Solanesol Mediated SIRT-1 Activation Prevents Neurobehavioral and Neurochemical Defects in Ouabain-Induced Experimental Model of Bipolar Disorder in Rats

Bidisha Rajkhowa Indo Soviet Friendship College of Pharmacy Sidharth Mehan (  sidh.mehan@gmail.com ) Indo Soviet Friendship College of Pharmacy https://orcid.org/0000-0003-0034-835X Pranshul Sethi Indo Soviet Friendship College of Pharmacy Sonalika Bhalla Indo Soviet Friendship College of Pharmacy Aradhana Prajapati Indo Soviet Friendship College of Pharmacy Sumit Kumar Indo Soviet Friendship College of Pharmacy Abdulrahman Alshammari King Saud University College of Pharmacy Metab Alharbi King Saud University College of Pharmacy Naif AlSuhaymi UQU College of Medicine: Umm Al-Qura University College of Medicine Abdullah Alghamdi Security Forces Hospital in Riyadh: Security Forces Hospital Program Abdulsalam A. Alqahtani Najran University Faculty of Pharmacy Yosif Almoshari Jazan University Faculty of Pharmacy


Experimental animals
Adult Wistar rats (220-250gm, nine weeks of age, either sex) were collected from the ISF College of Pharmacy Central Animal House in Moga, Punjab. These animals were evenly divided and housed in polyacrylic cages with a wire mesh top and soft bedding under typical husbandry circumstances of a 12hour reverse light cycle, free access to food and water, and a temperature of 23±2°C. According to the requirements of the Government of India, the experimental procedure was approved by the Institutional AnimalEthics Committee (IAEC) with a registration number.816/PO/ReBiBt/S/04/CPCSEAasprotocolno. ISFCP/IAEC/CPCSEA/Meeting No: 28/2020/Protocol No.463. Animals were acclimatized to laboratory conditions before being used in experiments.

Drugs and chemicals
OUA was purchased from Sigma-Aldrich (USA). Ex-gratia samples of SNL from BAPEX (India) and Lithium carbonate from Sun Pharma were provided. All of the other chemicals employed in the experiment were of analytical grade. Before use, the medication and chemical solutions were freshly made. Oral administration of SNL dissolved in water (with 2% ethanol) (p.o.) [59].

Experimental animal grouping
A total of 48 Wistar rats (either sex), nine weeks old, were employed during the course of the 28-day protocol schedule. These rats were kept in a polyacrylic cage with a wire mesh top and soft bedding (38 cm 32 cm 16 cm; 3-4 rats per cage) at a regulated temperature (22°C±2°C) and humidity (65-70 %) with arti cial illumination (12 h/12 h light/dark cycle, lights on at 6:00 AM). Their bedding consisted of residue-free wood shavings that had been sanitized. These animals had unrestricted access to a standard chow diet as well as puri ed water. To avoid the effects of the circadian rhythm, the entire experimental protocol schedule was completed between 9:00 AM and 1:00 PM. They were randomly divided into eight groups (n=6 per group). Group1 vehicle control; Group2 Sham control; Group3 SNL perse (80mg/kg p.o.); Group 4 OUA (1 mM/0.5µl/5min/Unilateral/ICV injection); Group5 OUA+SNL (40mg/kg, p.o.);Group 6 OUA+SNL (80mg/kg p.o.); Group7 OUA+Li (60mg/kg, i.p), and Group8 OUA+Li+SNL80. Several behavioral parameters were measured from the rst to the 28th day (Forced swim test, Open eld test, Locomotor activity). The 28th day was marked by collecting biological samples (CSF and blood plasma) from Wistar adult rats. The animals were fully anesthetized with sodium pentobarbital (270 mg/ mL, i.p.), and then fresh brains were preserved in ice-cold PBS (0.1 M) of PBS for further biochemical evaluation. The biochemical estimation of SIRT-1 level determination in brain homogenate, blood plasma, and CSF was performed on the 29th and 30th days. Oxidative indicators (MDA, GSH, SOD, Nitrite, AChE, LDH) were also measured in brain homogenates. Similarly, apoptotic markers (Caspase-3, Bax, Bcl-2) and mitochondrial ETC-complexes enzymes (Complex-I, II, IV, V, and CoQ10) in the brain homogenate and blood plasma were also examined. In ammatory markers (IL-1, TNF-α) and neurotransmitters (Ach, Dopamine, 5-HT, Glutamate) were also measured in brain homogenate and blood plasma. The protocol for the experiment is summarized in (Figure 1).

