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Possible nitric oxide modulation in the protective effects of rutin against experimental head trauma–induced cognitive deficits: behavioral, biochemical, and molecular correlates

  • Anil Kumar
    Correspondence
    Corresponding author. Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh 160014, India. Tel.: +91 172 2534106; fax +91 172 2543101.
    Affiliations
    Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh, India
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  • Puneet Rinwa
    Affiliations
    Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh, India
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  • Hitesh Dhar
    Affiliations
    Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh, India
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Published:January 06, 2014DOI:https://doi.org/10.1016/j.jss.2013.12.028

      Abstract

      Background

      Traumatic head injury is turning out to be a major cause of disability and death. Nitric oxide (NO), an intercellular messenger plays a crucial role in the pathophysiology of several neurologic disorders. Therefore, the present study was designed to investigate the effects of rutin, a well-known flavonoid against cognitive deficits and neuroinflammation associated with traumatic head injury and the probable role of NO pathway in this effect.

      Materials and methods

      Wistar rats were exposed to head trauma using weight drop method and kept for a postsurgical rehabilitation period of 2 wk. Later, animals were administered with rutin (20, 40, and 80 mg/kg; per oral) alone and in combination with NO modulators such as NG-nitro-L-arginine methyl ester and L-arginine, daily for another 2 wk.

      Results

      Head injury caused impaired spatial navigation in Morris water maze test and poor retention in elevated plus maze task. Furthermore, there was a significant rise in acetylcholinesterase activity, oxidative stress, neuroinflammation (tumor necrosis factor α), and neuronal apoptosis (caspase-3) in both cortex and hippocampal regions of traumatized rat brain. Rutin significantly attenuated these behavioral, biochemical, and molecular alterations associated with head trauma. Furthermore, pretreatment of NG-nitro-L-arginine methyl ester (10 mg/kg, intraperitoneally), a nonspecific nitric oxide synthase inhibitor, with subeffective dose of rutin (40 mg/kg) potentiated the protective effects; however, pretreatment of L-arginine (100 mg/kg; intraperitoneally), an NO donor, reversed the effects of rutin.

      Conclusions

      The present study suggests that NO modulation could possibly be involved in the neuroprotective effects of rutin against head trauma–induced cognitive deficits, neuroinflammation, and apoptotic signaling cascade.

