Ameliorative role of Phosphodiesterase-5 (PDE-5) inhibitor “Avanafil” via modulating cAMP & cGMP Pathway against Alzheimer’s disease

Ameliorative role of Phosphodiesterase-5 (PDE-5) inhibitor “Avanafil” via modulating cAMP & cGMP Pathway against Alzheimer’s disease | 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 Ameliorative role of Phosphodiesterase-5 (PDE-5) inhibitor “Avanafil” via modulating cAMP & cGMP Pathway against Alzheimer’s disease Mohd Talib -, Nazia Siddiqui, Prabhash Nath Tripathi, Ankit Chaudhary This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5911692/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Aug, 2025 Read the published version in Neurochemical Research → Version 1 posted 16 You are reading this latest preprint version Abstract Alzheimer's disease (AD) is the utmost age-linked neuro-degenerative conditions, marked via gradual deterioration of cognitive abilities and continues to be a significant worldwide health issue. Etiology of AD is linked to neurobehavioral variations, deposition of Aβ, p-Tau, activations of GSK-3β, and fluctuations in cyclic nucleotides including cAMP & cGMP. As per evidence, PDE-5 inhibitors are able to boost cAMP & cGMP levels and other etiological hallmarks, which could be a novel AD cure. The main objective of present study was to examine therapeutic potential of Avanafil in a rat model of AD induced by administering 60mg/kg of D-galactose (D-galac) and 10mg/kg of Aluminium chloride (AlCl 3 ) for a period of 42 days. Following this, 28 days of therapy with two different doses of Avanafil (3mg/kg and 6mg/kg) was given. Towards end of treatment, locomotor activity & Morris water maze were performed. Rats were then euthanized and hippocampus was isolated for biochemical parameters & histological investigation. Results revealed that both neurobehavioral parameters exhibits significant difference in treatment group as compared to toxic group. Alterations in level of AchE, Aβ (1–42), GSK-3β, p-Tau, TNF-α, IL-1β, & IL-6, cAMP, cGMP & BDNF, and oxidative stress were significantly reversed towards normal level in the treatment group when compared to toxic rats. Histopathological changes by H&E staining showed significant difference in treatment vs. toxic rats. The current investigation suggested that Avanafil improves memory by improving cAMP and cGMP pathways, implying that it may have therapeutic prospective in cognitive deficiencies linked with Alzheimer's disease. Alzheimer’s disease Phosphodiesterase Inhibitor cAMP cGMP Avanafil Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Alzheimer's disease (AD), a long-term neurodegenerative disorder characterized by an advancing decline in cognitive function, stands as a significant origin of dementia in the elderly globally. According to recent epidemiological estimates, the count of individuals affected by dementia is projected to triple by 2050 compared to 2010, with a substantial proportion stemming from AD [ 1 ]. Furthermore, it's been postulated that due to rising life expectancies and ongoing shifts in living conditions, the prevalence of AD could reach 1 in 85 individuals by 2050 [ 2 ]. Over the past several decades, scholars have suggested that AD closely links to the extreme production and accumulation of amyloid-β (Aβ) peptide within the brain, yet the precise underlying mechanism remains elusive. Particularly, most current investigations support the role of inflammation as a crucial catalyst in AD progression and the gradual erosion of cognitive abilities. This perspective has gained traction due to the recent revelation of the antimicrobial properties of the Aβ peptide, which opens up the possibility that infections role to the development of AD and proposes that the genesis and advancement of Aβ plaques might be triggered by infections [ 3 ]. The strong connection between AD development and oxidative stress has been acknowledged for quite some time [ 4 , 5 ]. Oxidative and nitrosative stresses play a crucial role in the beginning of AD pathological processes [ 6 , 7 ]. These stresses arise from the inequality between the generation of reactive nitrogen species (RNS), reactive oxygen species (ROS) and their elimination through internal antioxidant enzyme systems, such as superoxide dismutase (SOD) [ 8 ]. SOD acts as the primary defence against ROS produced during oxidative stress within the body [ 9 ]. The production of ROS is indirectly assessed by examining malondialdehyde (MDA), a by-product of lipid peroxidation (LPO) caused by free radicals. When nitric oxide (NO) is excessively produced, it leads to the formation of various harmful RNS that contribute to nitrosative stress [ 10 ]. Neuronal apoptosis, a significant aspect of AD development [ 11 , 12 ], is partially governed by the Bcl-2 family, with Bcl-2 itself being neuroprotective against apoptosis [ 13 , 14 ]. Neuroinflammation play a vital role in AD onset and progression [ 15 ], with TNF-α being a major regulator of proinflammatory responses in the brain [ 16 ]. Inhibiting TNF-α has shown potential in improving cognitive decline in patients [ 17 ]. Therefore, effective AD therapeutic strategies should focus on restoring cholinergic hypothesis, enhancing neuro-synaptic plasticity, and alleviating oxidative damage, neuronal apoptosis, and neuroinflammation. Recent research has revealed that chronic administration of a combination of D-galac and AlCl 3 promoted to impaired learning and memory functions [ 18 – 20 ]. This combination induces widespread neurodegeneration, including disruptions in the cholinergic coordination, mitochondrial dysfunction, apoptosis, and oxidative stress. It effectively replicates AD-like pathological changes, including the accumulation of amyloid plaques and the formation of neurofibrillary tangles. As a result, this approach has been used to create an animal model resembling AD, referred to as a non-transgenic AD animal model [ 21 – 26 ]. This particular animal model has demonstrated its worth in the exploration of neurodegenerative disease mechanisms such as AD and in conducting drug screening activities [ 27 ]. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), play a fundamental role as secondary messengers in brain signal transduction, governing PKA/BDNF and NO/PKG/BDNF pathways [ 28 ]. Elevating their concentrations activates brain-derived neurotrophic factor (BDNF) and added signaling molecules, contributing to spatial learning and memory functions [ 29 ]. Various compounds influencing the cAMP or cGMP signaling route have been studied in animal models of AD, with a particular emphasis on Phosphodiesterase (PDE) inhibitors due to their favourable safety profile [ 30 ]. Consequently, cGMP triggers its downstream protein kinase-G (PKG), which, in turn, activates miscellaneous substrates, including the transcription factor cyclic adenosine monophosphate (cAMP) response element-binding element (CREB). This activation promotes neurotransmission, synaptic plasticity, and the formation of memories [ 32 – 34 ]. PKG also stimulates the phosphatidylinositol 3-kinase (PI3k)/Akt signaling pathway, which exerts neuroprotective effects by impeding apoptosis [ 35 , 36 ].Accumulating evidence suggests that utilizing PDE inhibitors to reinstate cAMP and cGMP levels in the hippocampus could be a strategy to avert cognitive impairments. Previous research demonstrated that PDE inhibitors, definitely targeted at cAMP and cGMP, effectively saved memory and synaptic plasticity impairments in rodent models of AD. This study marks the initial exploration into the effects of Avanafil on Alzheimer's disease (AD) like characteristics, achieved via modulation of the cAMP and cGMP pathways. The study was conducted using an AD model induced by D-galac and AlCl3 in Albino Wistar rats. 2. Drugs and chemicals Female Wistar rats weight range 250gm and 300gm were obtained from the Animal House at Meerut Institute of Engineering and Technology, located at Baghpat Bypass, Meerut (UP), 250005, India. These rats were then accommodated in polypropylene cages, with eight rats housed together in pairs. They were placed under controlled environmental conditions, including a room temperature of 25 ± 2°C, a relative humidity range of 55–65%, and a 12-hour light/12-hour dark cycle, providing a regular day-night rhythm. The rats were provided unrestricted access to a standard commercial pellet diet and water. The entire experimental procedures adhered to the guidelines set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The Institutional Animal Ethics Committee (IAEC) of MIET, Meerut had reviewed and granted approval for the experimental protocol, bearing the registration number 711/PO/ReRc/S/02/CPCSEA 29/10/2002 under protocol number 88. 2.1 Drugs and chemicals Avanafil was purchased from Sigma Aldrich, USA. Donepezil was procured from Alkem Laboratory Limited Company, India. AlCl 3 , D-galac and Carboxymethyl cellulose was purchased from SISCO Research Laboratories. The Aβ, GSK-3β, cAMP, cGMP and BDNF Elisa kits were obtained from Genxbio in India, while the TNF-α, IL-1β, and IL-6 Elisa kits were acquired from Krishgen Biosystems, also based in India. Rats in the two different treatment groups received two dissimilar doses of Avanafil (3mg/kg and 6mg/kg) respectively. 3mg/kg and 6mg/kg dose was based on the prior evidence existing and dissolve 0.25% Carboxymethyl cellulose [ 36 ]. The dose of AlCl 3 10mg/kg + D-galac 60mg/kg is based on the evidence in previous study [ 37 ]. 2.2 Treatment schedule Rats were randomly and uniformly distributed into 8 groups, including 6 rats/group and groups were demonstrated as Group A (Control) : Rats were administered with water orally for 10 weeks; Group B (Sham) : Rats were given 10ml/kg, Carboxymethyl Cellulose (CMC) orally for 28 days; Group C (Toxic Group (AD)) : Rats were given AlCl 3 10mg/kg + D-galac 60mg/kg intraperitoneally (I.P) dissolved in distilled water to induce AD up to 42 days; Group D, and Group E (AD + Treatment) : Rats were given AlCl 3 10mg/kg + D-galac 60mg/kg intraperitoneally (I.P) dissolved in distilled water to induce AD up to 42 days and then administered Avanafil 3mg/kg and 6mg/kg orally for 28 days; Group F (AD + DNP) Rats given AlCl 3 10mg/kg + D-galac 60mg/kg intraperitoneally (I.P) dissolved in distilled water to induce AD up to 42 days and then administered Donepezil 5mg/kg orally for 28 days. Group F (Avanafil Perse) : Administered Avanafil 6mg/kg orally for 28days. Group H (DNP Perse) : Administered Donepezil 5mg/kg orally for 28days. 2.3 Behavioral Parameters 2.3.1 Locomotor Activity The assessment of motor impairment in all groups of rodents involved conducting locomotor activity (LA) using a video path analyzer. The LA data were collected during standard research facility hours from 8:30 a.m. to 1:30 p.m. The experiment took place in an open field enclosed by an acrylic box measuring 40.6 x 40.6 x 40.6 cm³, equipped with two sets of photo beams (16 beams in each dimension, spaced 2.5 cm apart). A True Scan 2.0 version computer interface (Coulbourn Instruments, Allentown, Pennsylvania, USA) connected to the activity meter recorded LA by counting beam breaks at a 100 ms sampling rate. To maintain cleanliness and prevent interference from olfactory cues, the cage underwent a 70% alcohol cleaning process between each test. Each rat in every group underwent a 30-minute acclimatization period, followed by a 20-minute recording of LA. Digital recording was done by detecting interruptions in the infrared beams caused by the movements of the rats within the activity meter. The recorded components of locomotor activity included parameters such as move time (s), rest time (s), horizontal activity (cm), mean velocity (cm/s), and total movement (#) for each group [ 38 ]. 2.3.2 Morris water maze test Morris water maze (MWM) assessment was coordinated to study the memory & learning of rodents. This endeavour was substituted in a dull concealed water tank of width (130 cm) and height (50cm), finished off by water to the significance of 30cm and saved up to a temperature of 26 ± 2°C to avoid hypo-thermia in rodents. Lights and sounds were additionally kept up with to stay away from any blunder. The tank was effectively divided into four quadrants, namely East, West, North and South. The time in both securing and test preliminary was estimated by physically process. In securing stage, the dark shaded rectangular departure foundation of 8×6 cm 2 region was lowered immersed in one quadrant (south-west) and remained immovable in a comparative quadrant all complete the examination. Rodents went through 4 successive preliminaries for 4 back to back days from various quadrants to investigate the secret stage (focus) inside the roof season of 60s and was permitted to sit onto the stage for 30Seconds. If rodents neglected to get away from the stage, were actually organized to the stage. During every procurement preliminary, get away from idleness period (time taken by each rodent to distribute the secret stage) was renowned for 4 days. Test preliminary was completed on fifth day to survey the orientation memory of each rodent. In this preliminary, the stage was eliminated from the quadrant (south-west) and every rat allowed to allocate the secret stage quadrant for 60s.The % stay time consumed by the rodent to investigate the secret stage in the objective quadrant (south-west) and stage passage recurrence to cross the objective quadrant was record [ 38 ]. 2.4. Biochemical parameters 2.4.1. Brain homogenate preparation After the 10-weeks experimental period, all rats within every group were anesthetized using light ether and euthanized. After euthanasia, each rat was individually perfused with phosphate buffer saline (PBS). The brains were immediately removed from the skulls and washed with cool saline solution. The hippocampus tissues were extracted from the whole brain and then blended with cold phosphate buffer (0.1M; pH-7.4) at a centrifugation speed of 800 X g for 5 minutes, all performed at a temperature of 4°C. The homogenization process was carried out using ten times the volume of the tissue. The resulting supernatants were subjected to further centrifugation at 10,000g for 10 minutes at 4°C. Ultimately, the supernatants obtained from this centrifugation step were collected and stored at a temperature of -80°C, in order to facilitate subsequent biochemical assessments [ 38 ]. 2.4.2 Determination of acetylcholinesterase (AChE) level The level of AChE (acetylcholinesterase) in the hippocampus region of the brain was determined using Ellman's method [ 39 ]. In this procedure, a reaction mixture was prepared, comprising 0.2 ml of acetylthiocholine iodide (75mM), 0.1 ml of buffered Ellman's reagent DTNB (5,5-dithio-bis-(2-nitrobenzoic acid)) at a concentration of 10 mM in 15 mM NaHCO3, and 3 ml of PBS (25mM, pH-7.4). This mixture was incubated at room temperature for 10 minutes. After the incubation period, 0.2 ml of the supernatant from the hippocampus homogenate was added to the mixture. The optical density of the resulting solution was measured at a wavelength of 412 nm over a 5-minute duration. Changes in absorbance were recorded at 30-second intervals using a Perkin Elmer Lambda 20 spectrophotometer. The results were expressed in terms of nanomoles of acetylthiocholine iodide hydrolyzed per minute per milligram of protein. 2.4.2 Determination of hippocampal soluble Aβ (1–42) and GSK-3β In all experimental groups, the levels of soluble Aβ (1–42) and GSK-3β in the hippocampal region of the brain were quantified using ELISA kits [ 40 ]. The quantification process followed the instructions provided with the respective analysis kits from the manufacturers. 2.4.3 Determination of pro-inflammatory cytokines TNF-α, IL-6 and IL-β All research groups had their level of the hippocampus neuroinflammatory cytokines TNF-α, IL-6, and IL-1β tested using the corresponding commercially presented ELISA kits [ 41 ], in accord with the manufacturer's guidelines. 2.4.4 Determination of cAMP, cGMP, and BDNF The quantification of hippocampal cAMP, cGMP, and BDNF levels were conducted [ 42 ] using ELISA kits in accordance with the instructions provided by the manufacturers. To determine the concentration of cAMP, cGMP, and BDNF in the samples, the optical density of each sample was compared to a standard curve. 