ICV-OUA induced experimental animal model of BD
The OUA-induced BD experimental model in rats was established using a well-known method [60]. Three days of OUA-ICV injection (1mM/0.5µl) were given to the rats in the experiment. According to Valvassori et al., OUA generates neurological damage similar to that shown in an experimental animal model of BD. It is a valid model for examining pathophysiological alterations similar to those seen in BD.
The rats were habituated to the laboratory environment. After acclimatization, all animals in the experimental groups were anaesthetized with ketamine (75 mg/kg, i.p.) before being placed in a stereotaxic frame [40]. After shaving the head and cutting a midline scalp incision, the skull was exposed. With the tooth bar set at 0 mm, each animal skin overlying the skull, as well as the coordinates for the striatum, must be precisely measured (AP-1.0mm; ML-2.5mm; DV-3.5mm) [60]. Then, according to the protocol schedule, all animals in the experimental groups received OUA (1mM/0.5µl/5min/Unilateral/ICV injection) for three days (1st, 3rd, and 7th days). The infusion was administered manually, using a Hamilton syringe, through the burr holes drilled onto the skull surface. The injection rate in the experimental groups was0.5µl/5min, with the needle remaining in place for a further 1 minute before being progressively removed. The cannula is sealed with a detachable plastic ear pin. The hole was lled with dental cement before being sutured with an absorbable surgical suture connected to a sterile surgical needle.
Rats were housed individually in a polyacrylic cage that usually contained a warm cloth for post-operative care. Special attention was given to them until they regained spontaneous movement, which generally occurred 2-3 hours after anaesthesia. The temperature in the room was kept at 25 ± 3°C. Milk and glucose water were kept in the cages for 2-3 days to avoid physical trauma after surgery. Gentamycin (35 mg/kg) was given intraperitoneally to rats for three days to prevent sepsis, and lignocaine gel was applied to the sutured area to relieve pain. Neosporin powder was dusted on them to prevent bacterial infection of the skin. Following surgery, the general health of the body and clinical symptoms such as dehydration were closely examined. After seven days, rats continued to eat healthy food and drink plenty of water, and their spontaneous mobility returned, indicating that they had healed. The protocol drug SNL at 40 and 80mg and the standard drug Lithium alone and Lithium in combination with SNL80 mg/kg were administered chronically beginning on day 8th and continuing until day 28th. Behavioural parameters such as locomotor activity, open eld test, and Forced Swimming Test were carried out in accordance with the protocol schedule. After completing the protocol schedule, all animals were decapitated on days 29th and 30th, and their brains were removed to perform biochemical, in ammatory, and neurochemical assessments [61].

Parameters assessed
Measurement of body weight According to the protocol schedule, body weight was measured on the 1st, 7th, 14th, 21st, and 28th days of the experiment [60].

Assessment of behavioural parameters
Open eld test (OFT) The animals exhibited manic-like behavior after a single injection of OUA for three days (1st, 3rd, and 7th). The rat was placed in a cage on the rst day and trained to explore an open eld for 5 minutes. During the test, a camera monitored each rat's activities, including an increase in the number of crossings, rearings, and time spent in the center. According to the protocol schedule, on days 1st, 7th, 14th, 21st, and 28th, an open eld test was used to measure the number of crossings, rearings, and time spent in the center in rats [62].

Locomotor activity
Increased locomotor activity is a sign of manic-like behaviour [63]. The device uses photocells to detect motor activity. The animals were placed in the activity room for 3 minutes prior to the recording for habituation. On the 1st, 9th, 18th, and 27th days after ICV administrations, locomotion was assessed using anactophotometer (

Forced swimming test (FST)
A Forced Swimming Test was used to evaluate the immobility time. Individual rats were placed in cylindrical tanks (height 50 cm; diameter 15 cm) with 30 cm of water at a temperature of 24±1°C. A camera lmed the rat's movements for 5 minutes. During the training session, rats are exposed to the tank for 15 minutes on the rst day and 5 minutes on the second day. The testing period for rats consists of a single 6-minute exposure, with the rst 2 minutes serving as a habituation period. Each animal was tested for its depressive-like behaviour on days 1st, 9th, 18th, and 27th following ICV injection. The immobility time was recorded for 5 minutes during each session. When the rat stopped struggling and stayed motionless in an upright position in the water, only making slight movements to keep its head above the water, it was determined to be immobile [64].

Neurochemical alterations evaluation
Collection and preparation of biological samples On day 29th of the experiment, 2.5 ml of blood was collected from anaesthetized rats through retro-bulbar puncture from the orbital venous plexus by inserting a capillary tube medially into the rat eye. Blood from the plexus was collected into a sterile Eppendorf tube via the capillary action through gentle rotation and retraction of the tube [65]. The blood samples were then centrifuged at 10,000×g for 15 minutes to separate the plasma, and the supernatant was carefully stored in a deep freeze (at -80C) for further use.
Following blood collection, rats were deeply anesthetized with sodium pentobarbital (270mg/ml, i.p.) and subjected to caudal incision, translucent duramater was exposed, and a 30gauge needle was gently placed at 30 angle into the cisterna magna [66]. Approximately 100µL CSF was carefully ejected into a 0.5ml sterile Eppendorf tube using the suction pressure of a 1ml tuberculin syringe attached to a needle. The collected sample was frozen at 80 C until analysed ELISA [67].
Immediately after CSF collection, rats were sacri ced by decapitation; whole brains were isolated from the skull with the utmost care, freshly weighed and washed with ice-cold, isotonic saline solution, and then homogenized with 0.1M (w/v) of chilled PBS (pH=7.4). The rat brain homogenate was then centrifuged at 10,000×g for 15 minutes, the supernatant was separated, and the aliquots were preserved. The samples were deep-freezed at -80ºC to be used as and when required for various biochemical estimations.

