-c5f7-4e0e-87af-606a87ab71fd","order_by":3,"name":"Olukayode O. Odubela","email":"","orcid":"","institution":"Olabisi Onabanjo University, Ago-Iwoye, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Olukayode","middleName":"O.","lastName":"Odubela","suffix":""},{"id":467210088,"identity":"24de7af3-25e2-4f4c-9617-47777f890007","order_by":4,"name":"Gideon O. Oluwatunase","email":"","orcid":"","institution":"University of Medical Sciences, Ondo State, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Gideon","middleName":"O.","lastName":"Oluwatunase","suffix":""},{"id":467210089,"identity":"89050599-dec3-4044-abfb-ee34a7be7186","order_by":5,"name":"Edgar Fernandez Mario","email":"","orcid":"","institution":"Kabale University, Uganda","correspondingAuthor":false,"prefix":"","firstName":"Edgar","middleName":"Fernandez","lastName":"Mario","suffix":""},{"id":467210090,"identity":"0a45676b-e927-4355-b49d-3b7d4334f0f2","order_by":6,"name":"Peter Etim Ekanem","email":"","orcid":"","institution":"Kabale University, Uganda","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"Etim","lastName":"Ekanem","suffix":""},{"id":467210091,"identity":"d4c4033e-451b-4fe6-84d4-e2784e683b49","order_by":7,"name":"Philemon D. Shallie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIiWNgGAWjYDACCQbGAw8MQAzGxgMgAX4QkVCAVwvDgQSIlgawFskGkBYDQlpgDBBtACFx65Cf3fzgQEKBDYO5dHPDgY85NnnG51cnfgA6VZ5f7ABWLQZ3jhkAHZbGYDnnYMPBmdvSis1uvN0sAXSY4czZCdi1AGWBWg4zGNxIbDjMu+1w4rYbZzeAtCQY3MauRX5G+geglv8QLX+BWjbPOLv5Bz4tDDdyQLYcgGhhBGrZwN+7Da8tBjdyCoBaknksZyQ2HOzdlpY44wbvNosEAwmcfgE6bOODD3/s5Mwl0h8++LnNJrG//+zmmz8qbOT5pXE4DAp4EBEhAVYpgVc5xIVwFv8BwqpHwSgYBaNgRAEA0hBt6WwrcOoAAAAASUVORK5CYII=","orcid":"","institution":"University of Missouri-Kansas City, USA","correspondingAuthor":true,"prefix":"","firstName":"Philemon","middleName":"D.","lastName":"Shallie","suffix":""}],"badges":[],"createdAt":"2025-06-05 12:40:07","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6829238/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6829238/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84182734,"identity":"5b08e71d-7ab3-424f-ae1b-54154bf2e129","added_by":"auto","created_at":"2025-06-09 04:24:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102135,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3.5: The Novel Object Recognition Test Arena: A = Familiar phase with two familiar objects (green). B = Discriminative phase with one familiar object (green) and one novel object (red).\u003c/p\u003e","description":"","filename":"3.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/4d8cd41dd7e593b34b87e258.png"},{"id":84182735,"identity":"7c68331a-e584-420f-a8bf-077eff2c7d4f","added_by":"auto","created_at":"2025-06-09 04:24:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94062,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/56a0172f26c9d0f5a6401d8c.png"},{"id":84183313,"identity":"9263f3ec-d362-4b16-b8d1-f382692bee7d","added_by":"auto","created_at":"2025-06-09 04:40:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128163,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/f3e02b6d2ffe02f99c7390df.png"},{"id":84183312,"identity":"88ac0aea-62a9-406b-a83a-a027804712f4","added_by":"auto","created_at":"2025-06-09 04:40:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127306,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/26349b236d2180e78b92818d.png"},{"id":84182743,"identity":"2558cc44-5af0-46cf-9873-43bba9545f6c","added_by":"auto","created_at":"2025-06-09 04:24:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109449,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/a7d673cf010dc888207d51f2.png"},{"id":84182861,"identity":"8e21aa0a-ce6a-4fcb-8944-8cc85ad0b6d6","added_by":"auto","created_at":"2025-06-09 04:32:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":99133,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/897b56cc8010828d0863e110.png"},{"id":84183315,"identity":"1b08ad1e-f029-48ac-96ed-a18f2d2bf175","added_by":"auto","created_at":"2025-06-09 04:40:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":168218,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/62a172c19639122fb6d96509.png"},{"id":84183846,"identity":"b382d809-a9fb-4c21-9017-075ab1c6e762","added_by":"auto","created_at":"2025-06-09 04:56:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1359858,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6829238/v1/e3d3c739-b085-407b-8873-5bcd99c1748e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eKaurenoic