Mechanistic Insight Into Diosmin-Induced Neuroprotection And Memory Improvement in Intracerebroventricular-Quinolinic Acid Rat Model: Resurrection Of Mitochondrial Functions And Antioxidants Part 1

Neurodegeneration is the final event after a cascade of pathogenic mechanisms in several brain disorders that lead to cognitive and neurological loss. Quinolinic acid (QA) is an excitotoxin derived from the tryptophan metabolism pathway and is implicated in several ailments, such as Alzheimer's, Parkinson's, Huntington's, and psychosis disease. 

As people age, neurodegeneration has gradually become a common phenomenon. Neurodegeneration can have adverse effects on people's physical and mental abilities, especially on memory. However, we can slow down neurodegeneration and improve memory through action.

First, maintaining a positive attitude toward life can help alleviate neurodegeneration. Studies have shown that a positive mindset can promote the growth and reconstruction of neurons, thereby enhancing people's cognitive functions. After all, a happy mood has a positive impact on the health of the brain.

Second, we can enhance memory and promote neural regeneration through exercise. Proper exercise can stimulate neurons in the brain to make new connections, thereby enhancing people's cognitive and memory abilities. In addition, for the elderly, exercise can also reduce the nervous appetite that causes neurodegeneration, which helps to maintain the health of the brain.

In addition, maintaining the diversity and challenge of daily activities is also an important way to improve memory. Regular and repetitive daily activities will make the brain's activities very mechanical and monotonous. On the contrary, diversified activities rejuvenate the brain and enhance people's cognitive and memory abilities.

In summary, although neurodegeneration can have adverse effects on people's memory, we can take corresponding actions to slow down this effect. Positive thinking, proper exercise, and diverse activities can improve memory and promote nerve regeneration. Let us cherish and protect our brains to keep them healthy and young. It can be seen that we need to improve memory. Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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Diosmin (DSM) is a natural flavonoid possessing such properties that may halt the course of neurodegenerative progression. In past studies, free radical scavenging, along with properties, such as antihyperglycemic, anti-inflammatory, and vasoactive properties, of DSM were pragmatic. Hence, in the current experimentations, the neuroprotective activity of DSM was investigated in the QA rat prototype. 

QA was administered through the intracerebroventricular route (QA-ICV) in rats on day one, and DSM (50 and 100 mg/kg, intraperitoneal route) was given from day 1 to 21. Memory, gait, sensorimotor functions, and biomarkers of oxidative mutilation and mitochondrial functions were evaluated in the whole brain. Results showed significant deterioration of sensorimotor performance, gait, and working- and long-term memory in rats by QA-ICV. These behavioral anomalies were significantly attenuated by DSM (50 and 100 mg/kg) and donepezil (standard drug). 

QA-ICV-induced decrease in body mass (g), diet, and water ingestion were also attenuated by DSM or donepezil treatments. QA-ICV inhibited mitochondrial complex I and II activities and caused an increase in oxidative and nitrosative stress along with a reduction in endogenous antioxidants in the brain. DSM dose-dependently ameliorated mitochondrial functions and decreased oxidative stress in QA-ICV-treated rats. DSM can be a possible alternative in treating neurodegenerative disorders with underlying mitochondrial dysfunction pathology.

1. Introduction

Progressive neurodegeneration with concomitant cognitive and neurological deficits are the major manifestations of several brain ailments, such as Alzheimer's (AD), Parkinson's (PD), and Huntington's disease (HD). 

Synaptic waning and impaired long-lasting potentiation because of the decreased expression of neurotrophins (e.g., neurotrophic factors, calcineurin, and neural development factors), neurochemical aberrations (e.g., acetylcholine, glutamate, monoamines, and c-aminobutyric acid), neuropeptides (e.g., oxytocin, substance P, somatostatin, and orexin), and changes in the internal milieu of the brain leads to deterioration of short term and long-term memory [1]. 

Excitatory pathways mediated by glutamatergic receptors are often allied with the consolidation of long-term memory in the hippocampus and cortex of the brain [2]. Receptors like N-methyl D-aspartate (NMDARs) are an essential component of long-lasting potentiation and depression, and calcium influx via NMDARs and voltage-gated calcium (Ca2+) channels (VGCCs) strengthens the synapse. 