Assessment of cellular and molecular markers
Measurement of SIRT-1 protein level  [70] and as ng/ml protein in blood plasma [71] and CSF [72].

Measurement of Bax and Bcl-2 levels
Commercial ELISA kits were used to determine the protein levels of Bax and Bcl-2 (E-EL-R0098/Bax/Bcl2 Elabsciences, Wuhan, Hubei, China). The level of Bax protein in brain homogenate [73] and blood plasma was measured [74]. Using ELISA commercial kits, the quantities of anti-apoptotic proteins such as Bcl-2 were evaluated in brain homogenate [37] and bloodplasma [74].

Assessment of mitochondrial ETC-complexes enzyme levels
Preparation of Post mitochondrial supernatant (PMS) from rat whole-brain homogenate The rat whole brain homogenate was centrifuged for 20 minutes at 5000 rpm at 4°C, and the resulting supernatant was used as rat brain PMS for further research. Differential centrifugation was used to prepare the crude mitochondrial fraction. By gently shaking at 4°C for 60 minutes, the pellet generated during the preparation of PMS was combined with 0.1M sodium phosphate buffer (pH 7.4) in a 1:10 proportion. The pellets were re-suspended in the same buffer containing extra sucrose at a concentration of 250 mmol/L after centrifugation at 16000 rpm at 0°C for 30 minutes. The centrifugation and resuspension steps were done three times, and the crude mitochondrial fraction produced in the buffered sucrose solution was used for further investigation [40,75].

Mitochondrial ETC complex-I enzyme activity (NADPH dehydrogenase)
To determine complex-I activity, the rate of NADH oxidation at 340 nm in an assay medium was measured spectrophotometrically at 37°C for 3 minutes. In the absence and presence of 2 µM rotenone, reactions were carried out, and the rotenone-sensitive activity was assigned to complex-I [40,76].
Mitochondrial ETC complex-II enzyme activity(Succinatedehydrogenase/SDH) At 490nm(Shimadzu, UV-1700), the absorbance of a 0.3 mL sodium succinate solution in a 50µl gradient fraction of homogenate was measured. The molar extinction coe cient of the chromophore (1.36×104 M−1 cm−1)was used to determine the results, which were reported as INT decreased µmol/mg protein [40,77].
Mitochondrial ETC complex-IV enzyme activity (cytochrome oxidase) Reduced cytochrome-C (0.3 mM) was added to the assay mixture in a 75 mM phosphate buffer. The process was started by adding a solubilized mitochondrial sample, and the absorbance change was measured for 2 minutes at 550 nm [40].
Mitochondrial ETC complex-V enzyme activity (ATP synthase) To inactivate the ATPases, aliquots of homogenates were sonicated immediately in ice-cold perchloric acid (0.1N). Supernatants containing ATP were neutralized with 1N NaOH and kept at -80°C until analysis after centrifugation (14.000 g, 4°C, and 5 min). A reverse-phase HPLC was used to measure the amount of ATP in the supernatants (PerkinElmer). The reference solution of ATP was made according to the dissolving standard, and the detecting wavelength was 254 nm [40,78].

Measurement of brain serotonin levels
The level of serotonin in brain homogenate was estimated using the method of Sharma et al. with minor modi cations. HPLC with an electrochemical detector and a C18 reverse-phase column was used to determine it. Sodium citrate buffer (pH 4.5) -acetonitrile (87: 13, v/v) is used in the mobile phase. Ten mmol/L citric acid, 25 mmol/L NaH2 HPO4, 25 mmol/L EDTA, and two mmol/L 1-heptane sulfonic acid made up the sodium citrate buffer. The electrochemical parameters in the experiments were +0.75 V, with sensitivity ranging from 5 to 50 nA. At a ow rate of 0.8 ml/min, the separation procedure was carried out. 20 µl of samples were manually injected. On the day of the experiment, brain samples were homogenized in 0.2 mol/L perchloric acid. The samples were then centrifuged for 5 minutes at 12,000 rpm. The supernatant was ltered via 0.22 mm nylon lters before being injected into the HPLC sample injector.
With the help of the breeze program, data were collected and evaluated. Using a standard with a 10-100 mg/ml concentration, serotonin concentrations were determined from the standard curve [40].
Assessment of brain dopamine levels Dopamine levels in striatal tissue samples were measured using Tiwari and colleague's technique. Dopamine activity in rat brain homogenate quanti ed as ng/mg protein [73].
Assessment of brain glutamate levels According to Alam et al., glutamate was measured in tissue samples after derivatization with ophthalaldehyde/β-mercaptoethanol (OPA/β-ME) and quantitative analysis in rat brain homogenates, glutamate activity is reported as ng/mg protein [39].

Assessment of brain acetylcholine levels
A diagnostic kit is used to measure acetylcholine (E-EL-0081/acetylcholine; Elabsciences, Wuhan, Hubei, China). All reagents and rat brain homogenate were produced according to the kit's normal procedure. In the microtiter plate, the optical density of the reaction mixture was determined at 540 nm [76].