Acid from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAnnona senegalensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAttenuates Hippocampal NMDA Receptor Overexpression and Enhances Cognitive Function in a Rat Model of PTZ-Induced Seizures\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eN-methyl-D-aspartate receptors (NMDARs) overexcitation has been linked to neurological conditions that induce neuronal death, including epilepsy, stroke, Alzheimer's disease (AD), and Parkinson's disease, according to related research (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Essiz et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The receptor is composed of 4 subunits derived from 3 gene families: GluN1-3. Each receptor is a tetramer composed of 2 GluN1 subunits and either 2 GluN2 or 2 GluN3 subunits. The NMDAR's pharmacological regulation is determined by the different subunit binding site combinations. The NR2 subunit contains the glutamate binding site, while the NR1 subunit has the glycine binding site. Different neuroanatomic manifestations are seen for these binding sites (Kapur, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sivakumar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn epilepsy, NMDAR regulation has drawn a lot of attention due to its role in the brain's neuroexcitatory and functional plasticity. NMDARs' functions and mechanisms in epilepsy are yet unknown, but they have been shown to contribute to seizures. During seizures in temporal lobe epilepsy (TLE), glutamate levels in the extracellular fluid increase which can directly activate NMDARs and result in neuroexcitatory injury. Cognitive impairment may be exacerbated by impaired NMDAR function. It should come as no surprise that NMDARs are a very desirable therapeutic target. Autoantibodies against NMDARs cause a distinctive condition that highlights the vital role these receptors play in behavior, cognition, motor, respiratory, and autonomic regulation (Benarroch, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUsing a range of techniques, including immunoblotting, binding affinities, and evaluation of subunit gene expression, some researchers have assessed the potential changes of NMDARs in epilepsy, given that NMDAR activity is crucial for neuronal excitation in the central nervous system. The primary excitatory input to the hippocampus is glutamatergic impulses from the entorhinal cortex, and a change in glutamate-mediated excitability may play a role in the pathophysiology of epileptic discharges (Carter et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Furthermore, as NMDAR antagonists offer defense against such damage, NMDARs may be in charge of the selective excitotoxic cell death of certain hippocampus neuronal populations brought on by seizures (Meldrum, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Research has demonstrated that certain AEDs influence glutamatergic synapses via NMDA receptors, which are typically excitatory amino acid (glutamate) receptors.\u003c/p\u003e \u003cp\u003eBy blocking these excitatory receptors, these AEDs lessen impulse conduction (Das et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Howard et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Numerous studies have examined the role of NMDARs in epileptic seizures and kaurenoic acid, a deterpenoids isolated from \u003cem\u003eAnnona senegalensis\u003c/em\u003e root back extract has shown an anticonvulsant potential and phenobarbital with notable effects (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Okoye et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e); however the relationship between kaurenoic acid activities and NMDARs with respect to epileptic seizure is unclear. This study is aimed to investigate the prophylactic potential of kaurenoic acid isolated from \u003cem\u003eAnnona senegalensis pers\u003c/em\u003e leaves on the level of expression of NMDARs in the hippocampus of PTZ-induced seizure in Wistar rats.