2.4.5 Protein estimation The total protein concentration in the hippocampus region of the brain was determined using Lowry's method, with bovine serum albumin (BSA) serving as the standard [ 43 ]. 2.4.6. Oxidative and Nitrosative stress biomarkers 2.4.6.1 Determination of lipid peroxidation The damage caused by the AlCl 3 + D-galactose combination in the hippocampus region of the brain due to free radical activity was assessed through lipid peroxidation, measured in terms of TBARS (thiobarbituric acid reactive substances) levels. The concentration of malondialdehyde (MDA) in the hippocampus region was determined using Will's method. In this process, a reaction mixture was prepared, consisting of 0.5 ml of the supernatant and 0.5 ml of tris-HCl. This mixture was then incubated at 37°C for a duration of 2 hours. After the incubation period, 1 ml of trichloroacetic acid (TCA) at a concentration of 10% was added to the reaction mixture, followed by centrifugation at 1000×g for 10 minutes. The resulting supernatant (1 ml) was mixed with 1 ml of thiobarbituric acid (TBA) solution with a concentration of 0.67%. Subsequently, the mixture was heated in boiling water for 10 minutes. After cooling, 1 ml of distilled water was added to the mixture, and the absorbance was measured at a wavelength of 532 nm. The TBARS level, indicative of lipid peroxidation, was expressed as nmol of MDA per milligram of protein [ 44 , 45 ]. This measurement provides insight into the extent of oxidative damage occurring in the hippocampus. 2.4.6.2 Estimation of Catalase (CAT) The level of CAT (catalase) was determined using the method outlined by Calibrne (1985). The procedure involved the creation of a reaction mixture by combining 0.05 ml of the supernatant with 1 ml of hydrogen peroxide (H 2 O 2 ) at a concentration of 0.019 M, and 1.95 ml of phosphate buffer at a concentration of 0.5 M and pH 7.0. This mixture was adjusted to a final volume of 3 ml. Subsequently, the absorbance of the reaction mixture was measured at a wavelength of 240 nm. The change in absorbance was recorded at 30-second intervals using a Perkin Elmer Lambda 20 spectrophotometer. The CAT activity was then quantified and expressed in terms of nanomoles of H 2 O 2 decomposed per minute per milligram of protein [ 45 , 46 ]. This measurement provides insights into the catalase enzyme ability to break down hydrogen peroxide, reflecting its anti-oxidative capacity. 2.4.6.3 Estimation of nitrite level The nitrite level in the hippocampus tissue was determined using the Griess reagent method. In this process, an equal volume of the supernatant and Griess reagent was mixed. The Griess reagent consists of 0.1% N-naphthyl ethylenediamine dihydrochloride, 1% sulfanilamide, and 5% phosphoric acid. This mixture was then incubated in the dark for 10 minutes at room temperature. Subsequently, the absorbance of the reaction mixture was measured at a wavelength of 540 nm using a Perkin Elmer Lambda 20 spectrophotometer. The nitrite level was quantified and expressed in micromoles per milligram of protein using a sodium nitrite standard curve [ 47 ]. This measurement provides insights into the levels of nitric oxide (NO) in the hippocampus tissue, indicating potential oxidative and inflammatory processes. 2.4.6.4 Estimation of Glutathione A 4% w/v sulfosalicylic acid precipitate (1 ml) was mixed with 100 µl of the supernatant obtained from the tissue homogenate. This mixture was stored in the refrigerator at a temperature between 2 and 8°C. After an hour, the samples were subjected to centrifugation at 1200 g for 15 minutes at 4°C. Following centrifugation, the supernatant was carefully removed, and any particles were discarded.100 µl of this supernatant was mixed with DTNB-Ellman's reagent. The reagent consisted of 200 µl of 5,5-dithiobis-2-nitrobenzoic acid, 2.7 ml of 0.1 M phosphate buffer (pH 8), and 2.7 ml of the supernatant from the previous step. This mixture resulted in the generation of a faint yellow color. A UV-visible spectrophotometer (Perkin Elmer, USA) was used to measure the absorbance of the generated color at a wavelength of 412 nm. The calculation was performed using the molar extinction coefficient of 1.36104 M − 1 cm − 1 , and the result was reported in units of micromoles of glutathione per milligram of protein (µM GSH/mg protein) [ 48 , 49 ]. This measurement provides insights into the antioxidant capacity of glutathione within the hippocampus tissue. 2.4.6.5 Estimation superoxide dismutase (SOD) The evaluation of superoxide dismutase (SOD) enzyme activity was conducted following the method established by Marklund and Marklund (1974), with certain modifications [ 50 , 51 ]. The assessment of SOD enzyme activity is based on its ability to hinder the autoxidation of pyrogallol. Here's a summary of the procedure. A volume of 10 µl of hippocampal homogenate was combined with 970µl of Tris-HCl buffer (100 mM Tris-HCl with 1 mM EDTA at pH 8.2).Subsequently, 20µl of pyrogallol at a concentration of 13 mM was introduced to the reaction mixture. This entire process took place at room temperature. After a lag time of 30 seconds, the test samples were subjected to spectrophotometric analysis at a wavelength of 420 nm to monitor changes in absorbance. The activity of the SOD enzyme was determined based on its ability to inhibit pyrogallol autoxidation. Specifically, one unit of SOD activity is defined as the amount of enzyme required to cause a 50% inhibition of pyrogallol autoxidation per minute. This activity was then expressed in units per minute per milligram of protein (U/min/mg protein).In essence, this procedure provides insights into the ability of the SOD enzyme to counteract oxidative stress by inhibiting the formation of superoxide radicals. 2.5 Histopathological analysis 2.5.1. Hematoxylin and Eosin (H & E) staining The entire brain was preserved in 10% neutral buffered formalin and subsequently embedded in paraffin. Standard coronal sections of the CA1 region of the hippocampus, each with a thickness of 5 µm, were meticulously sliced using a microtome. Silane-coated slides were then prepared, followed by a deparaffinization process using xylene. Afterward, the slides were rehydrated using an ethanol gradient and subsequently counter-stained with Hematoxylin and Eosin (H&E). To assess the morphology of neurons within the hippocampus CA1 region, the slides were observed under a Meiji fluorescent microscope at a magnification of 40X. Photomicrographs were captured to document the observed neuronal structures and characteristics [ 52 , 53 ]. This process enables the visual examination of the cellular composition and arrangement within the hippocampus region, providing insights into potential structural alterations or anomalies related to the experimental conditions. 2.6 Statistical Analysis The statistical analysis of the data derived from both the behavioral and biochemical parameters of all experimental study groups was conducted using Graph Pad Prism software version 8.02.The data were presented as mean ± SEM (standard error of the mean). To assess the significance of differences between the various experimental groups, the escape latency data in the Morris Water Maze (MWM) was subjected to a two-way ANOVA. For all other parameters, a one-way ANOVA was employed, followed by a post-hoc Tukey-Multiple Comparison test. These statistical analyses help identify and quantify any significant differences between the experimental groups' outcomes. 3. Results 3.1. Effect of Avanafil on D-galac + AlCl 3 induced motor impairment in the locomotor activity Locomotor responses, such as the time it takes for movement (measured in seconds), were recorded for all the experimental groups. In Alzheimer's disease (AD) rats treated with AlCl3 + D-galactose, there was a notable and statistically significant reduction in movement time (***P < 0.001) compared to both the control and sham groups, with a degree of significance indicated by the values (df = 7, 40 F = 16.38). On the other hand, Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP), demonstrated significant ( **p < 0.01, ***p < 0.001, ***p < 0.001) improvement in motor function in AlCl 3 + D-galactose-treated rats when compared to the toxic group (Table 1). 3.2. Effect of Avanafil on D-galac + AlCl3induced cognitive impairment in the Morris Water Maze (MWM) test Acquisition trials; In the Morris Water Maze (MWM) test, during the acquisition trials conducted over four days, rats administered with AlCl 3 + D-galactose showed a notable increase in escape latency on consecutive days (with the exception of Day 1, df = 7, 21 F = 5.743, ***p < 0.001) when compared to the control and sham groups. This increase in escape latency indicated memory impairment in these rats. However, in contrast, the rise in escape latency caused by AlCl3 + D-galactose was significantly reduced when Avanafil was orally administered at doses of 3mg/kg and 6mg/kg, as well as with the use of donepezil (DNP). This reduction demonstrates the memory- improving effect of Avanafil ( **p < 0.01, ***p < 0.001, and ***p < 0.001) (Fig. 1 A). Reference memory test or probe trial During the probe trial conducted on the fifth day, long-term memory or reference memory was assessed in all experimental groups of rats. It was observed that rats administered with AlCl 3 + D-galactose spent significantly less time in the target quadrant, as evidenced by a reduction in the percentage of dwell time, and also exhibited a decrease in the frequency of crossing the platform compared to the control and sham groups (*p < 0.05, **p < 0.01, ***p < 0.001). However, following the oral administration of Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP) for a duration of 28 days, the rats displayed significantly increased percentage of dwell time (df = 7, 40 F = 10.06, *p < 0.05, **p < 0.01, ***p < 0.001) and platform crossing frequency (df = 7, 40 F = 6.027, *p < 0.05, **p < 0.01, ***p < 0.001). This indicates an improvement in their long-term memory performance (Fig. 1 B and 1 C). 3.3. Avanafil decreased acetylcholinesterase (AChE) activity in hippocampus Effect of Avanafil on the level of AChE in the hippocampal region of the brain in all the experimental groups is summarised (Fig. 2 ). There was a significant (df = 7, 40 F = 135.9 ***p < 0.001) increase in the AchE level in the AlCl 3 + D-galac toxic group as compared to the control and sham groups. Whereas, a significant decrease was observed in the treatment group with Avanafil (3mg/kg and 6mg/kg) and DNP when compared with toxic group ( *p < 0.05, **p < 0.01, ***p < 0.001). 3.4. Avanafil dose-dependently prevented hippocampal GSK-3β stimulation It has been reported that the elevated GSK-3β leads to the Tau phosphorylation, which ultimately leads to neurodegeneration [ 55 ]. GSK-3β level was found to be significantly augmented (df = 7, 40 F = 27.39 ***p < 0.001) in rats induced with AlCl3 + D-galactose when compared with control and sham group. While Avanafil dose 3mg/kg, 6mg/kg and DNP exhibited a significant ( *p < 0.05, **p < 0.01, ***p < 0.001) decrease in the level of hippocampal GSK-3β when compared with toxic group (Fig. 3 ). 3.5. Effect Avanafil reduced the deposition of soluble Aβ (1–42) senile plaques in hippocampus Aβ (1–42) peptides are known to lead to the formation of senile plaques, which is a key pathological feature of Alzheimer's disease (AD) [ 54 ]. In the current study, we analyzed the levels of soluble Aβ (1–42) in the rat hippocampus using a rat-specific ELISA kit. Rats administered with AlCl 3 + D-galactose were found to exhibit a significant increase in the levels of Aβ (1–42) in the hippocampus (df = 7, 40 F = 12.11 ***p < 0.001). This increase in Aβ (1–42) was remarkably higher compared to the other groups. Furthermore, the administration of Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP), significantly reversed the elevated levels of Aβ (1–42) induced by the infusion of Aβ (1–42) in the rat hippocampus ( *p < 0.05, **p < 0.01, ***p < 0.001). This indicates that Avanafil and DNP had a marked effect in reducing the accumulation of Aβ (1–42) in the hippocampus, which is associated with AD pathology (Fig. 4 ). 3.6. Effect of Avanafil on hippocampal oxidative/nitrosative stress markers (MDA, Nitrite, CAT, GSH and SOD) In rats administered with AlCl3 + D-galactose, there was a dose-dependent and significant impact of Avanafil on oxidative and nitrosative stress markers in the hippocampus. The results indicated a substantial ( ***p < 0.001) increase in the levels of TBARS (Thiobarbituric Acid Reactive Substances) and Nitrite, along with a decrease in the activity of CAT (Catalase), SOD (Superoxide Dismutase), and GSH (Glutathione) enzymes (known for their antioxidant properties) when compared to the control and sham groups. These alterations in the levels of TBARS, Nitrite, CAT, SOD, and GSH enzyme were significantly reversed by Avanafil at doses of 3mg/kg and 6mg/kg. TBARS levels were significantly reduced by Avanafil (TBARS: df = 7, 40 F = 37.91 **p < 0.01, ***p < 0.001). Nitrite levels were significantly decreased with Avanafil treatment (Nitrite: df = 7, 40 F = 22.99 *p < 0.05, ***p < 0.001). CAT enzyme activity was significantly increased with Avanafil (CAT: df = 8, 45 F = 88.74 **p < 0.01, ***p < 0.001). GSH enzyme levels showed significant improvement with Avanafil (GSH: df = 7, 40 F = 6.949, *p < 0.05, **p < 0.01). SOD enzyme activity was also significantly increased with Avanafil treatment (SOD: df = 7, 40 F = 6.942, **p < 0.01, ***p < 0.001). These findings suggest that Avanafil had a dose-dependent protective effect against oxidative and nitrosative stress in the hippocampus of rats exposed to AlCl 3 + D-galactose, contributing to a reduction in the associated neurodegenerative effects (Table 2). 3.7. Effect of Avanafil on hippocampal cAMP, cGMP, and BDNF in rats The levels of cAMP, cGMP, and BDNF were assessed using specific ELISA kits. In the hippocampus of rats administered with AlCl3 + D-galactose, we observed a significant decrease in the levels of cAMP (df 7, 40; F = 10.06; ***p < 0.001), cGMP (df 7, 40; F = 6.146; ***p < 0.001), and BDNF (df 7, 40; F = 6.909; p < 0.01) when compared to the sham group, as indicated by one-way ANOVA. Conversely, in the treatment groups receiving Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP), there was a significant increase in the levels of cAMP compared to the toxic group (p > 0.5, ***p < 0.001, ***p < 0.001). Similarly, for cGMP, the treatment groups with Avanafil (3mg/kg and 6mg/kg) and DNP displayed a significant increase when compared to the toxic group ( **p < 0.01, ***p < 0.001, **p < 0.01). Additionally, BDNF levels in the treatment groups with Avanafil (3mg/kg and 6mg/kg) and DNP were significantly higher than in the toxic group ( **p < 0.01, ***p < 0.001, ***p < 0.001) (Table 3). 3.8. Avanafil dose-dependently inhibited hippocampal Tau phosphorylation Elevated GSK-3β level leads to the Tau phosphorylation, which ultimately results in neurodegeneration [ 55 ]. p-Tau level was found to be significantly augmented (df = 7, 40 F = 21.43 ****p < 0.0001) in rats induced with D-galactose + AlCl 3 when compared with control and sham group. While Avanafil dose 3mg/kg, 6mg/kg and DNP exhibited a significant ( *p < 0.05, **p < 0.01, ***p < 0.001) decrease in the level of hippocampal p-Tau when compared with toxic group (Fig. 5 ). 3.9. Avanafil attenuates the level of hippocampal TNF-α, IL-6 and IL-1β The levels of neuroinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the rat hippocampus were assessed using specific ELISA kits. It was observed that these cytokine levels were significantly increased in the rat hippocampus as a result of AlCl 3 + D-galactose toxicity, as indicated by the substantial ( ***p < 0.001) elevation compared to the control and sham groups. The administration of Avanafil at doses of 3mg/kg and 6mg/kg effectively reversed these alterations in the pro-inflammatory cytokine levels (TNF-α: df = 7, 40, F = 10.70, **p < 0.01, ***p < 0.001; IL-6: df = 7, 40, F = 7.042, **p < 0.01, ***p < 0.001; IL-1β: df = 7, 40, F = 5.712) (Fig. 6 ). 