Mechanistic Insight Into Diosmin-Induced Neuroprotection And Memory Improvement in Intracerebroventricular-Quinolinic Acid Rat Model: Resurrection Of Mitochondrial Functions And Antioxidants Part 3

2. Material and Methods

2.1. Experimental Animals. .is research was permitted by IAEC under protocol no. ASCB/IAEC/14/20/145. Albino Wistar rats (either sex, 200 g to 250 g, age 8 to 9 months old) were retained in typical-size polypropylene cuboidal enclosures under artificial settings of temperature (23 ± 2°C), 12:12 hours dark/light sequences, and humidity (40 ± 10%) within the institutional animal house. .e rodents were fed a standard nourishing foodstuff (Ashirwad Manufacturers, Punjab) and purified water at will. 

The relationship between males and females and memory has always attracted much attention. It is well known that male hormones play an important role in physical development, maintaining health, and promoting sexual development and sexual function. In addition, recent studies have shown that male hormones are closely related to memory.

Studies have shown that men perform better than women in certain memory tasks. For example, men usually perform better than women in spatial memory, and the reason is the effect of male hormones on the hippocampus. The hippocampus is the area in the brain that processes spatial memory. Male hormones can promote the function of the hippocampus, thereby improving men's spatial memory ability.

However, female hormones also have a positive effect on memory. Studies have found that female hormones can promote the expression of memory-related genes in the brain and increase the number and function of neurons in the hippocampus, thereby improving women's memory. In addition, female hormones can relieve stress, inhibit neuronal cell death, protect brain neurons, and further improve women's memory.

This shows that both male hormones and female hormones have a positive effect on improving memory. Men and women have different memory performances, each with its advantages and disadvantages, but it does not mean that men have a better memory than women or that women have a better memory than men. Therefore, we should not use gender as a measure of memory. Everyone should give full play to their advantage and constantly improve their memory.

Finally, whether men or women, we should pay attention to our physical health and mental condition and choose the right way to improve our memory. For example, constantly learning new things, developing regular living habits, and maintaining emotional stability can have a positive impact on improving memory. Let us work together to continuously explore and practice methods to improve memory and create a better future for ourselves. It can be seen that we need to improve our memory. Cistanche can significantly improve memory because Cistanche is a traditional Chinese medicine with many unique effects, one of which is to improve memory. The efficacy of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in many ways.

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All animal procedures are exclusively performed as per the guidelines of CPCSEA, GOI, New Delhi. .e animal custodians and handlers were blinded concerning different therapeutic regimens facilitated to animal cohorts. Investigative animal trials were executed, succeeding at least a single fortnight of familiarization duration. 

All investigations using animals were performed between 0900- and 1600-hours hours a day. 2.2. Drugs and Chemicals. Diosmin (DSM: 520-27-4), quinolinic acid (QA: 89-00-9), and standard analytes were acquired from Merck (India). 

Sodium dihydrogen phosphate (NaH2PO4), sodium hydroxide (NaOH), potassium phosphate dibasic (K2HPO4), nitrobluetetrazolium (NBT), phenazine methosulphate (5-methylphenazinium methyl sulphate), ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES), 1,2-bis[2-[bis(carboxymethyl) amino]ethoxy]ethane (EGTA), riboflavin, sodium cyanide (NaCN), natriumazid (NaN3), tetrasodium pyrophosphate, hydrogen peroxide (H2O2), NADH disodium (DPNH), NADPH tetrasodium (Coenzyme II reduced tetrasodium salt), phosphoric acid, Folin and Ciocalteu's phenol (FCR), and sulphosalicylic acid (5-SSA) reagent (HiMedia Laboratories, Maharashtra, India); diglycine, glacial acetic acid (CH3COOH), Ellman's reagent (3-Carboxy-4-nitrophenyl disulfide, DTNB), azabenzene (C5H5N), and sodium lauryl sulphate (SLS) (LobaChemie, Mumbai, India); 4,6-Dihydroxy-2-mercaptopyrimidine (2-TBA), disodium carbonate (Na2CO3), and (2-mercaptoethyl)trimethylammonium iodide acetate (TCI chemicals, India); zinc sulphate (ZnSO4), Rochelle salt (potassium sodium L(+)-tartrate), 2-(1- Naphthylamino)ethylamine dihydrochloride, nitrous acid sodium (NaNO2), and p-aminobenzenesulfonamide (Sisco Research Laboratories, India); butyl alcohol (Fisher Scientific, India) were used. 2.3. Intracerebroventricular Injection of Quinolinic Acid. 