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePlant Material, Extraction and Isolation of Kaurenoic Acid\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eAnnona senegalensis\u003c/em\u003e leaves were collected from the \u0026ldquo;Ishaka-Bushenyi\u0026rdquo; Municipality along Mbarara-Kasese Road, Western Uganda. Geographic coordinates are 0.5424\u0026deg; S and 30.1965\u0026deg; E. The Plant was identified harvested and processed to obtain 100g dry powder of the aqueous crude according to Dare et al, \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eTo isolate kaurenoic acid, solvent\u0026ndash;solvent fractionation by separation funnel method with methanol, ethyl acetate, and n-hexane in the increasing polarity order was employed to obtain the respective fractions. To carry out this process, the dried crude aqueous extract was transferred to a separation funnel, equal volume of n-hexane added and the funnel was shaken vigorously to partition non-polar compounds into the n-hexane layer. The funnel was allowed to settle then the upper (n-hexane) layer was separated from the lower (aqueous) layer. For ethyl acetate extraction, ethyl acetate was added to the remaining aqueous extract in the separation funnel, shaken vigorously to separate the semi-polar compounds including kaurenoic acid, which partitioned into the ethyl acetate phase and allowed to settle.\u003c/p\u003e\n \u003cp\u003eThe layer of ethyl acetate was gathered and allowed to evaporate at a lower pressure. A silica gel column was used for purification by column chromatography, with n-hexane serving as the initial elution solvent. By dissolving the concentrated ethyl acetate extract in a tiny amount of solvent (n-hexane acetate, 90:10), the sample was put onto the column. To obtain a sub-fraction (80:20), the ethyl acetate fraction was further eluted using gradient mixes of n-hexane and ethyl acetate (90:10 to 20:80). Based on the yield and potency, this sub-fraction was further identified as kaurenoic acid (Okoye et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mtunzi et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eHPLC Characterization of Kaurenoic Acid\u003c/h3\u003e\n\u003cp\u003eMethanol, ethyl acetate, and n-hexane extract of \u003cem\u003eAnnona senegalensis\u003c/em\u003e were subjected to HPLC analysis using a JASCO LC-4000 Series, Japan, HPLC system. The system contained employed an asymmetric twin-piston solvent delivery system SSQD (Slow Suction, Quick Delivery) which provides significantly better flow and pressure profiles and UV-Visible Detectors (UV-4070/4075). Reverse phase column chromatographic separation was performed using Octadecylsilane, Size: 4.6mm X 150\u0026ndash;250mm column with ethyl acetate mobile phase. For sample preparation, filtration was carried out to remove any particulates that could clog the column and the sample was diluted to an appropriate concentration suitable for analysis (50\u0026micro;m). For solvent selection, HPLC-graded solvent which are free from impurities (water, 99.8% methanol, 99.8% acetonitrile, 99.8% isopropanol, 99.8% ethanol and 99.9% ethyl acetate were used. Samples were stored in sterile samples and refrigerated to maintain stability. The standard concentrations used for analytical applications ranged from 10\u0026ndash;70 \u0026micro;g/mL, 1\u0026ndash;2% of the total column volume and 1 \u0026micro;g/\u0026micro;L concentration of the extract.\u003c/p\u003e\n\u003cp\u003eData collection and processing were done using ChromNAV Chromatography Data System (CDS) software and the detection wavelength was between 200 and 400 nm. The injection volume of samples and reference solutions was 3\u0026ndash;5\u0026micro;L, and the flow rate was 3.0 mL/min. By monitoring retention time and examining UV spectra, the peaks were found at 20 and 400nm Retension Time (5min) a little quantity of the reference standards.\u003c/p\u003e\n\u003ch3\u003eExperimental Animals and Design\u003c/h3\u003e\n\u003cp\u003eFor this investigation, thirty (30) adult male wistar rats were acquired and kept in the animal house in hygienic cages with a 12-hour light and 12-hour dark cycle at an average ambient temperature between 18\u003csup\u003e0\u003c/sup\u003eC and 26\u003csup\u003e0\u003c/sup\u003eC, fed with pelletized rat diet and given unlimited water. Two weeks to the start of the experiment, the animals were weighed (150\u0026ndash;250 grams) and grouped into five (5) groups at random (n\u0026thinsp;=\u0026thinsp;6) with identification marks and allowed to become familiar with their new environment. Group 1 (received distilled water as negative control) and Group 2 (received PTZ as positive control) while Group 3 (received low dose of KNA), Group 4 (received high dose of KNA) and Group 5 (received phenobarbital) before PTZ.