3.10. Avanafil attenuates the level of neurodegeneration in H & E staining (Histopathological analysis) Histopathological examination of the rats' hippocampal tissue from the various experimental groups was done using H&E staining, which is particular to the morphology of neurones. In the sham and control groups, the hippocampal region of the brain shows intact neurones with prominent nuclei, respectively, as shown by H&E staining. However, the group that was infused with AlCl 3 + D-galactose exhibits a toxic effect. In contrast to this toxic group, rat hippocampal H&E staining reveals a substantial increase in eosinophilic cytoplasm, pyknotic nuclei, and substantial vacuolization. Additionally, a significant decrease in eosinophilic stained neurones and insignificant neuronal toxicity were observed in H&E staining following treatment with Avanafil at doses of 3 mg/kg and 6 mg/kg (Fig. 7 ). 4. Discussion AD stands out as a prominent neurodegenerative condition associated with age, characterized by intricate cerebral impairments, synaptic and neuronal cell loss, and ultimately leading to fatal outcomes [ 56 , 57 ]. An effective initial treatment approach is necessary to address the latent phase of this neurodegenerative condition, aiming to manage severe dementia linked to AD, a persistent, long-standing ailment. Our comprehension of the disease's progression and the associated irregularities specific to AD has advanced over recent decades, owing to an extensive range of preclinical and clinical studies [ 58 ]. A successful early treatment strategy is essential to target the hidden stage of this neurodegenerative disorder, with the goal of mitigating severe dementia associated with AD, a chronic and enduring affliction. Our understanding of the disease's progression and the particular abnormalities linked to AD has made significant progress in recent decades, thanks to a wide array of preclinical and clinical investigations [ 59 , 60 ]. An effective initial treatment strategy is crucial for addressing the covert phase of this neurodegenerative disorder, aiming to alleviate the profound dementia connected with AD, a persistent and long-lasting condition. Our comprehension of the disease's advancement and the specific irregularities associated with AD has advanced significantly in recent years, owing to an extensive range of preclinical and clinical research endeavours [ 61 , 62 ]. The confluence of D-galac and AlCl3 leads to cerebral harm, inducing the deterioration of multiple functions. The concurrent administration of D-gal and AlCl3 disrupts cognitive abilities, modifies various biomarkers, and affects the structural integrity of the hippocampus in albino Wistar rats [ 62 ]. Through the cAMP/PKA/BDNF and NO/cGMP/PKG/BDNF signaling pathways, Avanafil effectively mitigates memory deficits, neuro-inflammation, oxidative stress, and brain histopathological changes induced by D-gal + AlCl3. This is evidenced by a notable decrease in locomotor activity growth, escape latency, path length during the spatial acquisition phase, and percentage dwell time in the reference memory test within the Morris Water Maze (MWM). Co-administration of D-galactose and AlCl3 orally for 42 days was found to impede spatial learning, memory capabilities, and memory association in mice, as demonstrated by decreased performance in both spatial and nonspatial learning and memory tasks in Wistar rats. This decline in cognitive functions following exposure to D-galactose + AlCl3 aligns with the findings of prior studies [ 63 ]. However, treatment of D-galac + AlCl 3 -exposed wistar rats with Avanafil (3mg/kg,6mg/kg) for 28 days markedly inverted this combination-induce reduction in intellectual abilities, and this outcome of Avanafil was in line with a previous report [ 37 ]. The administration of Avanafil resulted in improved performance by mice on the Actophotometer and Morris Water Maze (MWM), suggesting that Avanafil cognitive enhancement capabilities were comparable to those of DNP in terms of mitigating spatial and nonspatial memory deficits. To establish that these changes in cognitive abilities were not linked to motor impairment, an assessment of locomotor activity was also essential, in addition to the memory assessments. Impaired learning and memory, characteristic of Alzheimer's disease (AD), are often associated with disrupted cholinergic neurotransmission involving acetylcholine (ACh). ACh is acknowledged for its role in promoting cognitive processes, and training animals in diverse behavioral models can enhance these functions [ 64 ].Several acetylcholinesterase (AChE) inhibitors, such as donepezil, are available for the treatment of cognitive impairments and are widely regarded as highly effective medications for addressing the symptoms of Alzheimer's disease (AD) [ 65 , 66 ]. In a previous study, it was shown that Phosphodiesterase inhibitors exhibited acetylcholinesterase (AChE) inhibitory properties in a pre-clinical experiment, and the current study produced comparable results [ 67 ]. Avanafil countered the increased AChE concentration in wistar rats co-administered D-gal + AlCl 3 . The role of tau-tau interaction and hyper phosphorylation in Alzheimer's disease (AD) has been investigated using a range of molecular factors [ 68 ]. These excessively phosphorylated forms give rise to neurofibrillary tangles (NFTs), disrupting neural plasticity and leading to neuro-degeneration [ 69 , 70 ]. These neurofibrillary tangles (NFTs) emerge as a result of the binding between these excessively phosphorylated forms. The impact of D-gal and AlCl3-induced neuronal damage on increasing Tau hyper phosphorylation was demonstrated in a previous study [ 71 ]. Interestingly, this damage was found to be significantly mitigated and dissociated through the administration of Avanafil. Ageing and AD are situation that are related with arise in neuro-inflammation in addition to oxidative stress [ 72 ].Neuro-inflammatory cytokines have been linked to the aetiology of neuro-degenerative diseases like AD, according to experimental data [ 73 ].The release of neuroinflammatory cytokines and reduced neuronal survival and oxidative defence are only two examples of the negative effects that excessive glial cell activation can have on the brain [ 74 ]. Pro-inflammatory cytokines such as IL-6, IL-1β, and TNFα have been associated with disruptions in cholinergic and cognitive functions that resemble those observed in aging. The overexpression of these cytokines has also been connected to the administration of D-galactose and AlCl3 in mice, which has been demonstrated to induce significant neuro-inflammation [ 75 ]. In the present study, a similar increase in levels of TNF-α, IL-6, and IL-1β was observed in the hippocampus of mice following exposure to D-galactose + AlCl3. However, this increase was effectively diminished and reversed through the administration of Avanafil. Previous research had identified a Phosphodiesterase inhibitor's anti-inflammatory effect against pro-inflammatory cytokines in the brain tissue of Wistar rats, a finding that has now been corroborated in the current investigation [ 76 ]. Preclinical investigations have demonstrated that the combination of D-galactose and AlCl3 significantly elevates nitrite-nitrate and TBARS (thiobarbituric acid reactive substances) levels, while concurrently reducing the levels of antioxidant enzymes such as CAT (catalase), SOD (superoxide dismutase), and GSH (glutathione), which play a protective role against oxidative damage [ 77 ]. In addition to these findings, our study also observed oxidative stress in mice administered both D-galactose and AlCl 3 . This was evidenced by notably higher levels of nitrite-nitrate and TBARS and lower levels of CAT, SOD, and GSH. Interestingly, Avanafil appeared to alleviate the elevated levels of oxidative stress markers in the hippocampi of rats treated with D-galactose + AlCl 3 . In addition to these alterations mirroring neurodegenerative conditions associated with aging, such as Alzheimer's disease, as a consequence of the combined therapy of D-galactose and AlCl 3 in Wistar rats, we also focused on glycogen synthase kinase-3β (GSK-3β), a recognized contributor to neurodegeneration. Prolonged administration of AlCl 3 and/or D-galactose has been shown to elevate levels of AD-associated markers and total GSK-3β production [ 78 ]. Correspondingly, we observed a significant increase in GSK-3β levels in the hippocampus of mice following D-galactose + AlCl 3 -induced neurotoxicity, and this elevation was countered by the administration of Avanafil at the two respective doses 3mg/kg & 6mg/kg. Avanafil is categorized as a Phosphodiesterase inhibitor that specifically targets and inhibits PDE-5, designed to counteract erectile dysfunction. Nonetheless, previous research has highlighted the inhibitory effects of PDE-5, primarily in the context of AChE inhibition and mitigation of cognitive deficits. Furthermore, studies have shown that PDE-5 inhibition can effectively counteract oxidative stress, neuroinflammation, and the activation of reactive astrocytes in the hippocampus of mice, thereby safeguarding neurons from damage. These findings suggest that Avanafil might hold the potential to be employed as a therapeutic agent against neurodegenerative conditions such as Alzheimer's disease. 5. Conclusion This is the primary study to investigate the neuroprotective capability of Avanafil on the hippocampus cAMP/BDNF and NO/cGMP/BDNF flagging pathways in the D-galac + AlCl 3 rodent model of Promotion. Avanafil, when taken orally for 28 days, emphatically worked on spatial and nonspatial recollections in a few conduct standards and significantly switched variations in oxidative and neuro-inflammatory pressure signs produced by D-galac + AlCl3 in rodents. The discoveries of this study recommend that Avanafil good impact in Promotion treatment might be connected with its inhibitory consequences for amyloidosis and reclamation of the hippocampal cAMP, cGMP and BDNF flagging path-way, and subsequently it very well may be perceived as a promising helpful medication to treat Advertisement. These discoveries highlight Avanafil having a high restorative viability against neuronal degenerations like those found in Alzheimer's disease. Declarations Acknowledgements: The authors of current research express their gratitude to Meerut Institute of Engineering and Technology (MIET), Meerut for providing all the necessary facilities required during this research work. 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Tables Table-1: Effect of Avanafil on locomotor activity (LA) in D-galac + AlCl 3 administered rat. The group exposed to D-galac + AlCl3toxicity displayed not able alterations in locomotor activities when contrasted with both the control and sham groups. Furthermore, these significant changes were mitigated by the administration of Avanafil at doses of 3mg/kg and 6mg/kg, as well as by DNP at a dose of 5mg/kg, when compared to the toxic rats. Groups Move time(s) Rest time(s) Horizontal activity(cm) Mean velocity(cm/) Total movement(#) Control Group 615.6±26.65 169.9±14.71 5464±174.3 752.7±25.81 702±26.65 Sham Control 598.2±27.19 155.6±11.98 5231±172.7 746.7±24.81 664±28.76 AD(AlCl3 + D-galac) 168.5±16.41 $$$### 564.1±23.64 $$$### 1039±368.2 $$$## 276.7±17.67 $$$### 245±13.54 $$$### AD + Avanafil 3 mg/kg 306±19.55** 409.8±23.97** 2605±282.1** 436.4 ± 21.17* 392±17.65* AD + Avanafil 6 mg/kg 378±21.43*** 296±14.24*** 4658±333.2*** 482.4 ± 22.17*** 416±38.32*** AD + Donepezil 5mg/kg 527±24.42*** 209.6±10.91*** 4897±342.65*** 698±23.87*** 557±43.54*** Avanafil 6 mg/kg Perse 564±27.12*** 181.3±12.42*** 4952 ±361.6*** 721±24.87*** 614±34.8*** Donepezil 5 mg/kg Perse 588±32.1*** 184±13.52*** 5165±342.8*** 711±28.7*** 632±29.76*** The values are expressed as mean ± SEM. $$$### P < 0.001 vs control and sham group respectively **p < 0.01 vs D-galac + AlCl3administered toxic group ***p < 0.001 vs D-galac + AlCl3administered toxic group Table-2: Effect of Avanafil on hippocampal MDA, Nitrite, SOD, GSH and CAT in rats The data demonstrates the impact of Avanafil on hippocampal levels of MDA, Nitrite, SOD, GSH, and CAT in rats with Alzheimer's disease induced by D-galac + AlCl 3 . Statistical analysis was performed using a one-way ANOVA, followed by the post hoc Tukey-Kramer multiple comparison test to evaluate the results. Groups MDA (nMol/mg protein) Nitrite (µMol/mg protein) SOD (U/mg protein) GSH (µMol/mg protein) CAT(nmol H2O2/min/mg protein) Control Group 1.72 ± 0.243 11.68 ± 1.73 9.83 ± 0.7 5.34 ± 0.45 16.1 ± 0.39 Sham Control 1.92 ± 0.265 12.11 ± 1.89 9.23 ± 0.67 4.952 ± 0.34 15.23 ± 0.21 AD(D-galac +AlCl3) 6.101±0.2022 $$$### 29.12±2.64 $$$### 4.96±0.67 $$$### 1.65±0.24 $$$### 8.21±0.19 $$$### AD + Avanafil 3 mg/kg 4.88 ± 0.21** 21.83± 2.21* 6.74 ± 0.42** 3.53 ± 0.36* 10.21 ± 0.41** AD + Avanafil 6 mg/kg 3.62 ± 0.28*** 16.13 ± 1.92** 8.25 ±0.52*** 4.166 ± 0.46** 13.21 ± 0.47*** AD + Donepezil 5mg/kg 3.91± 0.31*** 14.13 ±1.68*** 8.65 ±0.61*** 4.63 ± 0.45*** 14.26 ± 0.391*** Avanafil 6 mg/kg Perse 2.89 ± 0.18 12.1 ± 1.32 9.26 ± 0.53 3.95 ± 0.498 15.51 ± 0.421 Donepezil 5 mg/kg Perse 2.68 ± 0.19 11.2 ± 1.45 9.11 ± 0.54 4.117 ± 0.54 15.91 ± 0.34 The values are expressed as mean ± SEM. $$$### P < 0.001 vs control and sham group respectively **p < 0.01 vs D-galac + AlCl3administered toxic group ***p< 0.001 vs D-galac + AlCl3administered toxic group Table-3: Effect of Avanafil on hippocampal cAMP, cGMP, and BDNF in rats. The data is showing the effect of Avanafil on the hippocampal cAMP, cGMP, and BDNF level in D-galac + AlCl 3 administered rats. The results were analysed by one-way ANOVA followed by the post hoc Tukey Kramer multiple comparison test and represented that Avanafil markedly increased cAMP, cGMP and BDNF level in the rat hippocampus. Data are presented as mean ± SEM (n=6). The markers indicate difference Groups cAMP (pMol/ml) cGMP(pmol/ml) BDNF (pg/ml) Control Group 57.291 ± 3.291 69.23 ± 4.924 346.23 ± 23.323 Sham Control 54.691 ± 4.179 67.9213 ± 3.94 342.43 ± 22.431 AD(D-galac + AlCl3) 23.0242 ± 3.425 $$$### 32.432 ± 3.57 $$$### 154.321 ± 20.261 $$$### AD + Avanafil 3 mg/kg 36.32 ± 2.621 ns 56.342 ± 5.68** 271.43 ± 29.12** AD + Avanafil 6mg/kg 46.6421 ± 3.645*** 66.643 ± 5.663*** 318.52 ± 30.546*** AD + Donepezil 5mg/kg 48.136 ± 4.323*** 58.932 ± 5.127** 308.235 ± 19.324*** Avanafil 6 mg/kg Perse 53.953 ± 3.137*** 66.32 ± 5.227*** 322.011 ± 17.322*** Donepezil 5 mg/kg Perse 54.911± 4.398*** 65.421 ± 4.684*** 319.236 ± 24.242*** $$$### P 0.05 as compared to administered toxic group **p < 0.01 vs D-galac + AlCl3administered toxic group ***p < 0.001 vs D-galac + AlCl3administered toxic group Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 13 Aug, 2025 Read the published version in Neurochemical Research → Version 1 posted Editorial decision: Revision requested 10 Mar, 2025 Reviews received at journal 03 Mar, 2025 Reviewers agreed at journal 13 Feb, 2025 Reviews received at journal 13 Feb, 2025 Reviewers agreed at journal 10 Feb, 2025 Reviewers agreed at journal 09 Feb, 2025 Reviewers agreed at journal 08 Feb, 2025 Reviewers agreed at journal 08 Feb, 2025 Reviewers agreed at journal 08 Feb, 2025 Reviews received at journal 07 Feb, 2025 Reviewers agreed at journal 07 Feb, 2025 Reviewers agreed at journal 07 Feb, 2025 Reviewers invited by journal 07 Feb, 2025 Editor assigned by journal 27 Jan, 2025 Submission checks completed at journal 27 Jan, 2025 First submitted to journal 27 Jan, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5911692","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":407803468,"identity":"a0b58240-fef4-4b8d-ade6-a39b722ce768","order_by":0,"name":"Mohd Talib -","email":"","orcid":"","institution":"Meerut Institute of Engineering and Technology, Meerut","correspondingAuthor":false,"prefix":"","firstName":"Mohd","middleName":"Talib","lastName":"-","suffix":""},{"id":407803469,"identity":"975fd00e-e015-493a-81ec-60e401871651","order_by":1,"name":"Nazia Siddiqui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYJCCAwwMEowNh4GsBAMJZn6QUEIB0VoqbNglG8B6CdvE2HAARJ1J4zcAM/BoMWc//vDAjz8Wsn3HmbdJPGw7LG18fnXihwcGDPL8YgewarHsSUg42NsmYTzzMFuZRGLbYWOzG283SwAdZjhzdgJWLQYHEg4c4G2QSNxwmMcMpCXZ7MbZDSAtCQa3cWg5/7Dh4J8/CC31m2ec3fwDr5YbyQyHedigWhLOpDEb8Pduw2/LjWcMh2Uhfim2AAYys8QN3m0WwAjC7Zfz6Y8/vvlTJ9t3/vDGmz9AUdl/dvPNHxU28vzS2LWgaIdQEmCVEgSVI2nhP0CU6lEwCkbBKBg5AAB5VWnVIC/21AAAAABJRU5ErkJggg==","orcid":"","institution":"Meerut Institute of Engineering and Technology, Meerut","correspondingAuthor":true,"prefix":"","firstName":"Nazia","middleName":"","lastName":"Siddiqui","suffix":""},{"id":407803470,"identity":"2af3022e-4bac-44fe-8a2e-7d1f07057603","order_by":2,"name":"Prabhash Nath Tripathi","email":"","orcid":"","institution":"Meerut Institute of Engineering and Technology, Meerut","correspondingAuthor":false,"prefix":"","firstName":"Prabhash","middleName":"Nath","lastName":"Tripathi","suffix":""},{"id":407803471,"identity":"fb083089-c81c-4cf4-9189-01ee0ea33093","order_by":3,"name":"Ankit Chaudhary","email":"","orcid":"","institution":"Meerut Institute of Engineering and Technology, Meerut","correspondingAuthor":false,"prefix":"","firstName":"Ankit","middleName":"","lastName":"Chaudhary","suffix":""}],"badges":[],"createdAt":"2025-01-27 11:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5911692/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5911692/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11064-025-04516-6","type":"published","date":"2025-08-13T15:57:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74985305,"identity":"2d39806a-a442-4bb0-8ba0-d99d4dc85afb","added_by":"auto","created_at":"2025-01-29 06:01:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Morris water maze (MWM) test was used to assess Alzheimer's disease induced by D-galac + AlCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e in rats.