Animals were subjected to anesthesia by administering intraperitoneally (i.p.) ketamine (90 mg/kg) and xylazine (10 mg/kg) cocktail using sterile water for injection. .e body was laid in the prone position on a warm heating cushion, and in the mount of a stereotaxic surgery instrument, the head was situated. .e scalp was incised at the midsagittal point, and the skull was uncovered by retracting the skin apart. 

Any one of the two lateral ventricles was arbitrarily chosen, and in the skull, the parietal bone was bored (stereotaxic coordinates -0.8 mm anteroposterior from bregma, ±1.5 mm mediolateral from midsagittal suture, and ±3.6 mm dorsoventral from the parietal bone surface) to make a burr hole [21]. 

On day one, quinolinic acid (QA) solution was freshly constituted (240 nmol) in PBS (Na+ -K+ [PO4] 2- buffered saline, pH 7.4) and was gradually injected using a Hamilton microsyringe at a flow rate of 1 µl/minute in the left or right cerebral ventricle of rats over 5 to 6 minutes duration with the volume of injection 5 µl ICV-vehicle [22]. 

After the inoculation of the whole drug, the microneedle was not dislodged for 4 to 5 minutes to enable the diffusivity of the drug in the cerebrospinal fluid and thwart its regurgitation. .e equivalent volume (10 µl) of PBS-vehicle was administered ICV in sham rats that were identically operated, however, QA was not injected. 

After drug injections, the holes were restored using a luting agent (zinc phosphate, PYRAX®), and the stitching of the skin was accomplished. To avert contamination (bacterial growth), Neosporin® was applied pro re nata. 

To evade postoperative sepsis, Orizolin (Zydus Cadila), a dose of 30 mg/kg (i.p.), was administered. Each rat was provided a warm environment (37 ± 0.5°C) to avert postsurgical hypothermia. Each rat was allowed semisolid food (inside the cage) and water gratis after surgery for seven days and housed discretely in a distinct cage (30 × 23 ×14 cm3 ). 

2.4. Experimental Protocol. DSM was injected at doses 50 and 100 mg/kg per body weight (b.w.) in rats through the intraperitoneal (i.p.) route using 0.5% dimethylsulfoxide vehicle in normal saline (dose-volume 5 ml/kg) [17]. 

Animals were randomly allocated in 5 clusters in a single-blind mode (n � 5): (i) sham (S), (ii) QA, (iii) QA + DSM50, (iv) QA + DSM100, and (v) QA + DNP. Rats were subjected to the intracerebroventricular administration of QA (QA-ICV) or sham surgery on the 1st day. 

DSM was administered for 21 consecutive days daily 120 minutes after QA-ICV from day one onwards. Donepezil (DNP) was employed as a standard drug in this study and injected (dose 3 mg/kg, i.p.) in QAICV-injected rats for 21 successive days. 

Animals in the sham and QA control groups were administered vehicle (sterile 0.5% dimethylsulfoxide in normal saline in dose-volume 5 ml/kg) from day 1 to 21. .e whole study was performed according to the scheme depicted in Figure 1. 2.5. Locomotor Activity. 

In all rat clusters, the mean locomotor activity was documented using an actophotometer device for 5 minutes. A separate animal was positioned in the actophotometer for 3 minutes of acclimatization. .e rats were then given 5 minutes, and the results were stated as counts per 5 minutes [11]. 2.6. Rotarod Test. 