\u003c/p\u003e\n\u003ch3\u003eSeizure Activity and Behavioural Study\u003c/h3\u003e\n\u003cp\u003ePTZ (Sigma\u0026ndash;Aldrich, P6500, Lot No. MKCM4261) was used to induce seizures and seizure-behaviour of individual animal was recorded with a PC Logitech B905 digital Webcam connected to a laptop in a square open-field standard box (100 x 100 x 50 cm) for 10 minutes. The seizure behavioural activities of the animals were scored according to Racine scale 3 and 6 characterized by myoclonic neck jerks with sharp spikes followed by spike-wave discharges EEG correlates and clonic, tonic-clonic seizures, and wild jumping with high amplitude, spike-wave discharges EEG correlate respectively. To investigate the prophylactic potential of KNA and PB against PTZ-induced seizure, 400mg/kg and 800mg/kg of KNA were administered orally to group 3 and group 4 respectively while group 5 received 10mg/kg of phenobarbital (PB) thirty minutes before administration of PTZ (40mg/kg), and these were compared with group 1 and group 2 which received 1m/kg orally and 40mg/kg PTZ intraperitoneally.\u003c/p\u003e\n\u003ch3\u003eThigmotaxis, Locomotor Behaviour and Discriminative Index\u003c/h3\u003e\n\u003cp\u003eTo determine the thigmotaxtic index, a metric used to assess anxiogenic and anxiolytic behaviour, the percentage of the 10-minute test duration that the individual stays next to the maze\u0026apos;s outer wall, a sign of anxiety-like behavior was measured (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) while the total ambulatory distance covered during the open field test (OFT), measured as an indicator of locomotor activity. To ascertain the non-spatial or recognition, novel object recognition test (NORT) was employed. The test was conducted 20 to 24 hours after the medicine was administered in a 100 \u0026times; 100 cm square open-field standard box with walls 50 cm high. A Logitech B905 2MP Portable Webcam was mounted on top of the box and linked to a laptop to capture the animals\u0026apos; exploratory activities.\u003c/p\u003e\n\u003cp\u003eThe test process was divided into three stages: recognition, familiarization, and habituation. During the habituation phase, each animal was carefully put in the object-free box and given five minutes to explore. The initial exploration session of the habituation phase was followed by the familiarization phase 24 hours later. Each animal was given ten minutes to investigate after two identical plastic items (A and B) were positioned in the box\u0026apos;s neighboring corners, about ten centimeters from the walls. After fifteen minutes, item B was used to start the recognition phase. It was then swapped out for a new object C that was different in color and form but had a comparable size, texture, and weight. For ten minutes, each animal was left alone in the box, and the amount of time it spent exploring the objects was noted (Fig. 1)\u003c/p\u003e\n\u003cp\u003eThe videos recorded were analyzed using ANY-maze 7.3 software to track the object exploration time which was used to calculate the percentage novel object preference index [TC / (TC\u0026thinsp;+\u0026thinsp;TA) x 100] and discrimination ratio [(TC \u0026ndash; TA) / (TC\u0026thinsp;+\u0026thinsp;TA)], where TA and TC signify time spent exploring object A and C respectively. The object exploration time was calculated by making two virtual zones around each object (A and C) using the software and the time spent in each zone was calculated (Antunes and Biala, \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e Mazumder et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eLevel of Expression of NMDA Receptor Using ELISA\u003c/h2\u003e\n \u003cp\u003eRat N-Methyl-d-aspartate (NMDA) Receptor 2 ELISA Kit, Bioassay Technology Laboratory, Zhejiang, China (Dhamad and Abdal Rhida, 2020; Tajima et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e) was used to investigate the level of the expression of NMDA Receptor 2 in the hippocampus of the animals. Following the bahavioural tests, the animals were anaesthetized with halothane (Piramal Pharma Limited, India), brain was extracted and the hippocampus removed by micro-dissection. The hippocampal tissue was homogenized with