Measurement of reduced glutathione levels
In the brain homogenate, the level of reduced glutathione was determined. 1 mL supernatant was precipitated with 1 mL 4% sulfosalicylic acid and cold digested for 1 hour at 4°C. The samples were centrifuged for 15 minutes at 1200 rpm. To 1 ml supernatant, 2.7 ml phosphate buffer (0.1M, pH 8) and 0.2 ml 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were added. A spectrophotometer was used to measure the yellow color that emerged at 412nm right away. Glutathione content in the supernatant, given as µM/mg protein [79].

Measurement of nitrite levels
A colorimetric assay utilizing Greiss reagent (0.1 % N-(1-naphthyl) ethylenediamine dihydrochloride, % sulfanilamide, and % phosphoric acid) determines the concentration of nitrite in the supernatant, which is indicative of the formation of nitric oxide (NO). Equal amounts of supernatant and Greiss reagent are mixed, the mixture is incubated at room temperature in the dark for 10 minutes, and the absorbance is measured spectrophotometrically at 540nm. A sodium nitrite standard curve is used to calculate nitrite concentration in the supernatant, which is given as µM/mg protein [79].

Measurement of malondialdehyde (MDA) levels
The MDA end product of lipid peroxidation was determined quantitatively in brain homogenates. A spectrophotometer was used to measure the quantity of MDA after its reaction with thiobarbituric acid at 532nm. MDA concentration is expressed in nM/mg of protein [80].
Measurement of superoxide dismutase (SOD) levels SOD activity was evaluated by auto-oxidation of epinephrine at pH 10.4 using spectrophotometry. The brain homogenate supernatant (0.2 ml) was combined with 0.8 ml of 50 mM glycine buffer, pH 10.4, and the reaction was begun with 0.02 ml epinephrine. The absorbance was spectrophotometrically measured at 480nm after 5 minutes. The activity of SOD was measured in nM/mg of protein [37].

Measurement of acetylcholinesterase (AChE) levels
The levels of acetylcholinesterase (AChE) were measured using spectrophotometry. The 0.05 ml supernatant, 3 ml 0.01M sodium phosphate buffer (pH 8), 0.10 ml acetylthiocholine iodide, and 0.10 ml DTNB were used in the test mixture (Ellman reagent). The absorbance change was spectrophotometrically recorded at 412 nm right away.In the supernatant, the enzymatic activity is represented as µM/mg protein [40].

Measurement of lactate dehydrogenase (LDH) assay
A diagnostic kit (Coral Diagnostics, India) was used to quantify the amount of LDH in the rat brain homogenate, and the amount of LDH was quanti ed as Units/L [81].
Evaluation of Na + /K + ATPase activity in rat brain homogenate The activity of the Na + /K + ATPase enzyme was measured using a spectrophotometer and a calorimetric method-based assay kit (E-BC-K539-M; Na + /K + ATPase ELabSciences, Wuhan, Hubei, China). The Na + /K + ATPase assay reaction mixture contains 5.0 mM MgCl2, 80.0 mM NaCl, 20.0 mM KCl, and 40.0 mM Tris-HCl in a nal volume of 200 l with a pH of 7.4. The reaction was begun after a 10-minute pre-incubation interval at 37°C by adding 3.0mM ATP and incubated for 20 minutes. Controls were carried out under identical conditions as before, but with the addition of 1.0 mM ouabain. The difference between the two assays was utilized to calculate Na + /K + ATPase activity. The speci c activity of the enzyme was measured in nmol of Pi released per minute per mg of protein [82].

Protein estimation
A Coral protein estimation kit (Biuret method) was used to determine the protein content.

Statistical analysis
The mean and standard error of the mean was used to express all of the ndings (SEM). The data were analyzed using a two-way ANOVA followed by a Bonferroni post hoc test and a one-way ANOVA followed by a Tukey's multi comparison test. It was determined that P<0.001 was statistically signi cant. The sample size was estimated after the data was con rmed to be normalized, and the normality distribution was checked using the Kolmogorov Smirnov test. GraphPad Prism version 5.03 for Windows was used to generate all statistical results (GraphPad Software, San Diego, CA, USA). The mean and standard error of the mean was used to express the statistical data (SEM).