In rodents, the rotarod test typically evaluates the equilibrium and muscle synchronization facets of sensorimotor functions. .e rats were presented to acquisition trials until their ability to run reached >60 seconds on the rod revolving at nine rotations per minute (rpm). 

After the acquisition trials, a separate rat was positioned on the cylindrical shaft, and the revolution velocity was boosted at a constant intermission of 10 seconds from 6 rpm (preliminary speed) to 30 rpm (concluding speed), spanning over 50 seconds. .e mean fall-off latency (in seconds) from the revolving cylindrical shaft was stated in the results. 2.7. 

Footprint Analysis. The principle behind performing footprint analysis in rats is assessing the gait abnormalities. 

For footprints, rat feet were immersed in four diverse colored nontoxic food dyes and were permitted to run on an inclined walkway (70 cm × 10 cm × 8 cm). The runway base was enclosed with a cellulose sheet of white color. .e rats were motivated to a dim uphill section at the end of the runway to obtain clear footprints. .e dye was gently removed from each animal using lukewarm water after the trials. .e footprints were scanned, and the "stride length" was measured using a standard ruler. Stride length was quantified by calculating the distance between the sequential placements of the identical rat's paw [11].

2.8. Novel Object Recognition Task (NORT). Standard protocol was followed as given by Kumar and Bansal [23]. 

ORT is an unprofitable and nonhostile exteroceptive archetype employed to assess the working type recollective memory exploited through the impulsive probing conduct of rodents. .e investigation is performed in a rooftop-open plywood cuboidal vessel (80 cm × 42 cm × 62 cm), positioned in a quiet area, illuminated by a 60W LED to manage consistent brightness in the vessel. 

Cylinder (white)-, pyramid (red)-, and cube (black)-shaped (12 cm tall) wooden items (in identical triplets) were solid and of enough weight to render them immobile by rodents. NORT was performed on the 16th day in 3 stages (S): acclimatization (S1), acquisition (S2), and novel object recognition examination (retention) stage (S3). 

During S1, three successive days before the trials were issued to discover the vacant floor base of the vessel (5 minutes) by the rats. Upon the completion of S1, the individual animal was habituated to any one set of solid items in the learning stage (S2). 

Twin-like things were positioned in 2 arbitrarily selected contrary angles of the vessel (9 cm to 11 cm gap from the side ramparts). Separately, a rodent was positioned at the center of the vessel facing opposite to the two solid items and was permitted to discover the two similar items for 5 minutes. Guiding the snout near the object at ≤2–3 cm distance or physical contact with the item with the muzzle was supposed as investigative conduct. 

After S2, the rodent was housed in a home cage trailed by an intertrial recess (ITR) of 60 minutes. 

Any single solid item offered in S2 was swapped by a different solid item, and the rodents were presented again to the twin items, i.e., a replica of the acquainted item and the different item. .e whole of the amalgamations and positions of the items were offset to abate likely prejudice instigated by a penchant for certain settings or items. .e vessel and solid items were meticulously wiped (ethyl alcohol 15% and dry cloth) after every investigation to curb the odorous signs. .e period expended discovering each item in S2 and S3 was documented using a stopwatch. .e duration expended investigating the two matching items in S2 (I1 � Ii1 + Ii2) and the duration expended investigating the two dissimilar items, i.e., acquainted and different, in S3 (I2 � Ii3 + Ib) was recorded. .e variance in duration expended investigating the different item and the duration of investigating the acquainted item (Ib–Ii3 � DI) discloses the retention of recollective memory. 

DI (discrimination index)/S3 duration (s) of investigating both the acquainted and new item (amended DI) improves the partialities by variances in the complete investigation and denotes the penchant for different items in contrast to acquainted ones {DI � (Ib–Ii3)/ (Ii3 + Ib)}. Recollective memory was appraised by quantifying the skill of rodents to single out the familiar/novel items in S3 and was stated as DI (amended for the overall investigation period in S3) [24].