buffer following established techniques or the manufacturer\u0026apos;s instructions, centrifuged and the supernatant collected. To carry out the assay, all the reagents, samples and standards were prepared brought to room temperature before use according to the user instruction. The sample and ELISA reagent were added into each well and incubate for 1 hour at 37\u0026deg;C. The plates were washed 5 times and the substrate solution A and B were added and incubated in the dark for 10 minutes at 37\u0026deg;C. Afterwards the stop solution was added and the blue color changed into yellow immediately. The optical density (OD value) of each well was determined immediately using a microplate reader set to 450 nm within 10 minutes after adding the stop solution.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eGraphPad (8.0.2) was used to perform an analysis of variance (ANOVA) and a Tukey post hoc test on the collected data (Geoff et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). The findings are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eEthical Consideration\u003c/h3\u003e\n\u003cp\u003eEthical Clearance was obtained from OOU Research Ethics Committee (OOU-REC) with reference: OOU/SCIENG/EC/0008/240924. Animals used were properly handled and cared for, with strict adherence to the standard protocol and institutional guidelines.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSeizure Severity Assessment\u003c/h2\u003e \u003cp\u003eKNA and phenobarbital demonstrated significant anticonvulsant effects in PTZ-induced seizure models, delaying the onset of seizure activities at different stages. Both compounds delayed the initial occurrence of first neck jerks (Fig.\u0026nbsp;1) and clonic, tonic-clonic seizure (lying on the side) (Fig.\u0026nbsp;2), corresponding to Racine scale 3 and 6 characterized by sharp spikes followed by spike-wave discharges and high-amplitude polyspikes spike-wave discharges EEG correlate respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Thigmotaxis, Locomotor Activities and Discriminative Index\u003c/h2\u003e \u003cp\u003eThe thigmotaxis index was reduced in the PTZ group compared to the control group. This reduction was ameliorated in the groups receiving low and high doses of KNA and PB. While the control, KNA, and PB groups spent more time near the walls, indicating a preference for the periphery, there was no statistically significant difference among the groups (Fig.\u0026nbsp;3)\u003c/p\u003e \u003cp\u003eThe total ambulatory distance covered during the OFT, measured as an indicator of locomotor activity, revealed no statistically significant differences among the groups. The PTZ, KNA, and PB groups showed a reduction in the distance traveled compared to the control (Fig.\u0026nbsp;4). The discriminative index, a measures learning and memory, was used to assess the animals' cognitive ability using the novel object recognition test (NORT). Except for the PB group, which had a statistically significant negative discriminative index (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), all the groups displayed a positive discriminative index. In contrast to the other groups, the high dosage of KNA showed a comparatively higher discriminative index, indicating enhanced cognitive function (Fig.\u0026nbsp;5)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the Level of Expression of NMDA Receptor in the Hippocampus\u003c/h2\u003e \u003cp\u003eThe level of NMDA receptor expression was upregulated in the PTZ group compared to the control but was relatively downregulated in the KNA and PB groups. The effect of the KNA high dose was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the PTZ group (Fig.\u0026nbsp;6)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe prophylactic potential of KNA and PB against PTZ-induced seizure in rats investigated in this study presented a delayed onset of myoclonic and clonic, tonic-clonic seizure with KNA showing a better performance than PB. The observed anticonvulsant activity of KNA is consistent with previous findings. Okoye et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported that KNA, isolated from the root bark of \u003cem\u003eAnnona senegalensis\u003c/em\u003e, significantly delayed the onset of myoclonic spasms and tonic-clonic phases in PTZ-induced seizures in mice, demonstrating a dose-dependent effect. Similarly, Lima et al. (2007) found that kaurenoic acid inhibited tonic hind-limb extension in spinal seizures induced by sudden cooling, with an effective dose (ED50) of 2.5 mg/kg. In PTZ-induced seizures, KNA increased the latency to seizure onset and provided 45% and 65% protection at doses of 0.625 and 1.25 mg/kg, respectively.