      Keywords

      1. Introduction

      Traumatic head injury due to accidents and violence is a major cause of death and disability, which accounts for approximately 2.5–6.5 million cases in the United States [
      • Maas A.I.
      • Stocchetti N.
      • Bullock R.
      Moderate and severe traumatic brain injury in adults.
      ]. Head injury can be classified as primary, which occurs immediately after trauma, and secondary, which includes a cascade of injuries that develop over a period of time after the initial traumatic impact. The most frequent consequences of head trauma include aggression, seizures, and cognitive problems [
      • Masel B.E.
      • DeWitt D.S.
      Traumatic brain injury: a disease process, not an event.
      ]. Head injury causes severe damage to cerebral cortex and hippocampus, which play a crucial role in processing of spatial learning and memory [
      • Tong W.
      • Igarashi T.
      • Ferriero D.M.
      • Noble L.J.
      Traumatic brain injury in the immature mouse brain: characterization of regional vulnerability.
      ]. A clinical report from Skelton et al. [
      • Skelton R.W.
      • Bukach C.M.
      • Laurance H.E.
      • Thomas K.G.
      • Jacobs J.W.
      Humans with traumatic brain injuries show place-learning deficits in computer-generated virtual space.
      ] explains that head trauma survivors face difficulty in spatial learning. Oxidative damage and apoptosis are the two main pathogenic mechanisms involved in secondary brain injury [
      • Bayir H.
      • Kochanek P.M.
      • Clark R.S.
      Traumatic brain injury in infants and children: mechanisms of secondary damage and treatment in the intensive care unit.
      ]. Oxidative stress has a critical role in the commencement of secondary injury cascade leading to increased lipid peroxidation and protein oxidation [
      • Marklund N.
      • Hillered L.
      Animal modelling of traumatic brain injury in preclinical drug development: where do we go from here?.
      ]. Secondary injury after head trauma has a significant role in the regulation of inflammatory responses leading to neurodegeneration [
      • Cederberg D.
      • Siesjo P.
      What has inflammation to do with traumatic brain injury?.
      ].
      Nitric oxide (NO), a free radical gas is known to exert its modulatory effects in both central and peripheral nervous system by acting as an intracellular messenger [
      • Garthwiate J.
      Glutamate, nitric oxide and cell-cell signalling in the nervous system.
      ]. NO is synthesized in response to the activation of N-methyl-D-aspartate receptors by a group of enzymes called as nitric oxide synthase (NOS) [
      • Palumbo A.
      • Poli A.
      • Di Cosmo A.
      • d'Ischia M.
      N-methyl-d-aspartate receptor stimulation activates tyrosinase and promotes melanin synthesis in the ink gland of the cuttlefish Sepia officinalis through the nitric oxide/cGMP signal transduction pathway.
      ]. NO participates in several neurobiological functions of brain including learning and memory [
      • Boehning D.
      • Snyder S.H.
      Novel neural modulators.
      ]. NO combines with superoxide anions to form peroxynitrite, a highly reactive species, which later causes nitration of proteins, lipid peroxidation, and produces carbonylation of proteins and ultimately leading to neuronal death [
      • Cahuana G.M.
      • Tejedo J.R.
      • Jimenez J.
      • Ramirez R.
      • Sobrino F.
      • Bedoya F.J.
      Nitric oxide-induced carbonylation of Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F cells.
      ]. Such deficits in neuronal functioning get aggregated by NO donors such as L-arginine [
      • Da Cunha I.C.
      • José R.F.
      • Orlandi Pereira L.
      • et al.
      The role of nitric oxide in the emotional learning of rats in the plus-maze.
      ]. On the other hand, NG-nitro-L-arginine methyl ester (L-NAME) reduces NOS activity and blocks deleterious effects of NO [
      • Yamada K.
      • Hiramatsu M.
      • Noda Y.
      • et al.
      Role of nitric oxide and cyclic GMP in the dizocilpine-induced impairment of spontaneous alternation behavior in mice.
      ]. Recent report suggests an abundant association of NOS with neurobiology of memory functions and its association with pathologic mechanisms of the disease [
      • Javadi-Paydar M.
      • Ghiassy B.
      • Ebadian S.
      • Rahimi N.
      • Norouzi A.
      • Dehpour A.R.
      Nitric oxide mediates the beneficial effect of chronic naltrexone on cholestasis-induced memory impairment in male rats.
      ].
      Even after an extensive work on head trauma, there are no reliable neuroprotective agents for the treatment of patients suffering from head injury. Since oxidative stress and neuroinflammation are the major contributors to the pathogenesis of head injury, therefore, search for a new antioxidant and anti-inflammatory agent is urgently required. Rutin (quercetin-3-O-rutinoside) is a common dietary flavonol glycoside found in many plants, such as buckwheat, passionflower, oranges, and grapes. It is often used as a natural pigment, stabilizer, and food preservative in various cosmetics and chemical industries [
      • Pu F.
      • Mishima K.
      • Irie K.
      • Egashira N.
      • Ishibashi D.
      • Matsumoto Y.
      Differential effects of buckwheat and kudingcha extract on neuronal damage in cultured hippocampal neurons and spatial memory impairment induced by scopolamine in an eight-arm radial maze.
      ]. Apart from this, rutin is known to possess diverse pharmacologic actions including antioxidant [
      • La Casa C.
      • Villegas I.
      • Alarcon de la Lastra C.
      • Motilva V.
      • Martin Calero M.J.
      Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions.
      ], anti-inflammatory [
      • Kim H.
      • Kong H.
      • Choi B.
      • Yang Y.
      • Kim Y.
      • Lim M.J.
      Metabolic and pharmacological properties of rutin, a dietary quercetin glycoside, for treatment of inflammatory bowel disease.
      ], neuroprotective [
      • Gupta R.
      • Singh M.
      • Sharma A.
      Neuroprotective effect of antioxidants on ischaemia and reperfusion-induced cerebral injury.
      ], antihyperglycemic [
      • Kamalakkannan N.
      • Prince P.S.M.
      Rutin improves the antioxidant status in streptozotocin-induced diabetic rat tissues.
      ], anticonvulsant [
      • Nassiri-Asl M.
      • Shariati-Rad S.
      • Zamansoltani F.
      Anticonvulsive effects of intracerebroventricular administration of rutin in rats.
      ], antiplatelet [
      • Sheu J.R.
      • Hsiao G.
      • Chou P.H.
      • Shen M.Y.
      • Chou D.S.
      Mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets.
      ], and vasoprotective [
      • Mellou F.
      • Loutrari H.
      • Stamatis H.
      • Roussos C.
      • Kolisis F.N.
      Enzymatic esterification of flavonoids with unsaturated fatty acids: effect of the novel esters on vascular endothelial growth factor release from K562 cells.
      ] properties. Recently, memory-enhancing effects of rutin have been extensively studied [
      • Nassiri-Asl M.
      • Zamansoltani F.
      • Javadi A.
      • Ganjvar M.
      The effects of rutin on a passive avoidance test in rats.
      ]. Studies from Koda et al. [
      • Koda T.
      • Kuroda Y.
      • Imai H.
      Rutin supplementation in the diet has protective effects against toxicant-induced hippocampal injury by suppression of microglial activation and pro-inflammatory cytokines: protective effect of rutin against toxicant-induced hippocampal injury.
      ] reported neuroprotective property of rutin against spatial memory impairment and neuronal loss in the hippocampal region. Rutin also inhibits the production of free radicals, and thus increases the activity of antioxidant enzymes, such as reduced glutathione (GSH) [
      • La Casa C.
      • Villegas I.
      • Alarcon de la Lastra C.
      • Motilva V.
      • Martin Calero M.J.
      Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions.
      ]. Rutin shows protection against several other biological antioxidants such as catalase, superoxide dismutase, and so forth [
      • Khan M.M.
      • Ahmad A.
      • Ishrat T.
      • et al.
      Rutin protects the neural damage induced by transient focal ischemia in rats.
      ]. Earlier, rutin was reported to attenuate the activation of microglial cells and neuroinflammatory cytokines [
      • Koda T.
      • Kuroda Y.
      • Imai H.
      Rutin supplementation in the diet has protective effects against toxicant-induced hippocampal injury by suppression of microglial activation and pro-inflammatory cytokines: protective effect of rutin against toxicant-induced hippocampal injury.
      ]. All these studies clearly explain the neuroprotective potential of rutin; however, its exact cellular and molecular pathway is still far from elucidation.
      The present study was designed with the aim to investigate the possible neuroprotective effects of rutin against traumatic head injury–induced cognitive impairment, neuroinflammation, and cell death cascade and to examine the possible involvement of NO pathway in this effect.