2.9. MorrisWater Maze (MWM). .e standard protocol was followed, as given by Kumar and Bansal [25]. MWM judges the spatial memory by swimming trials, in which the rodent finds an escape route to a concealed podium. 

A black-colored circular tank (2 m diameter, 0.6 m height) had water (25 ± 1°C) filled to a depth of 0.3 m. The aquatic reservoir was separated clockwise into 4 similar regions (R1, R2, R3, and R4) using two nylon fibers, secured perpendicularly on the upper perimeter of the tank. 

A dais (10.5 cm × 10.5 cm) was positioned underwater (1 cm underneath water) in the reservoir region R4. .e spot of the dais persisted intact all over the acquisition period. 

Every single rat was presented with four serial acquisition rounds (5 minutes ITR) every day. .e rodent was gently released into the aquatic reservoir facing the tank wall, with the site varying with every single trial from R1-R4, R2-R1, R3-R2, and R4-R3 on days 1 to 4, respectively, and it was permitted 120 seconds to detect the underwater podium. .e rodents continued to rest on the podium for 20 seconds. 

A failure to detect the platform within 120 seconds indicated the manual placement of rats on the platform, and then they were permitted 20 seconds on the platform. Escape latency time (ELT) is the duration (s) of discovering the concealed dais in the aquatic reservoir. 

Spatial learning was marked by day one vs. day four ELT. In the probe trial (5th day), the rodents investigated the reservoir for 120 seconds but were deprived of the podium. .e mean duration expended in the entire reservoir (4 regions) was recorded. .e mean duration expended in R4 (TSTQ: time spent in target quadrant) probing for the concealed podium was deemed as an index of reference memory. .e comparative setting of the tank relative to the items in the laboratory that act as visual signs and the investigator's position remained undisturbed [26]. 

2.10. Estimation of Biochemical Parameters. After completing the behavioral examinations, the complete brain of the rats was garnered and positioned on pulverized ice cubes, followed by bathing with freezing sterilized saline (isotonic 308 mOsmol/l NaCl) to remove the remains and blood. 

Homogenization of the entire brain was instantly accomplished in a freezing separation buffer (pH 7.4) with the composition 215 mM D-mannitol, 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 1 mM 1,2-bis[2- [bis(carboxymethyl)amino]ethoxy]ethane, 75 mM saccharose, and 0.1% BSA. .e homogenate was centrifugated at 4°C using 13000 × g force for 5 minutes. .e pellet was rejected, and the supernatant was separated into two portions and was recentrifuged (4°C) at 13000 × g force for 5 minutes. .e crude mitochondrial pellet was separated and again centrifuged in a separation buffer with 1,2-bis[2- [bis(carboxymethyl)amino]ethoxy]ethane at 12,500 × g for 11 minutes (4°C). .e semisolid deposit so obtained comprising uncontaminated mitochondria was resuspended in a separation buffer (pH 7.4) containing 75 mM saccharose, 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, and 215 mM D-mannitol [27]. 

Later, the mitochondrial fraction of the whole-brain homogenate was used to determine the biochemical markers using standard methods. 2.11. Estimation of Mitochondrial Complex.

2.11.1. NADH: Ubiquinone Oxidoreductase Activity. .e rate of complex I (NADH dehydrogenase) activity was quantified (nmol NADH oxidized/minute/mg protein) by following the technique of King and Howard [28]. .e oxidative generation of NAD+ from NADH is accompanied by cytochrome c reduction. .e assay blend consisted of cytochrome c (10.5 mM), 6 mM β-nicotinamide adenine dinucleotide (DPNH) dissolved using 2 mM diglycine buffer, and diglycine buffer (0.2 M, pH 8.5). 