\u003c/strong\u003e The results are presented as follows: \u003cstrong\u003e(A)\u003c/strong\u003e Escape Latencies during the acquisition phase for 4 consecutive days. \u003cstrong\u003e(B)\u003c/strong\u003e The amount of time spent in the target quadrant (in seconds). \u003cstrong\u003e(C)\u003c/strong\u003e The number of platform crossings during the probe trial. The values are expressed as mean ± SEM. \u003csup\u003e\u003cem\u003e$$$###\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 vs control and sham group respectively. \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01 vs D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e administered toxic group. \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/f28ecd3a4912e3b3b73a8792.png"},{"id":74986309,"identity":"3f5f5e54-e0ea-415f-ad0c-e594210bd5ff","added_by":"auto","created_at":"2025-01-29 06:09:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Avanafil on AChE in D-galac + AlCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u0026nbsp;\u0026nbsp; \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003einduced AD in rats. \u003c/strong\u003eThe values are expressed as mean ± SEM. \u003csup\u003e\u003cem\u003e$$$###\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 vs control and sham group respectively. \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01 vs D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e administered\u003cstrong\u003e \u003c/strong\u003etoxic group. \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/ac1ea74ee380eca472717a7a.jpg"},{"id":74985278,"identity":"238af587-67a2-43a9-8258-c7b91cd942e9","added_by":"auto","created_at":"2025-01-29 06:01:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAvanafil significantly attenuates the hippocampal GSK-3β level. \u003c/strong\u003eAll the values are expressed as mean ± SEM. \u003csup\u003e\u003cem\u003e###$$$\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 vs. control and sham group respectively. Significant difference was observed in treatment group with Avanafil (3mg/kg and 6mg/kg) **p \u0026lt; 0.01 and ***p \u0026lt; 0.001 vs. D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e administered\u003cstrong\u003e \u003c/strong\u003etoxic group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/c9e64442106af71c06e725a2.jpg"},{"id":74986300,"identity":"9b59218c-170f-4b65-bff8-181521f94f8a","added_by":"auto","created_at":"2025-01-29 06:09:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Avanafil on hippocampal Aβ1–42, in rats. \u003c/strong\u003eThe values are expressed as mean ± SEM. \u003csup\u003e\u003cem\u003e###$$$\u003c/em\u003e\u003c/sup\u003ep\u0026lt; 0.001 vs. control and sham groups respectively.*p \u0026lt; 0.05, **p \u0026lt; 0.01 and ***p \u0026lt; 0.001 vs. D-galac + AlCl3administered toxic group.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/46f60a4c5e0545641ad3d277.jpg"},{"id":74986296,"identity":"63a09325-9639-4d27-b38c-744f1a63657d","added_by":"auto","created_at":"2025-01-29 06:09:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":38575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Avanafil on hippocampal Tau Protein, in rats. \u003c/strong\u003eThe values are expressed as mean ± SEM. ###$$$p \u0026lt; 0.001 vs. control and sham groups respectively. Significant difference was observed in treatment group with Avanafil (3mg/kg and 6mg/kg) and DNP 5mg/kg \u003cem\u003e*p\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01 and \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001 vs.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/8542fb034df3b52c260255df.jpg"},{"id":74985325,"identity":"1a0c4f86-a908-42fd-bb77-81b47705ac0a","added_by":"auto","created_at":"2025-01-29 06:01:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":138689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Avanafil on pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in D-galac + AlCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e administered in rats. \u003c/strong\u003eAll the values in A, B and C are given as mean ± SEM. \u003csup\u003e\u003cem\u003e\u003cstrong\u003e###$$$\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 vs. control and sham group respectively. Significant difference was observed in treatment group with Avanafil (3mg/kg and 6mg/kg) and DNP 5mg/kg \u003cem\u003e*p\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01 and \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001 vs. D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e administered toxic group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/4f00aef0174fdf132dafd4dc.png"},{"id":74986304,"identity":"97dc5cbe-1c2c-4427-89c3-d04a1d84c372","added_by":"auto","created_at":"2025-01-29 06:09:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":107161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathology of hippocampus with H\u0026amp;E staining. (A)\u003c/strong\u003e Control group shows healthy neurons with prominent nuclei. \u003cstrong\u003e(B)\u003c/strong\u003e Sham group shows healthy neurons with prominent nuclei. \u003cstrong\u003e(C)\u003c/strong\u003e D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e (AD) infused group represents neuronal damage, neuronal shrinkage and eosinophilic stained cytoplasm. \u003cstrong\u003e(D)\u003c/strong\u003e AD + Avanafil 3mg/kg group shows moderate change in neurons morphology when compared to toxic group. \u003cstrong\u003e(E)\u003c/strong\u003e Treatment group with Avanafil 6mg/kg represents significant decreased in the number of eosinophilic stained neurons and \u003cstrong\u003e(F)\u003c/strong\u003e AD + DNP 5mg/kg treatment group shows mild neuronal injury with much more less number of eosinophilic stained neurons. \u003cstrong\u003e(G)\u003c/strong\u003e Avanafil Per se and \u003cstrong\u003e(H)\u003c/strong\u003e DNP per se groups shows intact neurons with prominent nuclei. DNP: Donepezil.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/ceab04a024827a55af0b9c6f.jpg"},{"id":89310543,"identity":"bb8e3e45-ce2e-4cef-939f-21435c06e1b2","added_by":"auto","created_at":"2025-08-18 16:07:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2680696,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5911692/v1/7b2cbeb2-1c72-4daf-b21a-392d18cb7c44.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ameliorative role of Phosphodiesterase-5 (PDE-5) inhibitor “Avanafil” via modulating cAMP \u0026 cGMP Pathway against Alzheimer’s disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlzheimer's disease (AD), a long-term neurodegenerative disorder characterized by an advancing decline in cognitive function, stands as a significant origin of dementia in the elderly globally. According to recent epidemiological estimates, the count of individuals affected by dementia is projected to triple by 2050 compared to 2010, with a substantial proportion stemming from AD [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Furthermore, it's been postulated that due to rising life expectancies and ongoing shifts in living conditions, the prevalence of AD could reach 1 in 85 individuals by 2050 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Over the past several decades, scholars have suggested that AD closely links to the extreme production and accumulation of amyloid-β (Aβ) peptide within the brain, yet the precise underlying mechanism remains elusive. Particularly, most current investigations support the role of inflammation as a crucial catalyst in AD progression and the gradual erosion of cognitive abilities. This perspective has gained traction due to the recent revelation of the antimicrobial properties of the Aβ peptide, which opens up the possibility that infections role to the development of AD and proposes that the genesis and advancement of Aβ plaques might be triggered by infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe strong connection between AD development and oxidative stress has been acknowledged for quite some time [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Oxidative and nitrosative stresses play a crucial role in the beginning of AD pathological processes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These stresses arise from the inequality between the generation of reactive nitrogen species (RNS), reactive oxygen species (ROS) and their elimination through internal antioxidant enzyme systems, such as superoxide dismutase (SOD) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. SOD acts as the primary defence against ROS produced during oxidative stress within the body [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The production of ROS is indirectly assessed by examining malondialdehyde (MDA), a by-product of lipid peroxidation (LPO) caused by free radicals. When nitric oxide (NO) is excessively produced, it leads to the formation of various harmful RNS that contribute to nitrosative stress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Neuronal apoptosis, a significant aspect of AD development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], is partially governed by the Bcl-2 family, with Bcl-2 itself being neuroprotective against apoptosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Neuroinflammation play a vital role in AD onset and progression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], with TNF-α being a major regulator of proinflammatory responses in the brain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Inhibiting TNF-α has shown potential in improving cognitive decline in patients [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, effective AD therapeutic strategies should focus on restoring cholinergic hypothesis, enhancing neuro-synaptic plasticity, and alleviating oxidative damage, neuronal apoptosis, and neuroinflammation.\u003c/p\u003e \u003cp\u003eRecent research has revealed that chronic administration of a combination of D-galac and AlCl\u003csub\u003e3\u003c/sub\u003e promoted to impaired learning and memory functions [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This combination induces widespread neurodegeneration, including disruptions in the cholinergic coordination, mitochondrial dysfunction, apoptosis, and oxidative stress. It effectively replicates AD-like pathological changes, including the accumulation of amyloid plaques and the formation of neurofibrillary tangles. As a result, this approach has been used to create an animal model resembling AD, referred to as a non-transgenic AD animal model [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This particular animal model has demonstrated its worth in the exploration of neurodegenerative disease mechanisms such as AD and in conducting drug screening activities [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), play a fundamental role as secondary messengers in brain signal transduction, governing PKA/BDNF and NO/PKG/BDNF pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Elevating their concentrations activates brain-derived neurotrophic factor (BDNF) and added signaling molecules, contributing to spatial learning and memory functions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Various compounds influencing the cAMP or cGMP signaling route have been studied in animal models of AD, with a particular emphasis on Phosphodiesterase (PDE) inhibitors due to their favourable safety profile [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consequently, cGMP triggers its downstream protein kinase-G (PKG), which, in turn, activates miscellaneous substrates, including the transcription factor cyclic adenosine monophosphate (cAMP) response element-binding element (CREB). This activation promotes neurotransmission, synaptic plasticity, and the formation of memories [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. PKG also stimulates the phosphatidylinositol 3-kinase (PI3k)/Akt signaling pathway, which exerts neuroprotective effects by impeding apoptosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].Accumulating evidence suggests that utilizing PDE inhibitors to reinstate cAMP and cGMP levels in the hippocampus could be a strategy to avert cognitive impairments. Previous research demonstrated that PDE inhibitors, definitely targeted at cAMP and cGMP, effectively saved memory and synaptic plasticity impairments in rodent models of AD. This study marks the initial exploration into the effects of Avanafil on Alzheimer's disease (AD) like characteristics, achieved via modulation of the cAMP and cGMP pathways. The study was conducted using an AD model induced by D-galac and AlCl3 in Albino Wistar rats.\u003c/p\u003e"},{"header":"2. Drugs and chemicals","content":"\u003cp\u003eFemale Wistar rats weight range 250gm and 300gm were obtained from the Animal House at Meerut Institute of Engineering and Technology, located at Baghpat Bypass, Meerut (UP), 250005, India. These rats were then accommodated in polypropylene cages, with eight rats housed together in pairs. They were placed under controlled environmental conditions, including a room temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, a relative humidity range of 55\u0026ndash;65%, and a 12-hour light/12-hour dark cycle, providing a regular day-night rhythm. The rats were provided unrestricted access to a standard commercial pellet diet and water. The entire experimental procedures adhered to the guidelines set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The Institutional Animal Ethics Committee (IAEC) of MIET, Meerut had reviewed and granted approval for the experimental protocol, bearing the registration number 711/PO/ReRc/S/02/CPCSEA 29/10/2002 under protocol number 88.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Drugs and chemicals\u003c/h2\u003e \u003cp\u003eAvanafil was purchased from Sigma Aldrich, USA. Donepezil was procured from Alkem Laboratory Limited Company, India. AlCl\u003csub\u003e3\u003c/sub\u003e, D-galac and Carboxymethyl cellulose was purchased from SISCO Research Laboratories. The Aβ, GSK-3β, cAMP, cGMP and BDNF Elisa kits were obtained from Genxbio in India, while the TNF-α, IL-1β, and IL-6 Elisa kits were acquired from Krishgen Biosystems, also based in India.\u003c/p\u003e \u003cp\u003eRats in the two different treatment groups received two dissimilar doses of Avanafil (3mg/kg and 6mg/kg) respectively. 