      2. Materials and methods

      2.1 Animals

      Adult male Wistar rats (250–300 g) were procured from Animal House of Panacea Biotec Ltd, Lalru (Panjab). Animals were housed under standard (25 ± 2°C, 60%–70% humidity) laboratory conditions, maintained on a 12-h natural day–night cycle, with free access to food and water. Animals were acclimatized to laboratory conditions before the experimental tests. The experimental protocols were approved by the Institutional Animal Ethical Committee of Panjab University (IAEC/282/UIPS/39) and conducted according to the Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines, Government of India on the use and care of experimental animals.

      2.2 Head trauma procedure

      A special weight-drop device developed by Marmarou et al. [
      • Marmarou A.
      • Foda M.A.
      • Van den Brink W.
      • Campbell J.
      • Kita H.
      • Demetriadou K.
      A new model of diffuse brain injury in rats. Part I: pathophysiology and biomechanics.
      ] and modified as described by Pandey et al. [
      • Pandey D.K.
      • Yadav S.K.
      • Mahesh R.
      • Rajkumar R.
      Depression-like and anxiety-like behavioural aftermaths of impact accelerated traumatic brain injury in rats: a model of comorbid depression and anxiety?.
      ] was used to deliver a standard diffuse traumatic impact. Under ketamine and xylazine anesthesia (75 and 5 mg/kg, intraperitoneally), a midline incision (1 cm) was made in the scalp of male Wistar rats, and the skin was retracted to expose the skull. A 10-mm diameter, 3-mm thick metallic disk designed to protect against skull fracture was surgically adhered between bregma and lambda suture lines of the skull. A cylindrical metallic weight of 450 g was dropped from a height of 2 m freely through a metal tube onto the disc. Foam bed (10 cm) was placed underneath the animal to absorb the impact due to weight drop. After impact, the metal disc was removed and skin was sutured with absorbable sutures (Ethicon 4-0, Absorbable surgical sutures USP [Catgut]; Johnson and Johnson, India). To prevent postsurgical infection, the animals also received Sulprim injection (each milliliter containing 200 and 40 mg of sulfadiazine and trimethoprim, respectively), intramuscularly (0.2 mL/300 g) once a day for 3 d of postsurgery. Sham operated rats were treated in the similar way, including midline incision, except brain injury. The animals were housed for 2 wk (14 d) and were given continuous care with daily observation and handling under the supervision of an experienced veterinarian. The animals were able to groom and did not display overt symptoms of pain or discomfort. Drug treatments were started after a 14 d surgical rehabilitation period, and pictogram of the entire protocol is represented in Figure 1.
      Figure thumbnail gr1
      Fig. 1Experimental design for traumatic head injury protocol.

      2.3 Drugs and treatment schedule

      Rutin, L-NAME, and L-Arginine were purchased from Sigma chemicals Co (St. Louis, MO). Enzyme-linked immunosorbent assay kit for tumor necrosis factor (TNF) α and caspase-3 were purchased from R&D Systems (Minneapolis, MN). All other chemicals used for biochemical estimations were of analytical grade. Total 90 animals were used in the study. The animals were randomly divided into nine experimental groups consisting of 10 animals in each (n = 10). First and second group was named as sham and head injury control, respectively. Rutin (20, 40, and 80 mg/kg; per oral) were treated as group 3–5, respectively. Pretreatment of L-NAME (10 mg/kg) and L-arginine (100 mg/kg) with rutin (40 mg/kg) served as group 6–7. L-NAME (10 mg/kg) and L-arginine (100 mg/kg) per se treatment was categorized as group 8 and 9, respectively. Rutin was dissolved in normal saline. Drugs were administered orally on the basis of body weight (0.5 mL/100 g), and drug solutions were made freshly at the beginning of each day of the study protocol. Two sets of animals (n = 5) were used for different behavioral tests and were studied independent of each other.