Neuroprotective potential of solanesol on weight variations in ouabain-induced bipolar disorder rats
Improvement in body weight after solanesol treatment Bodyweight was measured once a week, on days 1st, 7th, 14th, 21st, and 28th of the procedure schedule. Figure 2 depicts the differences in body weight caused by the toxin OUA compared to the treatment drugs over the protocol schedule. Compared to the vehicle, sham, and SNL80 perse treated groups, the administration of OUA for 1st, 3rd, and 7th days resulted in a consistent decline in body weight. From day 8th to day 28th, rats receiving prolonged oral treatment with SNL and Lithium demonstrate a remarkable restoration in body weight due to improvements in psychiatric behaviors such as decreased locomotor activity, rearing, stress, and increased food intake.
Compared to SNL40 and SNL80 mg/kg treated rats, the Li60 mg/kg treated rats showed a more signi cant improvement in body weight. In addition, compared to other drug treatment groups such as SNL40 mg/kg, SNL80 mg/kg, and Li60 mg/kg, standard drug Li60 mg/kg in combination with SNL80 mg/kg showed signi cant weight restoration. SNL 80 mg/kg is more effective than SNL40 mg/kg in recovering OUA-induced lower body weight,demonstrating that SNL has a dose-dependent impact on restoring body weight [Two-way ANOVA: F(28, 160)=903.4,p<0.001]. (Figure 2) 3.2 Neuroprotective potential of solanesol in the prevention of neurobehavioral abnormalities in ouabain-induced bipolar disorder rats Decrease manic-like behavior after solanesol treatment in the open eld task Three days (1st, 3rd, and 7th ) following a single OUA injection, the animals developed manic-like behaviors, as seen by the increased number of crossings, rearings, and time spent in the centre. Open eld parameters were conducted on days 1st, 7th, 14th, 21st, and 28th of the protocol period to determine the number of crossings, the number of rearings, and time spent in the centre in rats. a. Decrease number of crossing after solanesol treatment The number of boxes crossed by rats in an open eld is depicted in Figure 3a. There was no signi cant difference between the groups on the 1st day. The OUA-treated rats crossed more boxes than the vehicle, sham, and SNL80-treated rats. On the 7th day, there was no signi cant difference between the OUAtreated group and the other treatment groups. After 20 days of oral administration of the neurotoxic OUA, the SNL treatment group had a progressive reduction in the number of boxes crossing compared to the vehicle control, sham control, and SNL80 perse groups on days 14th, 21st, and 28th. At the 21st and 28th days, the Li60 mg/kg alone and combined with SNL80 mg/kg treated animals had considerably reduced the number of boxes crossing than the SNL80 mg/kg and SNL40 mg/kg treated groups. Furthermore, when comparing SNL80 mg/kg treatment to SNL40 mg/kg treatment in BD-like rats, animals showed a lesser number of boxes crossed [Two-way ANOVA:F(28,160)=190.0, p<0.001]. (Figure 3a) b. Decrease number of rearing after solanesol treatment In the open eld, the number of rearing behaviors in BD like rats is shown in Figure 3b.On the 1st day, there was no signi cant difference between the groups. The OUA-treated rats showed more rearing moves than the vehicle control, sham control, and SNL80 treated rats. There was no signi cant difference between the OUA treated and other treatment groups on the 7th day. On days 14th, 21st, and 28th, after 20 days of oral administration of the OUA, the number of rearings in the SNL treated groups decreased over time compared to the vehicle control, sham control, and SNL80 perse groups. The Li60 mg/kg alone and Li60 mg/kg along with SNL80 mg/kg treated animals showed a signi cantly lesser number of rearing on 21st and 28th days than the SNL80 mg/kg and SNL40 mg/kg treated groups. Furthermore, when BD-like rats were given SNL80 mg/kg versus SNL40 mg/kg, the animals showed a lesser number of rearing movements. signi cant difference between the groups. The OUA-treated rats stayed longer than vehicle, sham, and SNL80-treated rats. There was no signi cant difference between the OUA-treated group and the other treatment groups on the seventh day. On days 14th, 21st, and 28th compared to the vehicle control, sham control, and SNL80 perse groups, time spent in the center in the SNL treated groups reduced over time following 20days of oral administration of the OUA.The Li60 mg/kg alone and Li60 mg/kg combined with SNL80 mg/kg treated animals spent signi cantly less time in the center on the 21st and 28th days than the SNL80 mg/kg and SNL40 mg/kg treated groups. Moreover, BD-like rats administered SNL80 mg/kg spent less time in the center than rats given SNL40 mg/kg. Decreased manic-like behavior after solanesol treatment As illustrated in Figure 4, the results suggest that OUA signi cantly affects locomotor activity in BD-like rats. On the 1st day, there was no signi cant difference between the groups. Rats were given OUA on days 1st, 3rd, and 7th, demonstrating considerably higher locomotor activity during the protocol schedule compared to the vehicle control, sham control, and SNL80 treated rats. Locomotor activity decreased from day 8th to day 28th after SNL treatment, as observed with the mood stabilizer Li60 mg/kg treated rats. Compared to the SNL80 mg/kg and SNL40 mg/kg treatment groups, Li60 mg/kg administration, both alone and in combination with SNL80 mg/kg, signi cantly reduced locomotor activity. In addition, SNL80 mg/kg signi cantly reduced locomotor activity in actophotometer rats when compared to SNL40 mg/kg treated rats on day 27th [Two-way ANOVA: F(21,120)=244.1, p<0.001]. These results indicate that Lithium and SNL have an