3mg/kg and 6mg/kg dose was based on the prior evidence existing and dissolve 0.25% Carboxymethyl cellulose [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The dose of AlCl\u003csub\u003e3\u003c/sub\u003e 10mg/kg\u0026thinsp;+\u0026thinsp;D-galac 60mg/kg is based on the evidence in previous study [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Treatment schedule\u003c/h2\u003e \u003cp\u003eRats were randomly and uniformly distributed into 8 groups, including 6 rats/group and groups were demonstrated as \u003cb\u003eGroup A (Control)\u003c/b\u003e: Rats were administered with water orally for 10 weeks; \u003cb\u003eGroup B (Sham)\u003c/b\u003e: Rats were given 10ml/kg, Carboxymethyl Cellulose (CMC) orally for 28 days; \u003cb\u003eGroup C (Toxic Group (AD))\u003c/b\u003e: Rats were given AlCl\u003csub\u003e3\u003c/sub\u003e 10mg/kg\u0026thinsp;+\u0026thinsp;D-galac 60mg/kg intraperitoneally (I.P) dissolved in distilled water to induce AD up to 42 days; \u003cb\u003eGroup D, and Group E (AD\u0026thinsp;+\u0026thinsp;Treatment)\u003c/b\u003e: Rats were given AlCl\u003csub\u003e3\u003c/sub\u003e 10mg/kg\u0026thinsp;+\u0026thinsp;D-galac 60mg/kg intraperitoneally (I.P) dissolved in distilled water to induce AD up to 42 days and then administered Avanafil 3mg/kg and 6mg/kg orally for 28 days; \u003cb\u003eGroup F (AD\u0026thinsp;+\u0026thinsp;DNP)\u003c/b\u003e Rats given AlCl\u003csub\u003e3\u003c/sub\u003e 10mg/kg\u0026thinsp;+\u0026thinsp;D-galac 60mg/kg intraperitoneally (I.P) dissolved in distilled water to induce AD up to 42 days and then administered Donepezil 5mg/kg orally for 28 days. \u003cb\u003eGroup F (Avanafil Perse)\u003c/b\u003e: Administered Avanafil 6mg/kg orally for 28days. \u003cb\u003eGroup H (DNP Perse)\u003c/b\u003e: Administered Donepezil 5mg/kg orally for 28days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Behavioral Parameters\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Locomotor Activity\u003c/h2\u003e \u003cp\u003eThe assessment of motor impairment in all groups of rodents involved conducting locomotor activity (LA) using a video path analyzer. The LA data were collected during standard research facility hours from 8:30 a.m. to 1:30 p.m. The experiment took place in an open field enclosed by an acrylic box measuring 40.6 x 40.6 x 40.6 cm\u0026sup3;, equipped with two sets of photo beams (16 beams in each dimension, spaced 2.5 cm apart). A True Scan 2.0 version computer interface (Coulbourn Instruments, Allentown, Pennsylvania, USA) connected to the activity meter recorded LA by counting beam breaks at a 100 ms sampling rate.\u003c/p\u003e \u003cp\u003eTo maintain cleanliness and prevent interference from olfactory cues, the cage underwent a 70% alcohol cleaning process between each test. Each rat in every group underwent a 30-minute acclimatization period, followed by a 20-minute recording of LA. Digital recording was done by detecting interruptions in the infrared beams caused by the movements of the rats within the activity meter. The recorded components of locomotor activity included parameters such as move time (s), rest time (s), horizontal activity (cm), mean velocity (cm/s), and total movement (#) for each group [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Morris water maze test\u003c/h2\u003e \u003cp\u003eMorris water maze (MWM) assessment was coordinated to study the memory \u0026amp; learning of rodents. This endeavour was substituted in a dull concealed water tank of width (130 cm) and height (50cm), finished off by water to the significance of 30cm and saved up to a temperature of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C to avoid hypo-thermia in rodents. Lights and sounds were additionally kept up with to stay away from any blunder. The tank was effectively divided into four quadrants, namely East, West, North and South. The time in both securing and test preliminary was estimated by physically process. In securing stage, the dark shaded rectangular departure foundation of 8\u0026times;6 cm\u003csup\u003e2\u003c/sup\u003e region was lowered immersed in one quadrant (south-west) and remained immovable in a comparative quadrant all complete the examination. Rodents went through 4 successive preliminaries for 4 back to back days from various quadrants to investigate the secret stage (focus) inside the roof season of 60s and was permitted to sit onto the stage for 30Seconds. If rodents neglected to get away from the stage, were actually organized to the stage. During every procurement preliminary, get away from idleness period (time taken by each rodent to distribute the secret stage) was renowned for 4 days. Test preliminary was completed on fifth day to survey the orientation memory of each rodent. In this preliminary, the stage was eliminated from the quadrant (south-west) and every rat allowed to allocate the secret stage quadrant for 60s.The % stay time consumed by the rodent to investigate the secret stage in the objective quadrant (south-west) and stage passage recurrence to cross the objective quadrant was record [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Biochemical parameters\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Brain homogenate preparation\u003c/h2\u003e \u003cp\u003eAfter the 10-weeks experimental period, all rats within every group were anesthetized using light ether and euthanized. After euthanasia, each rat was individually perfused with phosphate buffer saline (PBS). The brains were immediately removed from the skulls and washed with cool saline solution. The hippocampus tissues were extracted from the whole brain and then blended with cold phosphate buffer (0.1M; pH-7.4) at a centrifugation speed of 800 X g for 5 minutes, all performed at a temperature of 4\u0026deg;C. The homogenization process was carried out using ten times the volume of the tissue. The resulting supernatants were subjected to further centrifugation at 10,000g for 10 minutes at 4\u0026deg;C. Ultimately, the supernatants obtained from this centrifugation step were collected and stored at a temperature of -80\u0026deg;C, in order to facilitate subsequent biochemical assessments [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Determination of acetylcholinesterase (AChE) level\u003c/h2\u003e \u003cp\u003eThe level of AChE (acetylcholinesterase) in the hippocampus region of the brain was determined using Ellman's method [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this procedure, a reaction mixture was prepared, comprising 0.2 ml of acetylthiocholine iodide (75mM), 0.1 ml of buffered Ellman's reagent DTNB (5,5-dithio-bis-(2-nitrobenzoic acid)) at a concentration of 10 mM in 15 mM NaHCO3, and 3 ml of PBS (25mM, pH-7.4). This mixture was incubated at room temperature for 10 minutes. After the incubation period, 0.2 ml of the supernatant from the hippocampus homogenate was added to the mixture. The optical density of the resulting solution was measured at a wavelength of 412 nm over a 5-minute duration. Changes in absorbance were recorded at 30-second intervals using a Perkin Elmer Lambda 20 spectrophotometer. The results were expressed in terms of nanomoles of acetylthiocholine iodide hydrolyzed per minute per milligram of protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Determination of hippocampal soluble Aβ (1\u0026ndash;42) and GSK-3β\u003c/h2\u003e \u003cp\u003eIn all experimental groups, the levels of soluble Aβ (1\u0026ndash;42) and GSK-3β in the hippocampal region of the brain were quantified using ELISA kits [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The quantification process followed the instructions provided with the respective analysis kits from the manufacturers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Determination of pro-inflammatory cytokines TNF-α, IL-6 and IL-β\u003c/h2\u003e \u003cp\u003eAll research groups had their level of the hippocampus neuroinflammatory cytokines TNF-α, IL-6, and IL-1β tested using the corresponding commercially presented ELISA kits [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], in accord with the manufacturer's guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 Determination of cAMP, cGMP, and BDNF\u003c/h2\u003e \u003cp\u003eThe quantification of hippocampal cAMP, cGMP, and BDNF levels were conducted [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] using ELISA kits in accordance with the instructions provided by the manufacturers. To determine the concentration of cAMP, cGMP, and BDNF in the samples, the optical density of each sample was compared to a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.4.5 Protein estimation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe total protein concentration in the hippocampus region of the brain was determined using Lowry's method, with bovine serum albumin (BSA) serving as the standard [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.6. Oxidative and Nitrosative stress biomarkers\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section4\"\u003e \u003ch2\u003e2.4.6.1 Determination of lipid peroxidation\u003c/h2\u003e \u003cp\u003eThe damage caused by the AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose combination in the hippocampus region of the brain due to free radical activity was assessed through lipid peroxidation, measured in terms of TBARS (thiobarbituric acid reactive substances) levels. The concentration of malondialdehyde (MDA) in the hippocampus region was determined using Will's method.\u003c/p\u003e \u003cp\u003eIn this process, a reaction mixture was prepared, consisting of 0.5 ml of the supernatant and 0.5 ml of tris-HCl. This mixture was then incubated at 37\u0026deg;C for a duration of 2 hours. After the incubation period, 1 ml of trichloroacetic acid (TCA) at a concentration of 10% was added to the reaction mixture, followed by centrifugation at 1000\u0026times;g for 10 minutes. The resulting supernatant (1 ml) was mixed with 1 ml of thiobarbituric acid (TBA) solution with a concentration of 0.67%. Subsequently, the mixture was heated in boiling water for 10 minutes. After cooling, 1 ml of distilled water was added to the mixture, and the absorbance was measured at a wavelength of 532 nm. The TBARS level, indicative of lipid peroxidation, was expressed as nmol of MDA per milligram of protein [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This measurement provides insight into the extent of oxidative damage occurring in the hippocampus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section4\"\u003e \u003ch2\u003e2.4.6.2 Estimation of Catalase (CAT)\u003c/h2\u003e \u003cp\u003eThe level of CAT (catalase) was determined using the method outlined by Calibrne (1985). The procedure involved the creation of a reaction mixture by combining 0.05 ml of the supernatant with 1 ml of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) at a concentration of 0.019 M, and 1.95 ml of phosphate buffer at a concentration of 0.5 M and pH 7.0. This mixture was adjusted to a final volume of 3 ml. Subsequently, the absorbance of the reaction mixture was measured at a wavelength of 240 nm. The change in absorbance was recorded at 30-second intervals using a Perkin Elmer Lambda 20 spectrophotometer. The CAT activity was then quantified and expressed in terms of nanomoles of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposed per minute per milligram of protein [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This measurement provides insights into the catalase enzyme ability to break down hydrogen peroxide, reflecting its anti-oxidative capacity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section4\"\u003e \u003ch2\u003e2.4.6.3 Estimation of nitrite level\u003c/h2\u003e \u003cp\u003eThe nitrite level in the hippocampus tissue was determined using the Griess reagent method. In this process, an equal volume of the supernatant and Griess reagent was mixed. The Griess reagent consists of 0.1% N-naphthyl ethylenediamine dihydrochloride, 1% sulfanilamide, and 5% phosphoric acid. This mixture was then incubated in the dark for 10 minutes at room temperature. Subsequently, the absorbance of the reaction mixture was measured at a wavelength of 540 nm using a Perkin Elmer Lambda 20 spectrophotometer. The nitrite level was quantified and expressed in micromoles per milligram of protein using a sodium nitrite standard curve [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This measurement provides insights into the levels of nitric oxide (NO) in the hippocampus tissue, indicating potential oxidative and inflammatory processes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section4\"\u003e \u003ch2\u003e2.4.6.4 Estimation of Glutathione\u003c/h2\u003e \u003cp\u003eA 4% w/v sulfosalicylic acid precipitate (1 ml) was mixed with 100 \u0026micro;l of the supernatant obtained from the tissue homogenate. This mixture was stored in the refrigerator at a temperature between 2 and 8\u0026deg;C. After an hour, the samples were subjected to centrifugation at 1200 g for 15 minutes at 4\u0026deg;C. Following centrifugation, the supernatant was carefully removed, and any particles were discarded.100 \u0026micro;l of this supernatant was mixed with DTNB-Ellman's reagent. The reagent consisted of 200 \u0026micro;l of 5,5-dithiobis-2-nitrobenzoic acid, 2.7 ml of 0.1 M phosphate buffer (pH 8), and 2.7 ml of the supernatant from the previous step. This mixture resulted in the generation of a faint yellow color. A UV-visible spectrophotometer (Perkin Elmer, USA) was used to measure the absorbance of the generated color at a wavelength of 412 nm. The calculation was performed using the molar extinction coefficient of 1.36104 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the result was reported in units of micromoles of glutathione per milligram of protein (\u0026micro;M GSH/mg protein) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This measurement provides insights into the antioxidant capacity of glutathione within the hippocampus tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section4\"\u003e \u003ch2\u003e2.4.6.5 Estimation superoxide dismutase (SOD)\u003c/h2\u003e \u003cp\u003eThe evaluation of superoxide dismutase (SOD) enzyme activity was conducted following the method established by Marklund and Marklund (1974), with certain modifications [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The assessment of SOD enzyme activity is based on its ability to hinder the autoxidation of pyrogallol. Here's a summary of the procedure. A volume of 10 \u0026micro;l of hippocampal homogenate was combined with 970\u0026micro;l of Tris-HCl buffer (100 mM Tris-HCl with 1 mM EDTA at pH 8.2).Subsequently, 20\u0026micro;l of pyrogallol at a concentration of 13 mM was introduced to the reaction mixture. This entire process took place at room temperature. After a lag time of 30 seconds, the test samples were subjected to spectrophotometric analysis at a wavelength of 420 nm to monitor changes in absorbance. The activity of the SOD enzyme was determined based on its ability to inhibit pyrogallol autoxidation. Specifically, one unit of SOD activity is defined as the amount of enzyme required to cause a 50% inhibition of pyrogallol autoxidation per minute. This activity was then expressed in units per minute per milligram of protein (U/min/mg protein).In essence, this procedure provides insights into the ability of the SOD enzyme to counteract oxidative stress by inhibiting the formation of superoxide radicals.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histopathological analysis\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Hematoxylin and Eosin (H \u0026amp; E) staining\u003c/h2\u003e \u003cp\u003eThe entire brain was preserved in 10% neutral buffered formalin and subsequently embedded in paraffin. Standard coronal sections of the CA1 region of the hippocampus, each with a thickness of 5 \u0026micro;m, were meticulously sliced using a microtome. Silane-coated slides were then prepared, followed by a deparaffinization process using xylene. Afterward, the slides were rehydrated using an ethanol gradient and subsequently counter-stained with Hematoxylin and Eosin (H\u0026amp;E).