      2.4 Behavioral tests

      2.4.1 Elevated plus maze paradigm

      Rats (n = 5) were examined in elevated plus maze (EPM), which consisted of two open (50 × 10 cm) and two closed (50 × 10 × 40 cm) arms facing each other with an open roof. The entire maze was elevated at a height of 50 cm, and each animal was placed at either end of the open arm facing outwards. Acquisition and retention of memory processes were assessed as previously described [
      • Sharma A.C.
      • Kulkarni S.K.
      Evaluation of learning and memory mechanisms employing elevated plus-maze in rats and mice.
      ]. Time taken by the animal to enter the closed arm in the first trial (acquisition trial) was noted and called as initial transfer latency (ITL). Cut-off time was fixed at 90 s and in case, an animal could not find the closed arm within this period, it was gently pushed into one of the closed arms and allowed to explore the maze for 30 s. Second trial (retention trial) was performed 24 h after the acquisition trial and retention transfer latency (RTL) was noted.

      2.4.2 Morris water maze (MWM) test (computer tracking using Ethovision software)

      Rats (n = 5) were tested in a spatial version of MWM test [
      • Morris R.
      Developments of a water-maze procedure for studying spatial learning in the rat.
      ] from day 24 to 28. The apparatus consisted of a circular water tank (180 cm in diameter and 60 cm high). A platform (12.5 cm in diameter and 38 cm high) invisible to the rats was set 2 cm below the water level inside the tank with water maintained at 28.5 ± 2°C at a height of 40 cm. The tank was located in a large room where there were several brightly colored cues external to the maze; these were visible from the pool and could be used by the rats for spatial orientation. The position of the cues remained unchanged throughout the study. The rats received four consecutive daily training trials in the following 5 d, with each trial having a ceiling time of 120 s and a trial interval of approximately 30 s. For each trial, each animal was put into the water at one of four starting positions, the sequence of which being selected randomly. During test trials, rats were placed into the tank at the same starting point, with their heads facing the wall. The animal had to swim until it climbed onto the platform submerged underneath the water. After climbing onto the platform, the animal remained there for 20 s before the commencement of the next trial. The escape platform was kept in the same position relative to the distal cues. If the animal failed to reach the escape platform within the maximally allowed time of 120 s, it was guided with the help of a rod and allowed to remain on the platform for 20 s. The time to reach the platform (escape latency time [ELT] in seconds) and total distance traveled to reach the hidden platform (path length in centimeter) were measured by using a computer tracking system with Ethovision software, Noldus Information Technology, The Netherlands.

      2.4.3 Memory retrieval test

      A probe trial was performed on day 28 wherein the extent of memory retention was assessed. In the probe trial, the rats were placed into the pool for a total duration of 120 s as in the training trial, except that the hidden platform was removed from the pool. Time spent in target quadrant (TSTQ) was measured by using a computer tracking system with Ethovision software. It indicates the degree of memory consolidation that has taken place after learning. Target quadrant is the quadrant where the platform was previously located before conducting retrieval trial.

      2.5 Dissection and homogenization

      Immediately after the last behavioral test, animals were sacrificed by decapitation and brain samples were rapidly removed and placed on dry ice for isolation of cerebral cortex and hippocampus. A 10% (wt/vol) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000g for 15 min. Aliquots of supernatants were separated and used for biochemical (n = 5) and molecular (n = 5) estimations.

      2.6 Estimation of oxidative–nitrosative stress markers

      2.6.1 Measurement of lipid peroxidation

      The extent of lipid peroxidation was determined quantitatively by performing the method as described by Wills [
      • Wills E.D.
      Mechanism of lipid peroxide formation in animal tissues.
      ]. The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using PerkinElmer Lambda 20 spectrophotometer (Norwalk, CT). The values were calculated using the molar extinction coefficient of chromophore (1.56 × 10 /M/cm).

      2.6.2 Estimation of nitrite

      The accumulation of nitrite in the supernatant, an indicator of the production of NO was determined by a colorimetric assay with Greiss reagent (0.1% N-(1-napthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 5% phosphoric acid) [
      • Green L.C.
      • Wagner D.A.
      • Glogowski J.
      • Skipper P.L.
      • Wishnok J.S.
      • Tannenbaum S.R.
      Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.
      ]. Equal volumes of the supernatant and the Greiss reagent were mixed, and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using PerkinElmer Lambda 20 spectrophotometer. The concentration of nitrite in the supernatant was determined from sodium nitrite standard curve.

      2.6.3 Estimation of reduced GSH

      Reduced GSH in the brain parts was estimated according to the method of Ellman et al. [
      • Ellman G.L.
      • Courtney K.D.
      • Andres Jr., V.
      • Feather-Stone R.M.
      A new and rapid colorimetric determination of acetylcholinesterase activity.
      ]. Homogenate (1 mL) was precipitated with 1.0 mL of 4% sulfosalicylic acid and the samples were immediately centrifuged at 1200g for 15 min at 4°C. The assay mixture contained 0.1 mL of supernatant, 2.7 mL of phosphate buffer of pH 8.0 and 0.2 mL of 0.01 M dithiobisnitrobenzoic acid (DTNB). The yellow color developed was read immediately at 412 nm using PerkinElmer lambda 20 spectrophotometer. The results were expressed as micromoles of reduced GSH per milligram of protein.