\u003c/p\u003e \u003cp\u003eThe thigmotaxis index was reduced insignificantly in the PTZ group compared to the control group which was ameliorated in the treatment groups receiving low and high doses of KNA and PB. The control, KNA, and PB groups spent more time near the walls, indicating a preference for the periphery and positive thigmotaxis while the PTZ group had a negative thigmotaxis showing less anxiety-related behaviour. A high thigmotaxis index indicates high axiety-like behaviour while low thigmotaxis index indicates a low anxiety-like behaviour (Kraeuter et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Leon, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study study, PTZ demonstrated an anxiolytic effect which is agreement with the report of Kajita and Mushiake (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) stating that anxiety-like behaviors decreased compared with those of the control group in the open field, light/dark transition, and elevated plus-maze tests after 10 PTZ injections.\u003c/p\u003e \u003cp\u003eAs for the locomotive behaviour, the total ambulatory distance covered by the animals during exploration period, a measurement of voluntary movement have been shown to be sensitive to drug treatment and disease progression (Gibbs and Crosbie-Watson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The decreased in the total ambulatory distance covered observed in the PTZ group compared with the control which was improved by KNA and PB suggests sedative and/or motor impairment effects of PTZ on the animals. In this study, although PTZ did not significantly raise the amount of NMDA receptor expression in the PTZ group as compared to the control group statistically, nevertheless, the upregulation observed in this finding is in line with other research showing that PTZ raises NMDA receptor subunit expression (Rahimi-Madiseh et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompared to PTZ, kaurenoic acid and phenobarbital decrease the amount of NMDA receptor subunit expression, which this study demonstrated to be statistically significant in the prophylactic trial with a greater dosage of KNA. This agrees with previous studies by (Kapur, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Prasad et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) which reported that NMDA receptor antagonist could play a crucial role in seizure reduction and/or termination when combined with benzodiazepines and neuroprotection against status epilepticus-induced cell loss in the hippocampus (Mazarati and Wasterlain, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Niquet et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kapur, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, kaurenoic acid outperformed phenobarbital in its ability to preserve cognitive function and successfully reduce hippocampus NMDAR expression. It also showed excellent anticonvulsant and neuroprotective effects. These results lend credence to its potential as a safer treatment option for seizure control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability statement\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"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntunes M, Biala G (2012) The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 13:93\u0026ndash;110\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenarroch EE (2011) NMDA receptors: recent insights and clinical correlations. Neurology 76(20):1750\u0026ndash;1757\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarter DS, Deshpande LS, Rafiq A, Sombati S, DeLorenzo RJ (2011) Characterization of spontaneous recurrent epileptiform discharges in hippocampal\u0026ndash;entorhinal cortical slices prepared from chronic epileptic animals. Seizure 20(3):218\u0026ndash;224\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Xu D, Fan L, Fang Z, Wang X, Li M (2022) Roles of N-Methyl-D-Aspartate Receptors (NMDARs) in Epilepsy. Front Mol Neurosci 14:797253\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDare SS, Folarin RO, Fakunle PB, Odubela OO, Oluwatunase GO, Ekanem PE, Shallie PD (2024) Evaluating the anticonvulsant potential of kaurenoic acid isolated from annona senegalensis leaves in pentylenetetrazole-induced seizures: behavioral, cognitive, and cellular insights in Wistar rats. J Experimental Clin Anat 21(2):235\u0026ndash;242\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas CP, Prabhakar S, Lal V, Kharbanda PS (2002) Scleroderma, stroke, optic neuropathy: a rare association. Neurol India 50(4):504\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhamad AE, Rhida MAA (2020) COVID-19: molecular and serological detection methods. PeerJ 8:e10180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEssiz S, Gencel M, Aktolun M, Demir A, Carpenter T, Servili B (2021) Correlated conformational dynamics of the human GluN1-GluN2A type N-methyl-D-aspartate (NMDA) receptor. J Mol Model 27:162\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeoff C, Fidler F, Vaux DL (2007) Error bars in experimental biology. J Cell Biol 177(1):7\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGibbs EM, Crosbie-Watson RH (2017) A simple and low-cost assay for measuring ambulation in mouse models of muscular dystrophy. J visualized experiments: JoVE, (130), 56772\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoward P, Twycross R, Shuster J, Mihalyo M, R\u0026eacute;mi J, Wilcock A (2011) Anti-epileptic drugs. J Pain Symptom Manag 42(5):788\u0026ndash;804\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKajita Y, Mushiake H (2024) Dynamic changes in seizure state and anxiety-like behaviors during pentylenetetrazole kindling in rats. Epilepsy Behav 159:110019\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapur J (2018) Role of NMDA receptors in the pathophysiology and treatment of status epilepticus. Epilepsia Open 3(S2):165\u0026ndash;168\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKraeuter AK, Guest PC, Sarnyai Z (2019) The Open Field Test for Measuring Locomotor Activity and Anxiety-Like Behavior. Methods in Molecular Biology (Clifton, N.J.), 1916, 99\u0026ndash;103\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeon Y (2023), November 21 Open Field Tests: Mastering Animal Behavior Research. Amuza Inc. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.amuzainc.com/blog/how-to-set-up-an-open-field-test-for-animal-behavior-research/\u003c/span\u003e\u003cspan address=\"https://www.amuzainc.com/blog/how-to-set-up-an-open-field-test-for-animal-behavior-research/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazarati AM, Wasterlain CG (1999) N-methyl-D-asparate receptor antagonists abolish the maintenance phase of self-sustaining status epilepticus in rat. Neurosci Lett 265(3):187\u0026ndash;190\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazumder AG, Sharma P, Patial V, Singh D (2017) Crocin attenuates kindling development and associated cognitive impairments in mice via inhibiting reactive oxygen species-mediated NF‐κB activation. 120(5):426\u0026ndash;433Basic \u0026amp; clinical pharmacology \u0026amp; toxicology\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeldrum BS (1993) Excitotoxicity and selective neuronal loss in epilepsy. Brain Pathol 3(4):405\u0026ndash;412\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMtunzi FM, Ejidike IP, Ledwaba I, Ahmed A, Pakade VE, Klink MJ, Modise SJ (2017) Solvent\u0026ndash;solvent fractionations of Combretum erythrophyllum (Burch.) leave extract: Studies of their antibacterial, antifungal, antioxidant and cytotoxicity potentials. Asian Pac J Trop Med 10(7):670\u0026ndash;679\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiquet J, Baldwin R, Norman K, Suchomelova L, Lumley L, Wasterlain CG (2017) Simultaneous triple therapy for the treatment of status epilepticus. Neurobiol Dis 104:41\u0026ndash;49\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkoye TC, Akah PA, Omeje EO, Okoye FBC, Nworu CS (2013) Anticonvulsant effect of kaurenoic acid isolated from the root bark of Annona senegalensis. Pharmacol Biochem Behav 109:38\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrasad A, Williamson JM, Bertram EH (2002) Phenobarbital and MK-801, but not phenytoin, improve the long-term outcome of status epilepticus. Ann Neurol 51(2):175\u0026ndash;181\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahimi-Madiseh M, Lorigooini