\u003c/p\u003e \u003cp\u003eTo assess the morphology of neurons within the hippocampus CA1 region, the slides were observed under a Meiji fluorescent microscope at a magnification of 40X. Photomicrographs were captured to document the observed neuronal structures and characteristics [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This process enables the visual examination of the cellular composition and arrangement within the hippocampus region, providing insights into potential structural alterations or anomalies related to the experimental conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis of the data derived from both the behavioral and biochemical parameters of all experimental study groups was conducted using Graph Pad Prism software version 8.02.The data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). To assess the significance of differences between the various experimental groups, the escape latency data in the Morris Water Maze (MWM) was subjected to a two-way ANOVA. For all other parameters, a one-way ANOVA was employed, followed by a post-hoc Tukey-Multiple Comparison test. These statistical analyses help identify and quantify any significant differences between the experimental groups' outcomes.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effect of Avanafil on D-galac\u0026thinsp;+\u0026thinsp;AlCl\u003csub\u003e3\u003c/sub\u003e induced motor impairment in the locomotor activity\u003c/h2\u003e \u003cp\u003eLocomotor responses, such as the time it takes for movement (measured in seconds), were recorded for all the experimental groups. In Alzheimer's disease (AD) rats treated with AlCl3\u0026thinsp;+\u0026thinsp;D-galactose, there was a notable and statistically significant reduction in movement time (***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to both the control and sham groups, with a degree of significance indicated by the values (df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;16.38). On the other hand, Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP), demonstrated significant (\u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,\u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001,\u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) improvement in motor function in AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose-treated rats when compared to the toxic group (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2. Effect of Avanafil on D-galac\u0026thinsp;+\u0026thinsp;AlCl3induced cognitive impairment in the Morris Water Maze (MWM) test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAcquisition trials; In the Morris Water Maze (MWM) test, during the acquisition trials conducted over four days, rats administered with AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose showed a notable increase in escape latency on consecutive days (with the exception of Day 1, df\u0026thinsp;=\u0026thinsp;7, 21 F\u0026thinsp;=\u0026thinsp;5.743, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) when compared to the control and sham groups. This increase in escape latency indicated memory impairment in these rats. However, in contrast, the rise in escape latency caused by AlCl3\u0026thinsp;+\u0026thinsp;D-galactose was significantly reduced when Avanafil was orally administered at doses of 3mg/kg and 6mg/kg, as well as with the use of donepezil (DNP). This reduction demonstrates the memory- improving effect of Avanafil (\u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eReference memory test or probe trial\u003c/strong\u003e \u003cp\u003eDuring the probe trial conducted on the fifth day, long-term memory or reference memory was assessed in all experimental groups of rats. It was observed that rats administered with AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose spent significantly less time in the target quadrant, as evidenced by a reduction in the percentage of dwell time, and also exhibited a decrease in the frequency of crossing the platform compared to the control and sham groups \u003cem\u003e(*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,\u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, following the oral administration of Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP) for a duration of 28 days, the rats displayed significantly increased percentage of dwell time (df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;10.06,\u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and platform crossing frequency (df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;6.027, \u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This indicates an improvement in their long-term memory performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Avanafil decreased acetylcholinesterase (AChE) activity in hippocampus\u003c/h2\u003e \u003cp\u003eEffect of Avanafil on the level of AChE in the hippocampal region of the brain in all the experimental groups is summarised (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). There was a significant (df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;135.9 \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase in the AchE level in the AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galac toxic group as compared to the control and sham groups. Whereas, a significant decrease was observed in the treatment group with Avanafil (3mg/kg and 6mg/kg) and DNP when compared with toxic group (\u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Avanafil dose-dependently prevented hippocampal GSK-3β stimulation\u003c/h2\u003e \u003cp\u003eIt has been reported that the elevated GSK-3β leads to the Tau phosphorylation, which ultimately leads to neurodegeneration [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. GSK-3β level was found to be significantly augmented (df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;27.39 ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in rats induced with AlCl3\u0026thinsp;+\u0026thinsp;D-galactose when compared with control and sham group. While Avanafil dose 3mg/kg, 6mg/kg and DNP exhibited a significant (\u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease in the level of hippocampal GSK-3β when compared with toxic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Effect Avanafil reduced the deposition of soluble Aβ (1\u0026ndash;42) senile plaques in hippocampus\u003c/h2\u003e \u003cp\u003eAβ (1\u0026ndash;42) peptides are known to lead to the formation of senile plaques, which is a key pathological feature of Alzheimer's disease (AD) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In the current study, we analyzed the levels of soluble Aβ (1\u0026ndash;42) in the rat hippocampus using a rat-specific ELISA kit. Rats administered with AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose were found to exhibit a significant increase in the levels of Aβ (1\u0026ndash;42) in the hippocampus (df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;12.11 \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This increase in Aβ (1\u0026ndash;42) was remarkably higher compared to the other groups. Furthermore, the administration of Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP), significantly reversed the elevated levels of Aβ (1\u0026ndash;42) induced by the infusion of Aβ (1\u0026ndash;42) in the rat hippocampus (\u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This indicates that Avanafil and DNP had a marked effect in reducing the accumulation of Aβ (1\u0026ndash;42) in the hippocampus, which is associated with AD pathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effect of Avanafil on hippocampal oxidative/nitrosative stress markers (MDA, Nitrite, CAT, GSH and SOD)\u003c/h2\u003e \u003cp\u003eIn rats administered with AlCl3\u0026thinsp;+\u0026thinsp;D-galactose, there was a dose-dependent and significant impact of Avanafil on oxidative and nitrosative stress markers in the hippocampus. The results indicated a substantial (\u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase in the levels of TBARS (Thiobarbituric Acid Reactive Substances) and Nitrite, along with a decrease in the activity of CAT (Catalase), SOD (Superoxide Dismutase), and GSH (Glutathione) enzymes (known for their antioxidant properties) when compared to the control and sham groups. These alterations in the levels of TBARS, Nitrite, CAT, SOD, and GSH enzyme were significantly reversed by Avanafil at doses of 3mg/kg and 6mg/kg.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eTBARS levels were significantly reduced by Avanafil (TBARS: df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;37.91 \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eNitrite levels were significantly decreased with Avanafil treatment (Nitrite: df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;22.99 \u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCAT enzyme activity was significantly increased with Avanafil (CAT: df\u0026thinsp;=\u0026thinsp;8, 45 F\u0026thinsp;=\u0026thinsp;88.74 \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGSH enzyme levels showed significant improvement with Avanafil (GSH: df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;6.949, \u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSOD enzyme activity was also significantly increased with Avanafil treatment (SOD: df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;6.942, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThese findings suggest that Avanafil had a dose-dependent protective effect against oxidative and nitrosative stress in the hippocampus of rats exposed to AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose, contributing to a reduction in the associated neurodegenerative effects (Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Effect of Avanafil on hippocampal cAMP, cGMP, and BDNF in rats\u003c/h2\u003e \u003cp\u003eThe levels of cAMP, cGMP, and BDNF were assessed using specific ELISA kits. In the hippocampus of rats administered with AlCl3\u0026thinsp;+\u0026thinsp;D-galactose, we observed a significant decrease in the levels of cAMP (df 7, 40; F\u0026thinsp;=\u0026thinsp;10.06; \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), cGMP (df 7, 40; F\u0026thinsp;=\u0026thinsp;6.146; \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and BDNF (df 7, 40; F\u0026thinsp;=\u0026thinsp;6.909; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) when compared to the sham group, as indicated by one-way ANOVA. Conversely, in the treatment groups receiving Avanafil at doses of 3mg/kg and 6mg/kg, as well as donepezil (DNP), there was a significant increase in the levels of cAMP compared to the toxic group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.5, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, for cGMP, the treatment groups with Avanafil (3mg/kg and 6mg/kg) and DNP displayed a significant increase when compared to the toxic group (\u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, BDNF levels in the treatment groups with Avanafil (3mg/kg and 6mg/kg) and DNP were significantly higher than in the toxic group (\u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,\u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Avanafil dose-dependently inhibited hippocampal Tau phosphorylation\u003c/h2\u003e \u003cp\u003eElevated GSK-3β level leads to the Tau phosphorylation, which ultimately results in neurodegeneration [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. p-Tau level was found to be significantly augmented (df\u0026thinsp;=\u0026thinsp;7, 40 F\u0026thinsp;=\u0026thinsp;21.43 ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in rats induced with D-galactose\u0026thinsp;+\u0026thinsp;AlCl\u003csub\u003e3\u003c/sub\u003e when compared with control and sham group. While Avanafil dose 3mg/kg, 6mg/kg and DNP exhibited a significant (\u003cem\u003e*p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decrease in the level of hippocampal p-Tau when compared with toxic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Avanafil attenuates the level of hippocampal TNF-α, IL-6 and IL-1β\u003c/h2\u003e \u003cp\u003eThe levels of neuroinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the rat hippocampus were assessed using specific ELISA kits. It was observed that these cytokine levels were significantly increased in the rat hippocampus as a result of AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose toxicity, as indicated by the substantial (\u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) elevation compared to the control and sham groups. The administration of Avanafil at doses of 3mg/kg and 6mg/kg effectively reversed these alterations in the pro-inflammatory cytokine levels (TNF-α: df\u0026thinsp;=\u0026thinsp;7, 40, F\u0026thinsp;=\u0026thinsp;10.70, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; IL-6: df\u0026thinsp;=\u0026thinsp;7, 40, F\u0026thinsp;=\u0026thinsp;7.042, \u003cem\u003e**p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003e***p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; IL-1β: df\u0026thinsp;=\u0026thinsp;7, 40, F\u0026thinsp;=\u0026thinsp;5.712) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e3.10. Avanafil attenuates the level of neurodegeneration in H \u0026amp; E staining (Histopathological analysis)\u003c/h2\u003e \u003cp\u003eHistopathological examination of the rats' hippocampal tissue from the various experimental groups was done using H\u0026amp;E staining, which is particular to the morphology of neurones. In the sham and control groups, the hippocampal region of the brain shows intact neurones with prominent nuclei, respectively, as shown by H\u0026amp;E staining. However, the group that was infused with AlCl\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;D-galactose exhibits a toxic effect. In contrast to this toxic group, rat hippocampal H\u0026amp;E staining reveals a substantial increase in eosinophilic cytoplasm, pyknotic nuclei, and substantial vacuolization. Additionally, a significant decrease in eosinophilic stained neurones and insignificant neuronal toxicity were observed in H\u0026amp;E staining following treatment with Avanafil at doses of 3 mg/kg and 6 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAD stands out as a prominent neurodegenerative condition associated with age, characterized by intricate cerebral impairments, synaptic and neuronal cell loss, and ultimately leading to fatal outcomes [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. An effective initial treatment approach is necessary to address the latent phase of this neurodegenerative condition, aiming to manage severe dementia linked to AD, a persistent, long-standing ailment. Our comprehension of the disease's progression and the associated irregularities specific to AD has advanced over recent decades, owing to an extensive range of preclinical and clinical studies [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. A successful early treatment strategy is essential to target the hidden stage of this neurodegenerative disorder, with the goal of mitigating severe dementia associated with AD, a chronic and enduring affliction. Our understanding of the disease's progression and the particular abnormalities linked to AD has made significant progress in recent decades, thanks to a wide array of preclinical and clinical investigations [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. An effective initial treatment strategy is crucial for addressing the covert phase of this neurodegenerative disorder, aiming to alleviate the profound dementia connected with AD, a persistent and long-lasting condition. Our comprehension of the disease's advancement and the specific irregularities associated with AD has advanced significantly in recent years, owing to an extensive range of preclinical and clinical research endeavours [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The confluence of D-galac and AlCl3 leads to cerebral harm, inducing the deterioration of multiple functions. The concurrent administration of D-gal and AlCl3 disrupts cognitive abilities, modifies various biomarkers, and affects the structural integrity of the