      2.6.4 Estimation of catalase

      Catalase activity was determined by the method of Luck [
      • Luck H.
      Catalase.
      ], wherein the breakdown of hydrogen peroxide (H2O2) is measured at 240 nm. Briefly, the assay mixture consisted of 3 mL of H2O2, phosphate buffer, and 0.05 mL of supernatant of tissue homogenate (10%), and the change in absorbance was recorded at 240 nm using PerkinElmer lambda 20 spectrophotometer. The results were expressed as micromoles of H2O2 decomposed per minute per milligram of protein.

      2.6.5 Estimation of protein

      The protein content was estimated by biuret method [
      • Gornall A.G.
      • Bardawill C.J.
      • David M.M.
      Determination of serum proteins by means of the biuret reaction.
      ] using bovine serum albumin as a standard.

      2.7 Estimation of acetylcholinesterase (AChE) activity

      AChE activity was assessed as described by Ellman et al. [
      • Ellman G.L.
      • Courtney K.D.
      • Andres Jr., V.
      • Feather-Stone R.M.
      A new and rapid colorimetric determination of acetylcholinesterase activity.
      ]. The assay mixture contained 0.05 mL of supernatant, 3 mL of sodium phosphate buffer (pH 8), 0.1 mL of acetylthiocholine iodide, and 0.1 mL of dithiobisnitrobenzoic acid (Ellman reagent). The change in absorbance was measured for 2 min at 30 s intervals at 412 nm using PerkinElmer lambda 20 spectrophotometer. Results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per minute per milligram of protein.

      2.8 Molecular estimations

      2.8.1 Estimation of pro-inflammatory cytokine (TNF-α)

      The quantification of TNF-α was done by rat TNF-α immunoassay kit (R&D Systems). The Quantikine rat TNF-α immunoassay is a 4.5 h solid phase enzyme-linked immunosorbent assay designed to measure rat TNF-α levels. The assay used the sandwich enzyme immunoassay technique. A monoclonal antibody specific for rat TNF-α was precoated in the microplate. Standards control and samples were pipetted into the wells, and any rat TNF-α present was bound by the immobilized antibody. After washing away any unbound substance, an enzyme-linked polyclonal antibody specific for rat TNF-α was added to the wells. After a wash to remove any unbound antibody–enzyme reagent, a substrate solution was added to the wells. The enzyme reaction yielded a blue product, which turned yellow when the stop solution was added. The intensity of the color measured was in proportion to the amount of rat TNF-α bound in the initial steps. The sample values were then read off the standard curve.

      2.8.2 Estimation of apoptotic factor (caspase-3)

      Caspase-3, also known as CPP-32, is an intracellular cysteine protease that exists as a proenzyme and becomes activated during the cascade of events associated with apoptosis. The tissue homogenates can be tested for protease activity by the addition of a caspase-specific peptide, which is conjugated to the color reporter molecule p-nitroaniline. The cleavage of the peptide by the caspase releases chromophore p-nitroaniline, which can be quantified spectrophotometrically at a wavelength of 405 nm. The level of caspase enzymatic activity in the cell lysate/homogenate is directly proportional to the color reaction. The enzymatic reaction for caspase activity was carried out using R&D systems caspase-3 colorimetric kit.

      2.9 Statistical analysis

      All the values were expressed as mean ± standard error of mean. Behavioral data were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni post test. All other test data were analyzed using one-way ANOVA followed by post-hoc Tukey's test. The criterion for statistical significance was P < 0.05. All statistical procedures were carried out using sigma stat Graph Pad Prism (Graph Pad Software, San Diego, CA).

      3. Results

      3.1 Effect of rutin and its modification with NO modulators on latency time in EPM

      Control animals performed poorly throughout the experiment and showed no change in RTL on day 28 as compared with ITL on day 27, thereby showing head injury–induced memory impairment (P < 0.05). Treatment with rutin (40 and 80 mg/kg) significantly lowered RTL in head-injured rats signifying improvement in learning and memory (P < 0.05). Pretreatment of L-NAME (10 mg/kg) with subeffective dose of rutin (40 mg/kg) potentiated its protective effects, which was significant as compared with their effects alone. However, L-arginine (100 mg/kg) pretreatment reversed the protective effect of rutin (40 mg/kg), which was significant as compared with rutin (40 mg/kg) alone. Besides, the effects of L-NAME (10 mg/kg) and L-arginine (100 mg/kg) per se treatments did not show any significant effect as compared with control (F [9, 44] = 34.11 [P < 0.05]; Fig. 2).
      Figure thumbnail gr2
      Fig. 2Effect of rutin and its modification with NO modulators on latency time in EPM. Data expressed as mean ± standard error of mean. aP < 0.05 as compared with sham; bP < 0.05 as compared with control; cP < 0.05 as compared with R (40); dP < 0.05 as compared with L-NAME (10) (Two-way ANOVA followed by Bonferroni post test). Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg).