Z, Boroujeni SN, Taji M, Amini-Khoei H (2022) The Role of the NMDA Receptor in the Anticonvulsant Effect of Ellagic Acid in Pentylenetetrazole-Induced Seizures in Male Mice. Behavioural Neurology, 2022(1), 9015842\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSivakumar S, Ghasemi M, Schachter SC (2022) Targeting NMDA receptor complex in management of epilepsy. Pharmaceuticals 15(10):1297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTajima T, Hata K, Kusakabe J, Miyauchi H, Badshah JS, Kageyama S, Hatano E (2023) Anti-complement 5 antibody ameliorates antibody-mediated rejection after liver transplantation in rats. Front Immunol 14:1186653\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XY, Diaz-delCastillo M, Kong L, Daniels N, MacIntosh-Smith W, Abdallah A, Domanski D, Sofrenovic D, Yeung TP, Skel), Valiente D, Vollert J, Sena E, Rice AS, Soliman N (2023) A systematic review and meta-analysis of thigmotactic behaviour in the open field test in rodent models associated with persistent pain. PLoS ONE, 18(9), e0290382\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Olabisi Onabanjo University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"NMDA receptor, epileptic seizures, kaurenoic acid, hippocampus, cognition, neuroprotection","lastPublishedDoi":"10.21203/rs.3.rs-6829238/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6829238/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003cbr\u003e\nOveractivation of N-methyl-D-aspartate receptors (NMDARs) is implicated in seizure-induced excitotoxicity and neurodegeneration. Kaurenoic acid, a diterpenoid from \u003cem\u003eAnnona senegalensis\u003c/em\u003e, has shown anticonvulsant potential, but its modulatory effect on NMDARs in epilepsy remains unclear. This study investigates the neuroprotective and cognitive effects of kaurenoic acid and its impact on hippocampal NMDAR expression in a PTZ-induced seizure model in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003e\u003cbr\u003e\nKaurenoic acid (KNA) was isolated from the ethyl acetate fraction of \u003cem\u003eA. senegalensis\u003c/em\u003e leaves. Thirty male Wistar rats were divided into five groups (n=6): control, PTZ only, KNA (400 or 800 mg/kg, p.o) + PTZ, and phenobarbital (PB, 10 mg/kg, i.p) + PTZ. Seizure activity was scored using Racine’s scale. Cognitive performance was assessed via the Open Field and Novel Object Recognition tests. Hippocampal NMDAR levels were measured using ELISA. Data were analyzed with ANOVA and Tukey’s post hoc test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003cbr\u003e\nKNA significantly increased seizure thresholds in myoclonic (173±33 s) and tonic-clonic seizures (444±41 s), comparable to PB (233±43 s and 534±42 s), p\u0026lt;0.0001. KNA improved thigmotaxis (98%) and preserved cognitive function with a positive discrimination index (44±17%), whereas PB impaired cognition (−61±18%). Locomotor activity decreased with both KNA and PB. PTZ elevated NMDAR expression (27±1 µmol) compared to control (22.8±0.2 µmol), while both KNA and PB reduced NMDAR levels (17±3 and 23±1 µmol, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003cbr\u003e\nKaurenoic acid demonstrated strong anticonvulsant and neuroprotective effects, effectively lowering hippocampal NMDAR expression and preserving cognitive function better than phenobarbital. These findings support its therapeutic potential as a safer alternative for seizure management.\u003c/p\u003e","manuscriptTitle":"Kaurenoic Acid from Annona senegalensisAttenuates Hippocampal NMDA Receptor Overexpression and Enhances Cognitive Function in a Rat Model of PTZ-Induced Seizures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 04:24:54","doi":"10.21203/rs.3.rs-6829238/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":"4520fdc2-0ad3-4db7-894e-f0558cc5731e","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49586350,"name":"Molecular Biology"},{"id":49586351,"name":"Cellular \u0026 Molecular Neuroscience"},{"id":49586352,"name":"Neurobiology of Disease"},{"id":49586353,"name":"Animal Behavior"}],"tags":[],"updatedAt":"2025-06-09T04:24:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 04:24:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6829238","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6829238","identity":"rs-6829238","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}