A dissolvable mitochondrial fraction was incorporated into the assay concoction to trigger the reaction. .e variation in optical density (O.D.) at λmax � 550 nm was followed for 120 seconds.

2.11.2. Succinate: Ubiquinone Oxidoreductase Activity. .e rate of succinate dehydrogenase (complex II) activity was quantified (nmol succinate oxidized/minute/mg protein) by following the technique of King [29]. 

Succinic acid oxidation is triggered by a mock electron receiver, potassium cyanoferrate (K3Fe(CN)6). .e assay blend comprised of succinic acid (0.63 M), 1% BSA, K3Fe(CN)6 (0.036 M), and Na+ -K+ [PO4] 2- buffer (0.23 M, pH 7.6). A dissolvable mitochondrial fraction was incorporated into the assay concoction to trigger the reaction. .e variation in O.D. at λmax � 420 nm was followed for 120 seconds. 2.12. Determination of Oxidative Stress Biomarkers 2.12.1. iobarbituric Acid Reactive Substances (TBARS). 

To evaluate TBARS (nmol per mg protein) [30], the analyze combination (concluding quantity ∼4 ml) comprising 0.10 ml homogenized brain, 1.51 ml 4,6-dihydroxy-2-mercaptopyrimidine (0.8%), 200 µl SLS (8.18%), 1.49 ml glacial acetic acid (21%, pH 3.51), and 0.71 ml deionized water was subjected to water-bath heating at 96°C for 60 minutes. 

A 15: 1 ratio of butyl alcohol/azabenzene (5.1 ml) was supplemented in an analysis concoction that was centrifugated at 4,000 × g power (10 minutes), and the supernatant was secluded. 

With a twin-beam UV1700 spectrophotometer (Shimadzu, Japan), chromophore malondialdehyde-4,6-dihydroxy-2- mercaptopyrimidine O.D. was appraised at a wavelength (λmax � 532 nm), and ε � 1.56 ×105 /M/cm (molar extinction coefficient) was applied to compute 4,6-dihydroxy-2-mercaptopyrimidine adducts. 

2.12.2. Reduced Glutathione (L-c-Glutamyl-L-Cysteinyl-glycine) Levels. Ellman's [31] procedure was implemented to appraise L-glutathione (GSH) content. .e test concoction encompassing homogenate (1.1 ml) and 1 ml of 4% 2-hydroxy-5-sulfobenzoic acid (5-SSA) was centrifugated (4°C) for 11 minutes at 2,500 × g power. 

Later, 2.8 ml Na+ -K+ [PO4] 2- buffer (51.2 mM, pH 7.77) and 0.21 ml 3-carboxy-4- nitrophenyl disulfide (0.12 mM, pH 7.89) were blended with the above-separated supernatant (0.12 ml). 

Tripeptide (µmol GSH per mg protein) was quantified with the twin-beam UV1700 spectrophotometer (λmax � 412 nm). applying ε � 1.36 ×104 /M/cm. 2.12.3. 

Glutathione Peroxidase Activity. .e activity of glutathione peroxidase (GPx) (EC 1.11.1.9) was appraised by implementing the technique of Mohandas et al. [32]. .e analyze blend comprised of 100 µl of 10% homogenate, 100 µl sodium azide (1.11 mM), 100 µl EDTA (1.13 mM), 40 µl glutathione-disulfide reductase (GSR, 1 IU/ml) (EC 1.8.1.7), 10 µl H2O2 (0.28 mM), 40 µl L-c-glutamyl-L-cysteinyl-glycine (1.2 mM), 100 µl coenzyme II reduced tetrasodium salt (0.22 mM), and 0.12 M 1.49 ml Na+ -K+ [PO4] 2- buffer (pH 7.4) in an entire quantity of 2000 µl. .e loss of coenzyme II-reduced tetrasodium at λmax � 340 nm was documented at a temperature of 25°C. GPx rate was computed as nmol NADPH oxidized/minute/mg protein using ε � of 6.22 ×103 /M/cm.

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