hippocampus in albino Wistar rats [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThrough the cAMP/PKA/BDNF and NO/cGMP/PKG/BDNF signaling pathways, Avanafil effectively mitigates memory deficits, neuro-inflammation, oxidative stress, and brain histopathological changes induced by D-gal\u0026thinsp;+\u0026thinsp;AlCl3. This is evidenced by a notable decrease in locomotor activity growth, escape latency, path length during the spatial acquisition phase, and percentage dwell time in the reference memory test within the Morris Water Maze (MWM). Co-administration of D-galactose and AlCl3 orally for 42 days was found to impede spatial learning, memory capabilities, and memory association in mice, as demonstrated by decreased performance in both spatial and nonspatial learning and memory tasks in Wistar rats. This decline in cognitive functions following exposure to D-galactose\u0026thinsp;+\u0026thinsp;AlCl3 aligns with the findings of prior studies [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. However, treatment of D-galac\u0026thinsp;+\u0026thinsp;AlCl\u003csub\u003e3\u003c/sub\u003e-exposed wistar rats with Avanafil (3mg/kg,6mg/kg) for 28 days markedly inverted this combination-induce reduction in intellectual abilities, and this outcome of Avanafil was in line with a previous report [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The administration of Avanafil resulted in improved performance by mice on the Actophotometer and Morris Water Maze (MWM), suggesting that Avanafil cognitive enhancement capabilities were comparable to those of DNP in terms of mitigating spatial and nonspatial memory deficits. To establish that these changes in cognitive abilities were not linked to motor impairment, an assessment of locomotor activity was also essential, in addition to the memory assessments. Impaired learning and memory, characteristic of Alzheimer's disease (AD), are often associated with disrupted cholinergic neurotransmission involving acetylcholine (ACh). ACh is acknowledged for its role in promoting cognitive processes, and training animals in diverse behavioral models can enhance these functions [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].Several acetylcholinesterase (AChE) inhibitors, such as donepezil, are available for the treatment of cognitive impairments and are widely regarded as highly effective medications for addressing the symptoms of Alzheimer's disease (AD) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In a previous study, it was shown that Phosphodiesterase inhibitors exhibited acetylcholinesterase (AChE) inhibitory properties in a pre-clinical experiment, and the current study produced comparable results [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Avanafil countered the increased AChE concentration in wistar rats co-administered D-gal\u0026thinsp;+\u0026thinsp;AlCl\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe role of tau-tau interaction and hyper phosphorylation in Alzheimer's disease (AD) has been investigated using a range of molecular factors [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. These excessively phosphorylated forms give rise to neurofibrillary tangles (NFTs), disrupting neural plasticity and leading to neuro-degeneration [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. These neurofibrillary tangles (NFTs) emerge as a result of the binding between these excessively phosphorylated forms. The impact of D-gal and AlCl3-induced neuronal damage on increasing Tau hyper phosphorylation was demonstrated in a previous study [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Interestingly, this damage was found to be significantly mitigated and dissociated through the administration of Avanafil.\u003c/p\u003e \u003cp\u003eAgeing and AD are situation that are related with arise in neuro-inflammation in addition to oxidative stress [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].Neuro-inflammatory cytokines have been linked to the aetiology of neuro-degenerative diseases like AD, according to experimental data [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].The release of neuroinflammatory cytokines and reduced neuronal survival and oxidative defence are only two examples of the negative effects that excessive glial cell activation can have on the brain [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Pro-inflammatory cytokines such as IL-6, IL-1β, and TNFα have been associated with disruptions in cholinergic and cognitive functions that resemble those observed in aging. The overexpression of these cytokines has also been connected to the administration of D-galactose and AlCl3 in mice, which has been demonstrated to induce significant neuro-inflammation [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. In the present study, a similar increase in levels of TNF-α, IL-6, and IL-1β was observed in the hippocampus of mice following exposure to D-galactose\u0026thinsp;+\u0026thinsp;AlCl3. However, this increase was effectively diminished and reversed through the administration of Avanafil. Previous research had identified a Phosphodiesterase inhibitor's anti-inflammatory effect against pro-inflammatory cytokines in the brain tissue of Wistar rats, a finding that has now been corroborated in the current investigation [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePreclinical investigations have demonstrated that the combination of D-galactose and AlCl3 significantly elevates nitrite-nitrate and TBARS (thiobarbituric acid reactive substances) levels, while concurrently reducing the levels of antioxidant enzymes such as CAT (catalase), SOD (superoxide dismutase), and GSH (glutathione), which play a protective role against oxidative damage [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. In addition to these findings, our study also observed oxidative stress in mice administered both D-galactose and AlCl\u003csub\u003e3\u003c/sub\u003e. This was evidenced by notably higher levels of nitrite-nitrate and TBARS and lower levels of CAT, SOD, and GSH. Interestingly, Avanafil appeared to alleviate the elevated levels of oxidative stress markers in the hippocampi of rats treated with D-galactose\u0026thinsp;+\u0026thinsp;AlCl\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIn addition to these alterations mirroring neurodegenerative conditions associated with aging, such as Alzheimer's disease, as a consequence of the combined therapy of D-galactose and AlCl\u003csub\u003e3\u003c/sub\u003e in Wistar rats, we also focused on glycogen synthase kinase-3β (GSK-3β), a recognized contributor to neurodegeneration. Prolonged administration of AlCl\u003csub\u003e3\u003c/sub\u003e and/or D-galactose has been shown to elevate levels of AD-associated markers and total GSK-3β production [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Correspondingly, we observed a significant increase in GSK-3β levels in the hippocampus of mice following D-galactose\u0026thinsp;+\u0026thinsp;AlCl\u003csub\u003e3\u003c/sub\u003e-induced neurotoxicity, and this elevation was countered by the administration of Avanafil at the two respective doses 3mg/kg \u0026amp; 6mg/kg.\u003c/p\u003e \u003cp\u003eAvanafil is categorized as a Phosphodiesterase inhibitor that specifically targets and inhibits PDE-5, designed to counteract erectile dysfunction. Nonetheless, previous research has highlighted the inhibitory effects of PDE-5, primarily in the context of AChE inhibition and mitigation of cognitive deficits. Furthermore, studies have shown that PDE-5 inhibition can effectively counteract oxidative stress, neuroinflammation, and the activation of reactive astrocytes in the hippocampus of mice, thereby safeguarding neurons from damage. These findings suggest that Avanafil might hold the potential to be employed as a therapeutic agent against neurodegenerative conditions such as Alzheimer's disease.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis is the primary study to investigate the neuroprotective capability of Avanafil on the hippocampus cAMP/BDNF and NO/cGMP/BDNF flagging pathways in the D-galac\u0026thinsp;+\u0026thinsp;AlCl\u003csub\u003e3\u003c/sub\u003e rodent model of Promotion. Avanafil, when taken orally for 28 days, emphatically worked on spatial and nonspatial recollections in a few conduct standards and significantly switched variations in oxidative and neuro-inflammatory pressure signs produced by D-galac\u0026thinsp;+\u0026thinsp;AlCl3 in rodents. The discoveries of this study recommend that Avanafil good impact in Promotion treatment might be connected with its inhibitory consequences for amyloidosis and reclamation of the hippocampal cAMP, cGMP and BDNF flagging path-way, and subsequently it very well may be perceived as a promising helpful medication to treat Advertisement. These discoveries highlight Avanafil having a high restorative viability against neuronal degenerations like those found in Alzheimer's disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors of current research express their gratitude to Meerut Institute of Engineering and Technology (MIET), Meerut for providing all the necessary facilities required during this research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eAll the authors of the present research work have reviewed the manuscript and declared no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eAll the enquiries regarding availability of the data should be directed to the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eAll the authors declare that there is no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eAll the authors of present study declare that there is no potential conflicts of interest in conducting this research and publishing article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl-Chalabi, A., Allen, C., Counsell, C., Farrin, A., Dickie, B., \u0026amp; Kelly, J. 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Neuroproteomics Chip-Based Mass Spectrometry and Other Techniques for Alzheimer\u0026rsquo;s Disease Biomarkers\u0026ndash;Update. \u003cem\u003eCurrent Pharmaceutical Design\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(14), 1124-1151. https://doi.org/10.2174/1381612828666220413094918.\u003c/li\u003e\n\u003cli\u003eYalman, K., Şen, A., \u0026Ccedil;evik, \u0026Ouml;., Kadioğlu-Yaman, B., Ertaş, B., Yildiz, S., \u0026amp; Şener, G. (2022). Investigation of the protective and therapeutic efficacy of Myrtus communis extract in aluminum chloride and D-galactose-induced Alzheimer\u0026apos;s disease in rats. \u003cem\u003eJournal of Research in Pharmacy\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(5), 1363-1374. http://dx.doi.org/10.29228/jrp.242.\u003c/li\u003e\n\u003cli\u003eAlsegiani, A. S., \u0026amp; Shah, Z. A. (2022). The influence of gut microbiota alteration on age-related neuroinflammation and cognitive decline. \u003cem\u003eNeural Regeneration Research\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(11), 2407-2412. 10.4103/1673-5374.335837\u003c/li\u003e\n\u003cli\u003eSochocka, M., Zwolinska, K., \u0026amp; Leszek, J. (2017). The infectious etiology of Alzheimer\u0026apos;s disease. \u003cem\u003eCurrent neuropharmacology\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(7), 996-1009. https://doi.org/10.2174/1570159X15666170313122937\u003c/li\u003e\n\u003cli\u003eQuincozes-Santos, A., Santos, C. L., de Souza Almeida, R. R., da Silva, A., Thomaz, N. K., Costa, N. L. F., ... \u0026amp; Bobermin, L. D. (2021). Gliotoxicity and glioprotection: the dual role of glial cells. \u003cem\u003eMolecular neurobiology\u003c/em\u003e, \u003cem\u003e58\u003c/em\u003e(12), 6577-6592. https://doi.org/10.1007/s12035-021-02574-9\u003c/li\u003e\n\u003cli\u003eZameer, S., Alam, M., Hussain, S., Vohora, D., Ali, J., Najmi, A. K., \u0026amp; Akhtar, M. (2020). Neuroprotective role of alendronate against APP processing and neuroinflammation in mice fed a high fat diet. \u003cem\u003eBrain Research Bulletin\u003c/em\u003e, \u003cem\u003e161\u003c/em\u003e, 197-212. https://doi.org/10.1016/j.brainresbull.2020.04.010.\u003c/li\u003e\n\u003cli\u003eKeskin, H., Keskin, F., Tavaci, T., Halici, H., Yuksel, T. N., Ozkaraca, M., ... \u0026amp; Halici, Z. (2021). Neuroprotective effect of roflumilast under cerebral ischaemia/reperfusion injury in juvenile rats through NLRP‐mediated inflammatory response inhibition. \u003cem\u003eClinical and Experimental Pharmacology and Physiology\u003c/em\u003e, \u003cem\u003e48\u003c/em\u003e(8), 1103-1110. https://doi.org/10.1111/1440-1681.13493.\u003c/li\u003e\n\u003cli\u003eHong, X. P., Chen, T., Yin, N. N., Han, Y. M., Yuan, F., Duan, Y. J., ... \u0026amp; Chen, Z. B. (2016). Puerarin ameliorates D-galactose induced enhanced hippocampal neurogenesis and tau hyperphosphorylation in rat brain. \u003cem\u003eJournal of Alzheimer\u0026apos;s Disease\u003c/em\u003e, \u003cem\u003e51\u003c/em\u003e(2), 605-617. 10.3233/JAD-150566.\u003c/li\u003e\n\u003cli\u003eAltaf, A., Zargham, M. K., Hussain, A., Lail, A., Sheikh, F. S., \u0026amp; Bangash, M. H. (2021). Inhibition of Tau by Phytocompounds and Extracts from Medicinal Plants. \u003cem\u003ePhytopharmacological Communications\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(1), 27-47. https://doi.org/10.55627/ppc.001.01.0101.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable-1: Effect of Avanafil on locomotor activity (LA) in D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e administered rat.\u0026nbsp;\u003c/strong\u003eThe group exposed to D-galac + AlCl3toxicity displayed not able alterations in locomotor activities when contrasted with both the control and sham groups. Furthermore, these significant changes were mitigated by the administration of Avanafil at doses of 3mg/kg and 6mg/kg, as well as by DNP at a dose of 5mg/kg, when compared to the toxic rats.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"709\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMove \u0026nbsp;time(s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRest time(s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHorizontal activity(cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean velocity(cm/)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal movement(#)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e615.6\u0026plusmn;26.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e169.9\u0026plusmn;14.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e5464\u0026plusmn;174.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e752.7\u0026plusmn;25.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e702\u0026plusmn;26.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSham Control\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e598.2\u0026plusmn;27.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e155.6\u0026plusmn;11.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e5231\u0026plusmn;172.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e746.7\u0026plusmn;24.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e664\u0026plusmn;28.