      3.2 Effects of rutin and its modification with NO modulators on ELT in MWM

      Head injury rats showed a significant delay in ELT to reach the hidden platform as compared with the sham group, showing a poor learning performance (P < 0.05). Rutin (40 and 80 mg/kg) treatment significantly shortened the ELT as compared with control (P < 0.05). However, rutin (20 mg/kg) did not show any significant improvement in memory performance. L-NAME (10 mg/kg) pretreatment with subeffective dose of rutin (40 mg/kg) potentiated their protective effect, which was significant as compared with their effects alone; however, L-arginine (100 mg/kg) reversed the protective effect of rutin (40 mg/kg; F [9, 44] = 23.45, 35.46 [P < 0.05]; Table 1).
      Table 1Effects of rutin and its modification with NO modulators on ELT in MWM.
      Treatment (mg/kg)Day 1 ELT (s)Day 2 ELT (s)Day 3 ELT (s)Day 4 ELT (s)
      Sham93.2 ± 5.3449.2 ± 5.4540.8 ± 3.4232.6 ± 4.12
      Control97.6 ± 4.3493.5 ± 5.34
      P < 0.05 as compared with sham.
      88.4 ± 6.12
      P < 0.05 as compared with sham.
      85.3 ± 4.56
      P < 0.05 as compared with sham.
      R (20)95.3 ± 6.2288.5 ± 6.1282.5 ± 2.6778.6 ± 5.65
      R (40)90.3 ± 5.2370.2 ± 5.34
      P < 0.05 as compared with control.
      64.2 ± 4.32
      P < 0.05 as compared with control.
      56.4 ± 6.21
      P < 0.05 as compared with control.
      R (80)93.3 ± 4.4661.4 ± 4.27
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      53.5 ± 3.23
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      41.4 ± 4.62
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      L-NAME (10)+R (40)93.6 ± 4.6263.5 ± 4.45
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (Two-way ANOVA followed by Bonferroni post test).
      50.5 ± 7.23
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (Two-way ANOVA followed by Bonferroni post test).
      44.2 ± 3.44
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (Two-way ANOVA followed by Bonferroni post test).
      L-ARG (100)+ R (40)90.2 ± 4.3186.2 ± 2.11
      P < 0.05 as compared with R (40).
      80.4 ± 3.12
      P < 0.05 as compared with R (40).
      76.6 ± 6.12
      P < 0.05 as compared with R (40).
      L-NAME (10) per se94.2 ± 4.5694.2 ± 4.56
      P < 0.05 as compared with sham.
      90.5 ± 4.2
      P < 0.05 as compared with sham.
      84.4 ± 5.12
      P < 0.05 as compared with sham.
      L-ARG (100) per se96.1 ± 5.2693.2 ± 5.34
      P < 0.05 as compared with sham.
      90.1 ± 4.44
      P < 0.05 as compared with sham.
      89.1 ± 4.56
      P < 0.05 as compared with sham.
      Data expressed as mean ± standard error of mean.
      Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg).
      P < 0.05 as compared with sham.
      P < 0.05 as compared with control.
      P < 0.05 as compared with R (40).
      § P < 0.05 as compared with L-NAME (10) (Two-way ANOVA followed by Bonferroni post test).

      3.3 Effects of rutin and its modification with NO modulators on total distance traveled to reach the hidden platform (path length)

      Progressive decrease in path length to reach the hidden platform from day 24–27 in water maze task is associated with intact memory of animals. The total distance traveled to reach the hidden platform did not differ between any of the groups on the first day of testing in MWM, but from second day onward there was a significant difference in path length of head trauma rats as compared with sham animals. Treatment with rutin (40 and 80 mg/kg) significantly shortened the path length as compared with control (P < 0.05). Furthermore, L-NAME (10 mg/kg) pretreatment with the subeffective dose of rutin (40 mg/kg) potentiated its protective effect; however, L-arginine (100 mg/kg) reversed the effects of rutin (40 mg/kg). Furthermore, per se treatment of L-NAME (10 mg/kg) and L-arginine (100 mg/kg) did not show any significant effect on the path length when compared with control (F [9, 44] = 74.32, 85.12 [P < 0.05]; Table 2).
      Table 2Effects of rutin and its modification with NO modulators on total distance traveled to reach the hidden platform (path length).
      TreatmentDay 1 (mg/kg)Day 2 PL (cm)Day 3 PL (cm)Day 4 PL (cm)
      Sham2345.2 ± 156.41321.4 ± 123.4982.3 ± 122.2812.5 ± 78.3
      Control2284.2 ± 182.12142.2 ± 153.4
      P < 0.05 as compared with sham.
      2045.2 ± 125.4
      P < 0.05 as compared with sham.
      1976.4 ± 147.8
      P < 0.05 as compared with sham.
      R (20)2234.4 ± 123.42105.7 ± 135.81927.4 ± 156.71855.3 ± 165.5
      R (40)2312.4 ± 124.41823.1 ± 137.61564.5 ± 178.9
      P < 0.05 as compared with control.
      1432.4 ± 147.0
      P < 0.05 as compared with control.
      R (80)2302.3 ± 143.31523.4 ± 112.3
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      1156.1 ± 133.1
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      1054.5 ± 123.4
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      L-NAME (10)+R (40)2250.4 ± 178.91588.2 ± 153.3
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (two-way ANOVA followed by Bonferroni's posttest).
      1203.2 ± 143.5
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (two-way ANOVA followed by Bonferroni's posttest).
      1112.3 ± 168.9
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (two-way ANOVA followed by Bonferroni's posttest).
      L-ARG (100)+R (40)2254.2 ± 157.32082.4 ± 177.4
      P < 0.05 as compared with R (40).
      1845.5 ± 112.6
      P < 0.05 as compared with R (40).
      1823.4 ± 105.2
      P < 0.05 as compared with R (40).
      L-NAME (10) per se2344.5 ± 144.22135.2 ± 141.2
      P < 0.05 as compared with sham.
      2089.5 ± 155.7
      P < 0.05 as compared with sham.
      2045.6 ± 147.4
      P < 0.05 as compared with sham.
      L-ARG (100) per se2342.4 ± 134.82150.2 ± 135.1
      P < 0.05 as compared with sham.
      2095.2 ± 133.2
      P < 0.05 as compared with sham.
      2075.4 ± 157.5
      P < 0.05 as compared with sham.
      Data expressed as mean ± standard error of mean.
      Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg), PL = Path length.
      P < 0.05 as compared with sham.
      P < 0.05 as compared with control.
      P < 0.05 as compared with R (40).
      § P < 0.05 as compared with L-NAME (10) (two-way ANOVA followed by Bonferroni's posttest).