antimanic effect when given alone and a more signi cant enhancement in antimanic action when given together during OUA-induced BD like rats on days 18th and 27th. (Figure 4) Decreased depression-like behavior after solanesol treatment As shown in Figure 5, the results reveal that OUA has a considerable in uence on immobility time in BDlike rats. On the 1st day, there was no signi cant difference between the groups. Rats were given OUA on days 1st, 3rd, and 7th had signi cantly prolonged immobility time during the protocol schedule compared to the vehicle control, sham control, and SNL80 perse treated rats. From day 8th to day 28th, immobility time was signi cantly reduced with SNL treatment, as reported with the mood stabilizer Li60 mg/kg.Li60 mg/kg treatment, both alone and combined with SNL80 mg/kg, signi cantly reduced immobility time compared to the SNL80 mg/kg and SNL40 mg/kg treatment groups. Furthermore, compared to SNL40 .001] samples were elevated after continuous oral administration of SNL at doses of 40 mg/kg and 80 mg/kg. In rat brain homogenate, blood plasma, and CSF samples, SNL80 mg/kg was more effective than SNL40 mg/kg in restoring SIRT-1 protein expression. Furthermore, the Li60 mg/kg alone and Li60 mg/kg combined with SNL80 mg/kg treated groups were more effective in restoring SIRT-1 protein expression in rat brain homogenate, blood plasma, and CSF samples than the SNL80 mg/kg and SNL40 mg/kg treated groups. (Table 1)  Decreased level of caspase-3, Bax, and increased Bcl-2 levels after long-term administration of solanesol The levels of cell death indicators such as Caspase-3, Bax, and Bcl-2 were measured in rat brain homogenate and blood plasma samples after the protocol schedule. In rat brain homogenate and blood plasma samples, ICV injection of OUA treatment resulted in a signi cant increase in pro-apoptotic markers such as caspase-3 and Bax. In contrast, the ICV injection of OUA for three days (1st, 3rd, and 7th) resulted in a signi cant decrease in anti-apoptotic Bcl-2 protein levels in rat brain homogenate and blood .001] samples with respect to the OUA-treated BD like rats. Also, SNL80 mg/kg treatment was more effective than SNL40 mg/kg treatment in restoring abnormal levels of apoptotic markers in BD-like rats. Furthermore, in rat brain homogenate and blood plasma, the Li60 mg/kg alone and Li60 mg/kg combined with SNL80 mg/kg treated groups showed more signi cance in restoring the altered levels of apoptotic markers than the SNL80 mg/kg and SNL40 mg/kg treated groups. (Table 2) Restoration of mitochondrial ETC-complexes enzyme level after long-term administration of solanesol After the experiment protocol schedule, the enzyme activity of mitochondrial ETC-complexes was evaluated in rat brain homogenate. In OUA-treated rats, twenty days of chronic administration with SNL40mg/kg and SNL80 mg/kg substantially and dose-dependently recovers and increases mitochondrial ETC complex enzymatic activity. The signi cant restoration was observed with a high dose of SNL80 mg/kg group in mitochondrial ETC complexes-I, II, IV, V, and CoQ10 compared to a low dose of SNL40 mg/kg. The most signi cant improvements in mitochondrial ETC complexes-I, II, IV, V, and CoQ10 in rat brain homogenate were seen in the Li60 mg/kg alone and Li60 mg/kg in combination with SNL80 mg/kg treated groups, which were more effective than the SNL80 mg/kg and SNL40 mg/kg treated groups. (Table 3) Table3:Neuroprotective potential of solanesol on TNF-α and IL-1β level in ouabain-induced bipolar disorder in rats S. no. .001] in rat brain homogenate. Moreover, SNL80 mg/kg versus SNL40 mg/kg treated rats re-establish lower neurotransmitter levels. The Li60 mg/kg alone and Li60 mg/kg combined with SNL80 mg/kg treated groups were more effective than the SNL80 mg/kg, and SNL40 mg/kg treated groups in restoring the altered levels of neurotransmitters in rat brain homogenate. (Table 4) homogenate and blood plasma samples, the Li60 mg/kg alone and Li60 mg/kg in conjunction with SNL80 mg/kg treated groups exhibited a substantial improvement in lowering the level of these in ammatory mediators compared to the SNL80 mg/kg, and SNL40 mg/kg treated groups at the end of protocol schedule. (Table 5)  Increased Na + /K + ATPase enzyme activity after long-term administration of solanesol The enzyme activity of Na + /K + ATPase in rat brain homogenate was assessed immediately afterward the experiment protocol schedule. Compared to the vehicle control, sham control, and SNL80 perse groups, ICV injection of OUA resulted in a substantial decrease in Na+/K+ ATPase activity. The activity of Na+/K+ ATPase in rat brain homogenate was increased after continuous oral administration of SNL at dosages of 40 mg/kg and 80 mg/kg [One-way ANOVA: F(7,35)=2.236, P<0.001]. SNL80 mg/kg restored Na+/K+ ATPase activity more effectively than SNL40 mg/kg in rat brain homogenate. Furthermore, the Li60 mg/kg alone and combined with SNL80 mg/kg treated groups restored Na+/K+ ATPase more e ciently than the SNL80 mg/kg, and SNL40 mg/kg treated groups (Table 7; Figure 6)  Several investigations have found that rats exhibit manic behaviours such as increased locomotor activity, rearing, and crossing after ICV injection of OUA [13,92]. We observed the same result in our experiment, where locomotor activity, number of boxes traversed, number of rearing movements, and time spent at the center signi cantly increased after seven days of protocol schedule in ICV-OUA induced BDlike rats. In addition, following OUA administration, the current investigation exhibited manic and depressive-like behaviours in the same animal. This study aims to show that SNL can prevent BD-like behavioural and neurochemical abnormalities in OUA-induced BD in rats by upregulating SIRT-1 protein levels.