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD(AlCl3 + D-galac)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e168.5\u0026plusmn;16.41\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e564.1\u0026plusmn;23.64\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e1039\u0026plusmn;368.2\u003csup\u003e$$$##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e276.7\u0026plusmn;17.67\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e245\u0026plusmn;13.54\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Avanafil 3 mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e306\u0026plusmn;19.55**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e409.8\u0026plusmn;23.97**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e2605\u0026plusmn;282.1**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e436.4 \u0026plusmn; 21.17*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e392\u0026plusmn;17.65*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Avanafil 6 mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e378\u0026plusmn;21.43***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e296\u0026plusmn;14.24***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e4658\u0026plusmn;333.2***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e482.4 \u0026plusmn; 22.17***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e416\u0026plusmn;38.32***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Donepezil 5mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e527\u0026plusmn;24.42***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e209.6\u0026plusmn;10.91***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e4897\u0026plusmn;342.65***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e698\u0026plusmn;23.87***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e557\u0026plusmn;43.54***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAvanafil 6 mg/kg Perse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e564\u0026plusmn;27.12***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e181.3\u0026plusmn;12.42***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e4952 \u0026plusmn;361.6***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e721\u0026plusmn;24.87***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e614\u0026plusmn;34.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.3357%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDonepezil 5 mg/kg Perse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2327%;\"\u003e\n \u003cp\u003e588\u0026plusmn;32.1***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.323%;\"\u003e\n \u003cp\u003e184\u0026plusmn;13.52***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e5165\u0026plusmn;342.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6432%;\"\u003e\n \u003cp\u003e711\u0026plusmn;28.7***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.8223%;\"\u003e\n \u003cp\u003e632\u0026plusmn;29.76***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe values are expressed as mean \u0026plusmn; SEM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003e$$$###\u003c/sup\u003e\u003c/em\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 vs control and sham group respectively\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01 vs D-galac + AlCl3administered\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003etoxic group\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001 vs D-galac + AlCl3administered toxic group\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable-2: Effect of Avanafil on hippocampal MDA, Nitrite, SOD, GSH and CAT in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data demonstrates the impact of Avanafil on hippocampal levels of MDA, Nitrite, SOD, GSH, and CAT in rats with Alzheimer\u0026apos;s disease induced by D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e. Statistical analysis was performed using a one-way ANOVA, followed by the post hoc Tukey-Kramer multiple comparison test to evaluate the results.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"669\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDA (nMol/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNitrite (\u0026micro;Mol/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSOD (U/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGSH (\u0026micro;Mol/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAT(nmol H2O2/min/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Group\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e1.72 \u0026plusmn; 0.243\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e11.68 \u0026plusmn; 1.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e9.83 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e5.34 \u0026plusmn; 0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e16.1 \u0026plusmn; 0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSham Control\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e1.92 \u0026plusmn; 0.265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e12.11 \u0026plusmn; 1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e9.23 \u0026plusmn; 0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e4.952 \u0026plusmn; 0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e15.23 \u0026plusmn; 0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD(D-galac +AlCl3)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e6.101\u0026plusmn;0.2022\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e29.12\u0026plusmn;2.64\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e4.96\u0026plusmn;0.67\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e1.65\u0026plusmn;0.24\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e8.21\u0026plusmn;0.19\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Avanafil 3 mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e4.88 \u0026plusmn; 0.21**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e21.83\u0026plusmn; 2.21*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e6.74 \u0026plusmn; 0.42**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e3.53 \u0026plusmn; 0.36*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e10.21 \u0026plusmn; 0.41**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Avanafil 6 mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e3.62 \u0026plusmn; 0.28***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e16.13 \u0026plusmn; 1.92**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e8.25 \u0026plusmn;0.52***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e4.166 \u0026plusmn; 0.46**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e13.21 \u0026plusmn; 0.47***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Donepezil 5mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e3.91\u0026plusmn; 0.31***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e14.13\u0026nbsp;\u0026plusmn;1.68***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e8.65 \u0026plusmn;0.61***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e4.63 \u0026plusmn; 0.45***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e14.26 \u0026plusmn; 0.391***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAvanafil 6 mg/kg Perse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e2.89 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e12.1 \u0026plusmn; 1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e9.26 \u0026plusmn; 0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e3.95 \u0026plusmn; 0.498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e15.51 \u0026plusmn; 0.421\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.7612%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDonepezil 5 mg/kg Perse\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.3134%;\"\u003e\n \u003cp\u003e2.68 \u0026plusmn; 0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.2687%;\"\u003e\n \u003cp\u003e11.2 \u0026plusmn; 1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.2239%;\"\u003e\n \u003cp\u003e9.11 \u0026plusmn; 0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.9701%;\"\u003e\n \u003cp\u003e4.117 \u0026plusmn; 0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4627%;\"\u003e\n \u003cp\u003e15.91 \u0026plusmn; 0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe values are expressed as mean \u0026plusmn; SEM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e$$$###\u003c/sup\u003eP \u0026lt; 0.001 vs control and sham group respectively\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e**p \u0026lt; 0.01 vs D-galac + AlCl3administered toxic group\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e***p\u0026lt; 0.001 vs D-galac + AlCl3administered toxic group\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable-3: Effect of Avanafil on hippocampal cAMP, cGMP, and BDNF in rats.\u0026nbsp;\u003c/strong\u003eThe data is showing the effect of Avanafil on the hippocampal cAMP, cGMP, and BDNF level in D-galac + AlCl\u003csub\u003e3\u003c/sub\u003e administered\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erats. The results were analysed by one-way ANOVA followed by the post hoc Tukey Kramer multiple comparison test and represented that Avanafil markedly increased cAMP, cGMP and BDNF level in the rat hippocampus. Data are presented as mean \u0026plusmn; SEM (n=6). The markers indicate difference\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"667\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecAMP (pMol/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecGMP(pmol/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBDNF (pg/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e57.291 \u0026plusmn; \u0026nbsp;3.291\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e69.23 \u0026plusmn; 4.924\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e346.23 \u0026plusmn; 23.323\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSham Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e54.691 \u0026plusmn; 4.179\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e67.9213 \u0026plusmn; 3.94\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e342.43 \u0026plusmn; 22.431\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD(D-galac + AlCl3)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e23.0242 \u0026plusmn; 3.425\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e32.432 \u0026plusmn; 3.57\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e154.321 \u0026plusmn; 20.261\u003csup\u003e$$$###\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Avanafil 3 mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e36.32 \u0026plusmn; 2.621\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e56.342 \u0026plusmn; 5.68**\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e271.43 \u0026plusmn; 29.12**\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Avanafil 6mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e46.6421 \u0026plusmn; \u0026nbsp;3.645***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e66.643 \u0026plusmn; 5.663***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e318.52 \u0026plusmn; 30.546***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAD + Donepezil 5mg/kg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e48.136 \u0026plusmn; 4.323***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e58.932 \u0026plusmn; 5.127**\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e308.235 \u0026plusmn; 19.324***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAvanafil 6 mg/kg Perse\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e53.953 \u0026plusmn; 3.137***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e66.32 \u0026plusmn; 5.227***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e322.011 \u0026plusmn; 17.322***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 166px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDonepezil 5 mg/kg Perse\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 172px;\"\u003e\n \u003cp\u003e54.911\u0026plusmn; 4.398***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 167px;\"\u003e\n \u003cp\u003e65.421 \u0026plusmn; 4.684***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 162px;\"\u003e\n \u003cp\u003e319.236 \u0026plusmn; 24.242***\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003e$$$###\u003c/sup\u003e\u003c/em\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 vs control and sham group respectively\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ens p\u003c/em\u003e\u0026gt;0.05 as compared to administered toxic group\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01 vs D-galac + AlCl3administered toxic group\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001 vs D-galac + AlCl3administered toxic group\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alzheimer’s disease, Phosphodiesterase Inhibitor, cAMP, cGMP, Avanafil, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-5911692/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5911692/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlzheimer's disease (AD) is the utmost age-linked neuro-degenerative conditions, marked via gradual deterioration of cognitive abilities and continues to be a significant worldwide health issue. Etiology of AD is linked to neurobehavioral variations, deposition of Aβ, p-Tau, activations of GSK-3β, and fluctuations in cyclic nucleotides including cAMP \u0026amp; cGMP. As per evidence, PDE-5 inhibitors are able to boost cAMP \u0026amp; cGMP levels and other etiological hallmarks, which could be a novel AD cure. The main objective of present study was to examine therapeutic potential of Avanafil in a rat model of AD induced by administering 60mg/kg of D-galactose (D-galac) and 10mg/kg of Aluminium chloride (AlCl\u003csub\u003e3\u003c/sub\u003e) for a period of 42 days. Following this, 28 days of therapy with two different doses of Avanafil (3mg/kg and 6mg/kg) was given. Towards end of treatment, locomotor activity \u0026amp; Morris water maze were performed. Rats were then euthanized and hippocampus was isolated for biochemical parameters \u0026amp; histological investigation. Results revealed that both neurobehavioral parameters exhibits significant difference in treatment group as compared to toxic group. Alterations in level of AchE, Aβ (1\u0026ndash;42), GSK-3β, p-Tau, TNF-α, IL-1β, \u0026amp; IL-6, cAMP, cGMP \u0026amp; BDNF, and oxidative stress were significantly reversed towards normal level in the treatment group when compared to toxic rats. Histopathological changes by H\u0026amp;E staining showed significant difference in treatment vs. toxic rats. The current investigation suggested that Avanafil improves memory by improving cAMP and cGMP pathways, implying that it may have therapeutic prospective in cognitive deficiencies linked with Alzheimer's disease.\u003c/p\u003e","manuscriptTitle":"Ameliorative role of Phosphodiesterase-5 (PDE-5) inhibitor “Avanafil” via modulating cAMP \u0026amp; cGMP Pathway against Alzheimer’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-29 06:01:01","doi":"10.21203/rs.3.rs-5911692/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-10T05:53:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-03T08:08:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285097315335708817421628751118693104994","date":"2025-02-13T09:39:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-13T08:22:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66344358680026990861592000398206391642","date":"2025-02-10T09:03:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31769740124263041726266907954976761859","date":"2025-02-09T08:17:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251217453067585986535733779835222130007","date":"2025-02-08T20:19:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196283809990713916930618779906620448851","date":"2025-02-08T07:29:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152366927326513379370620015040576617196","date":"2025-02-08T05:01:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-08T04:18:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224018506672645004952376829281426281642","date":"2025-02-08T03:24:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109183951762155836068491602622605774965","date":"2025-02-08T02:24:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-08T02:21:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-27T16:43:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-27T13:58:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2025-01-27T10:52:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c7c0875c-e2bc-4201-be27-9f1dedd700bb","owner":[],"postedDate":"January 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-18T16:01:21+00:00","versionOfRecord":{"articleIdentity":"rs-5911692","link":"https://doi.org/10.1007/s11064-025-04516-6","journal":{"identity":"neurochemical-research","isVorOnly":false,"title":"Neurochemical Research"},"publishedOn":"2025-08-13 15:57:30","publishedOnDateReadable":"August 13th, 2025"},"versionCreatedAt":"2025-01-29 06:01:01","video":"","vorDoi":"10.1007/s11064-025-04516-6","vorDoiUrl":"https://doi.org/10.1007/s11064-025-04516-6","workflowStages":[]},"version":"v1","identity":"rs-5911692","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5911692","identity":"rs-5911692","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}