      3.4 Effects of rutin and its modification with NO modulators on TSTQ

      During retention trial, with platform removed, control animals failed to remember the location of the platform, thus spending significantly less time in the target quadrant as compared with sham animals (P < 0.05). However, rutin (40 and 80 mg/kg) treatment significantly increased the TSTQ as compared with control group, indicating retention in memory performance (P < 0.05). Pretreatment of L-NAME (10 mg/kg) with subeffective dose of rutin (40 mg/kg) potentiated its memory retrieval effects, which were significant when compared with their effects alone. However, L-arginine (100 mg/kg) reversed the protective effect of rutin (40 mg/kg). Besides, the per se effects of L-NAME (10 mg/kg) and L-arginine (100 mg/kg) in both acquisition and retention trials did not show any significant effect when compared with control (F [9, 44] = 42.93 [P < 0.05]; Fig. 3).
      Figure thumbnail gr3
      Fig. 3Effects of rutin and its modification with NO modulators on TSTQ in MWM. Data expressed as mean ± standard error of mean. aP < 0.05 as compared with sham; bP < 0.05 as compared with control; cP < 0.05 as compared with R (40); dP < 0.05 as compared with L-NAME (10) (One-way ANOVA followed by Tukey test). Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg).

      3.5 Effects of rutin and its modification with NO modulators on computer tracking of the path traveled to reach the hidden platform

      During the probe trial, the path traveled by animals in each group was monitored by Ethovision software. Path traveled was significantly increased in head injury control rats as compared with sham group; showing head injury–induced memory loss. Sham group did not show any change in path traveled and was similar to naïve animals (not shown). However, rutin (40 and 80 mg/kg) treatment significantly shortened the path length as compared with control. Furthermore, L-NAME (10 mg/kg) pretreatment with subeffective dose of rutin (40 mg/kg) further decreased the path traveled suggesting improvement in learning and memory, which was significant as compared with their effects alone. However, pretreatment of L-arginine (100 mg/kg) with rutin (40 mg/kg) significantly reversed the protective effect of rutin (40 mg/kg). Besides, L-NAME (10 mg/kg) and L-arginine (100 mg/kg) treatment alone did not show any significant effect as compared with control (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Effects of rutin and its modification with NO modulators on computer tracking of the path traveled to reach the hidden platform. Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg). (Color version of figure is available online.)

      3.6 Effect of rutin and its modification with NO modulators on lipid peroxidation (LPO), reduced GSH, nitrite concentration, and catalase enzymes level