The rats did not show any behavioural alterations in the open eld test, forced swimming, or locomotor activity following OUA treatment compared to the vehicle control, sham control, and SNL80 perse groups. As a result, rats may have a calm episode nine days after OUA injection [60, 93,94]. Differences in rat strains and experimental conditions could explain the variation between experiments. The methodology was repeated in the current study for biochemical analysis, and we obtained identical results in the openeld test [95].
Several studies suggest that lithium medication can alleviate manic-like behaviour in rats given OUA-ICV injections [96,8]. In contrast, lithium treatment signi cantly reversed the immobility time. Although previous preclinical studies have shown lithium's antidepressant properties [97,98], the current study replicated the depression and manic-like behaviours in a selective BD animal model.
The development of an animal model of BD using OUA is based on the premise that decreased Na+/K+ATPase activity is essential in starting manic and depressive mood episodes [60,99]. Seven and nine days after ICV injection, OUA decreased Na+/K+ATPase activity in the rodent's brain. The role of Na+/K+ATPase in BD physiopathology was hypothesized more than 50 years ago [100]. A meta-analysis study found that Na+/K+ATPase activity is lower in the erythrocytes of BD patients [101]. Even a slight reduction in this enzyme activity can put the resting membrane potential near the threshold, enhancing neuronal excitability and delaying the onset of Ca2+ depuration [102]. Hyperactivity, associated with manic episodes in bipolar disorder, may be induced by increased neuronal excitability. Long-term suppression of Na+/K+-ATPase, which increases neuronal excitability, may reduce resting potential regulation, making subsequent neuronal depolarization more challenging. These events may reduce neuronal transmission velocity and, as a result, neuronal synaptic e ciency, resulting in BD depressive episodes [103]. Increasing the activity of the Na+/K+ATPase may be one of lithium's anti-oxidative mechanisms. OUA-induced oxidative damage in rats resembled the pathophysiological characteristics of BD patients. Indeed, reduced anti-oxidant glutathione enzymes in the brain have been reported in mania and depression animal models [104]. One of lithium's possible therapeutic effects is to modulate these anti-oxidant enzymes, which helps to maintain redox balance in the brain [105]. According to research ndings, decreased activity of Na+/K+ATPase in BD patients may be associated with increased production of dopamine and glutamate neurotransmitters, as well as oxidative damage, resulting in mood swings [106].
Lithium, as a mood stabilizer, acts to counteract these pathological changes, which helps to reduce BD symptoms. The proposed OUA model could be used to investigate the disorder's pathophysiology and assess potential mood stabilizers. In addition to decreased ATP synthesis, chronic OUA treatment of the brain resulted in increased oxidative stress-mediated by ROS and RNS, glial cell overactivation, and lower SIRT-1 protein level [60]. SIRT-1 deacetylation is dependent on NAD+ and ATP production in cells and regulates its levels in mitochondria and other areas of the brain. SIRT-1 dysregulation also causes memory impairment, and oxidative markers have been employed to identify the excessive production of ROS and RNS in the brain [107]. An increase in oxidative stress has been associated with a decrease in the activity of the Na+/K+ATPase in bipolar patients [108].
According to current ndings, OUA-treated rats had lower body weight on days 14th, 21st, and 28th. Furthermore, on days 9th, 18th, and 27th, there was an increase in locomotor activity in the actophotometer, which was responsible for manic-like behaviour. This manic-like activity was seen by OFT on the 7th, 14th, 21st, and 28th days, demonstrating a progressive rise in the number of rearing, the number of boxes crossing, and time spent in the center. FST on the 9th, 18th, and 27th days indicated an increase in immobility time.
This study investigates the effect of OUA on the protein level of SIRT-1 in the brain, which was found to be lower in brain homogenate, blood plasma, and CSF samples. In addition, the levels of the apoptotic markers caspase-3, Bax, and Bcl-2 were measured, and OUA-treated rats showed greater levels of caspase-3, Bax, and lower levels of Bcl-2. Reduction in mitochondrial ETC complex enzymes, on the other hand, has been associated with a signi cant increase in in ammatory cytokines TNF-α and IL-1β. Furthermore, this study looked into the effect of OUA on Na+/K+ ATPase activity, which was found to be decreased after the OUA injection. Our investigation demonstrated that when rats were repeatedly exposed to OUA, the amounts of neurotransmitters changed. Neurotransmitters have a variety of diverse effects on the brain. Several neurons in the brain release acetylcholine, which has been connected to memory and learning [109,110], circadian rhythms [111], antinociception [112,113], locomotion [114,115], and the sleep-wake cycle (116,117). Serotonin is a neurotransmitter that has several effects in the brain that are regulated by various serotonergic receptors [118], involved in cognition [119], learning, memory, and attention [120,121], emotions [122], stress, mood [123,124], movement [125], and sleep [126]. Glutamate, a primary excitatory neurotransmitter in the brain, is also implicated in long-term potentiation and long-term depression (synaptic plasticity). These two processes are associated with memory and learning [127] and neurogenesis [128]. Dopamine is a monoamine neurotransmitter that is involved in a variety of brain functions, including motor function control and learning new motor skills [129,130], pleasure and reward-seeking behavior [131,132], addiction [133], cognition [134,135], pain process [136,137], gastrointestinal motility [138,139]. Neurotoxic effects by OUA in rats are shown by decreased serotonin and acetylcholine levels and increased dopamine and glutamate levels. Oxidative stress is a major cause of neurodegenerative disorders. Treatment with OUA raises MDA, Nitrite, AChE, and LDH levels while decreasing antioxidant enzymes SOD and GSH levels.