However, excessive excitatory drive in the brain culminates in brain atrophy via free radicals, proinflammatory cytokines, and activation of cell death pathways [3, 4]. 

Quinolinic acid (QA) is a product of the kynurenine pathway of tryptophan metabolism and is an endogenous ligand of NMDARs [5]. Although tryptophan is obligatory for serotonin and tryptamine biosynthesis, > 95% of tryptophan is metabolized through the kynurenine pathway [6]. Kynurenine pathway metabolites (e.g., kynurenic acid) are neuroactive, including QA, and they are implicated in schizophrenia, AD, and HD [7]. 

QA activates the immune system (microglia and astrocytes), increasing the expression of chemotactic factors (e.g., monocyte chemoattractant protein-1, RANTES) and instigating free radicals. An increase in the blood-brain barrier (BBB) penetrability prevents the shielding effect against QA, which predisposes the brain to excess QA influx. QA is a metabolic inhibitor that makes it a potent neurotoxin [8]. 

QA inhibits monoamine oxidase-B (MAO-B), gluconeogenesis (via phosphoenolpyruvate carboxykinase), creatine kinase, mitochondrial complexes, and cellular respiration, and decreases ATP levels [9]. 

QA can augment oxidative stress and decline antioxidants in an NMDAR-dependent or -independent manner. QA-Fe2+ interaction instigates free radicals, leading to lipid peroxidation and DNA mutilation substantiated by an upsurge in hydroxyl radicals, poly (ADP-ribose) polymerase (PARP) activity, and lactate dehydrogenase (LDH) activity [10]. 

Clinical findings also revealed that QA is enhanced in the brain, blood, and cerebrospinal fluid (CSF) of AD and HD patients [5]. Findings in the past indicate that QA can induce cognitive deficits and other behavioral abnormalities in experimental animals [11]. 

Recent studies revealed that natural products could ameliorate the symptoms of cognitive dysfunction and improve the therapeutic outcome in neurodegenerative disorders [12, 13]. A flavonoid glycoside, diosmin (3′,5,7-trihydroxy-4′- methoxy flavone-7-rhamnoglucoside), is frequently extant in the pericarp of citrus fruits (Rutaceae) [14]. 

Diosmin (DSM) consists of a disaccharide group (6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl) attached with the aglycone moiety (diosmetin) through glycosidic linkage and can be biosynthesized from hesperidin. 

Intestinal flora transforms DSM glycoside into aglycone moiety, which is then rapidly absorbed through the gastrointestinal tract. In humans, the half-life of DSM is 26 to 43 hours when given through the oral route [15]. 

It is a vasoactive drug that improves microcirculation, and lymphatic drainage, and enhances the flexibility of veins by attenuating norepinephrine metabolism by catechol-O-methyl transferase. DSM abrogates microvascular permeability, leukocyte extravasation, and the appearance of adhesion molecules, such as ICAM-1 and VCAM-1 [14, 15]. 

Several studies indicated free radical rummaging and immune harmonizing properties of DSM in the brain [16, 17]. Clinical evidence recommends that DSM is a well-tolerable, safe, and nontoxic drug [15]. In nutraceuticals, DSM (Daflon) is often proposed to treat venous disorders, including hemorrhoids and hyperglycemic conditions. 

Previous findings indicated that DSM could stimulate insulin release from the β-cells, carbohydrate metabolism, and the expression of glucose transporters (GLUTs). Also, it decreases diabetic complications [15]. 

It attenuates dyslipidemia and hepatic gluconeogenesis [16]. In previous studies, DSM improved cognitive functions, attenuated the symptoms of schizophrenia, and showed neuroprotective effects in experimental animals [16–19]. 

Sawmiller et al. [20], in a study, noted a DSM-mediated decrease in amyloid-β and tau hyperphosphorylation by attenuating glycogen synthase kinase 3β in the 3 × Tg-AD mouse model. These findings aptly signify that DSM has the potential to ameliorate brain dysfunctions against QA. In this study, QA was used to induce dementia and other neurological deficits in rats. 

QA can act as a potent neurotoxin that inhibits several pathways and molecular mechanisms in the brain to induce progressive neurodegeneration and brain atrophy. .e contemporary investigation was designed to explore the outcomes of DSM in the QA-ICV rat prototype.

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. 

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 sulfate), 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].

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