Our ndings revealed that twenty days of chronic treatment with SNL40, 80 mg/kg in ICV injection to OUA-treated rats resulted in a signi cant improvement in body weight. In addition, there was a reduction in locomotor activity measured by the actophotometer. The high dose-response of SNL shows a signi cant improvement in behavioural abnormalities. In contrast, the standard drug lithium alone and in combination with SNL high dose exhibited a signi cant improvement in behavioural alterations compared to SNL alone treated rats. Current research indicates that SIRT-1 levels in CSF, brain homogenate, and blood plasma samples increase after continuous treatment with SNL40 and SNL80 mg/kg. Furthermore, Li-treated groups restored SIRT-1 protein levels more e ciently than SNL-treated groups in rat brain homogenate, blood plasma, and CSF samples. The apoptotic marker level in blood plasma and brain homogenate, on the other hand, shows a decrease in caspase-3, Bax and an increase in Bcl-2. Furthermore, the results show that continuous SNL treatment recovers mitochondrial ETC-complexes enzyme levels Complex I, II, IV, and V, as well as CoQ10 in brain homogenate. SNL administration reduces neuronal in ammation, as evidenced by lower levels of TNF-α and IL-1β in blood plasma and rat brain homogenate. Furthermore, SNL increased serotonin and acetylcholine levels while lowering dopamine and glutamate levels in rat brain homogenates.
Oxidative damage in OUA-treated rats treated with SNL40 and 80 mg/kg, on the other hand, shows a reduction in oxidative stress as seen by a signi cant decrease in MDA, Nitrite AChE, and LDH levels. In addition, there was a signi cant rise in the amount of anti-oxidant markers SOD and GSH in brain homogenate. Additionally, after continuous treatment with SNL40 and SNL80 mg/kg, Na+/K+ ATPase enzyme activity increased in rat brain homogenate, although Li-treated groups restored activity more effectively than SNL-treated groups. The Li60 mg/kg alone and Li60 mg/kg in conjunction with SNL80 mg/kg treated groups restored the altered Na+/K+ ATPase enzyme levels more successfully than the SNL80 mg/kg SNL40 mg/kg treated groups in brain homogenate samples.
As a result, the current study indicates that ICV-OUA administration reduces SIRT-1 protein level and neuronal death in rats. Furthermore, there was a reduction of mitochondrial ETC complexes in the disease condition and an increase in in ammation and oxidative stress. Prolonged SNL and Li therapy produces improvements and signi cant dose-dependent restorations. As a result, these SIRT-1 and SNL activators exerted neuroprotective effects following OUA-mediated BD rat model ICV injections.
Although the current ndings are just correlations, they suggest that SNL reduced SIRT-1 protein level in rats with BD-like behavioural and neurochemical symptoms in OUA-induced BD. Our ndings suggest that SIRT-1 levels in brain tissue, blood plasma, and CSF can be used as an effective and reliable early diagnostic biomarker for predicting neurological dysfunctions. Lithium works as a mood stabilizer drug to counteract these pathological changes that assist in alleviating BD symptoms. The proposed OUA model could explore disease etiology and screen potential mood stabilizer drug candidates. Overall, a mechanistic approach must be validated using sirtuin gene knock-in or knock-out experiments. A correlative study, such as Western Blot for cellular markers, is also necessary to offer molecular support for this hypothesis. Despite these limitations, the neuroprotective potential of SNL gives the possibility to develop a new disease-modifying treatment for the neurodegenerative disease by SIRT-1 signalling activation in the brain.

Conclusion
Page 28/47 Finally, the research con rms that SNL protects rats from developing BD caused by OUA. This is the rst study to link SNL's antioxidant, anti-in ammatory, and anti-apoptotic properties to its potential neuroprotective bene t as a therapy for the management of BD. The amounts of several neurochemicals in brain homogenate, blood plasma, and CSF were examined, revealing that SNL had a central and peripheral protective impact by reducing BD-like alterations. According to the ndings, this study can be used as strong evidence that SIRT-1 downregulation and serotonin evaluation can be employed as a potential biomarker for the early detection of BD. The primary limitation of this study is the lack of gross pathology and immunohistology research on the area-speci c molecular mechanistic effect of SNL. As a result, more preclinical research on the knock-in and knock-out of the SIRT-1 gene is required to better understand the molecular mechanism.  The Government of India supported this work, the experimental procedure was approved by the Institutional AnimalEthics Committee (IAEC) with a registration number.816/PO/ReBiBt/S/04/CPCSEA as protocol no. ISFCP/IAEC/CPCSEA/Meeting No: 28/2020/Protocol No.463.
Competing Interests "The authors declare no con ict of interest." "The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results".