      Head trauma caused significant increase in oxidative and nitrosative damage as evidenced by increase in LPO and nitrite levels, along with depletion of reduced GSH and catalase levels as compared with sham group. Rutin (40 and 80 mg/kg) significantly attenuated the oxidative stress markers (reduced LPO, nitrite levels, restoration of reduced GSH, and catalase levels) as compared with head trauma control. However, rutin (20 mg/kg) showed no significant improvement in oxidative damage. Furthermore, L-NAME (10 mg/kg) pretreatment with subeffective dose of rutin (40 mg/kg) potentiated their antioxidant effect, which was significant as compared with their effects alone; however, L-arginine (100 mg/kg) reversed the antioxidant effect of rutin (40 mg/kg). Besides, the per se effects of L-NAME (10 mg/kg) and L-arginine (100 mg/kg) did not show any significant effect on LPO (F [9, 44] = 65.22 [P < 0.05], GSH [F {9, 44}] = 49.33 [P < 0.01]), Nitrite (F [9, 44] = 14.56 [P < 0.05]), and Catalase (F [9, 44] = 56.77 [P < 0.01]) activity as compared with sham treatment (Table 3).
      Table 3Effects of rutin and its modification with NO modulators on oxidative-nitrosative stress parameters.
      Treatment (mg/kg)LPO (n mol of MDA/mg Pr)GSH (μmol of GSH/mg Pr)Nitrite (μg/mL)Catalase (μmol of H2O2 hydrolyzed/min/mg Pr)
      Sham
       Cerebral cortex0.162 ± 0.040.073 ± 0.004342.4 ± 9.50.89 ± 0.07
       Hippocampus0.117 ± 0.050.052 ± 0.003224.2 ± 4.90.64 ± 0.04
      Control
       Cerebral cortex0.520 ± 0.07
      P < 0.05 as compared with sham.
      0.018 ± 0.004
      P < 0.05 as compared with sham.
      733.7 ± 11.6
      P < 0.05 as compared with sham.
      0.23 ± 0.04
      P < 0.05 as compared with sham.
       Hippocampus0.390 ± 0.06
      P < 0.05 as compared with sham.
      0.011 ± 0.003
      P < 0.05 as compared with sham.
      590.2 ± 11.2
      P < 0.05 as compared with sham.
      0.17 ± 0.03
      P < 0.05 as compared with sham.
      R (20)
       Cerebral cortex0.486 ± 0.070.028 ± 0.008692.4 ± 8.20.29 ± 0.05
       Hippocampus0.361 ± 0.080.014 ± 0.004545.0 ± 9.80.23 ± 0.03
      R (40)
       Cerebral cortex0.396 ± 0.05
      P < 0.05 as compared with control.
      0.043 ± 0.006
      P < 0.05 as compared with control.
      577.2 ± 6.4
      P < 0.05 as compared with control.
      0.46 ± 0.04
      P < 0.05 as compared with control.
       Hippocampus0.253 ± 0.05
      P < 0.05 as compared with control.
      0.029 ± 0.003
      P < 0.05 as compared with control.
      402.8 ± 5.8
      P < 0.05 as compared with control.
      0.37 ± 0.01
      P < 0.05 as compared with control.
      R (80)
       Cerebral cortex0.208 ± 0.02
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      0.063 ± 0.002
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      405.2 ± 8.7
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      0.72 ± 0.06
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
       Hippocampus0.133 ± 0.01
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      0.045 ± 0.004
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      272.4 ± 6.7
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      0.58 ± 0.05
      P < 0.05 as compared with control.
      ,
      P < 0.05 as compared with R (40).
      L-NAME (10)+R (40)
       Cerebral cortex0.222 ± 0.08
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
      0.058 ± 0.003
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
      428.0 ± 7.9
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
      0.68 ± 0.04
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
       Hippocampus0.137 ± 0.04
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
      0.049 ± 0.002
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
      265.8 ± 6.4
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
      0.56 ± 0.01
      P < 0.05 as compared with R (40).
      ,
      P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).
      L-ARG (100)+R (40)
       Cerebral cortex0.501 ± 0.03
      P < 0.05 as compared with R (40).
      0.024 ± 0.002
      P < 0.05 as compared with R (40).
      681.2 ± 9.8
      P < 0.05 as compared with R (40).
      0.39 ± 0.03
      P < 0.05 as compared with R (40).
       Hippocampus0.350 ± 0.05
      P < 0.05 as compared with R (40).
      0.015 ± 0.001
      P < 0.05 as compared with R (40).
      542.1 ± 9.5
      P < 0.05 as compared with R (40).
      0.22 ± 0.04
      P < 0.05 as compared with R (40).
      L-NAME (10) per se
       Cerebral cortex0.508 ± 0.08
      P < 0.05 as compared with sham.
      0.019 ± 0.007
      P < 0.05 as compared with sham.
      722.2 ± 11.2
      P < 0.05 as compared with sham.
      0.28 ± 0.05
      P < 0.05 as compared with sham.
       Hippocampus0.362 ± 0.01
      P < 0.05 as compared with sham.
      0.013 ± 0.002
      P < 0.05 as compared with sham.
      594.1 ± 8.2
      P < 0.05 as compared with sham.
      0.20 ± 0.09
      P < 0.05 as compared with sham.
      L-ARG (100) per se
       Cerebral cortex0.576 ± 0.04
      P < 0.05 as compared with sham.
      0.016 ± 0.004
      P < 0.05 as compared with sham.
      785.7 ± 12.4
      P < 0.05 as compared with sham.
      0.24 ± 0.03
      P < 0.05 as compared with sham.
       Hippocampus0.386 ± 0.06
      P < 0.05 as compared with sham.
      0.009 ± 0.001
      P < 0.05 as compared with sham.
      581.2 ± 11.8
      P < 0.05 as compared with sham.
      0.15 ± 0.01
      P < 0.05 as compared with sham.
      LPO = lipid peroxidation. Data expressed as mean ± standard error of mean.
      Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg).
      P < 0.05 as compared with sham.
      P < 0.05 as compared with control.
      P < 0.05 as compared with R (40).
      § P < 0.05 as compared with L-NAME (10) (one-way ANOVA followed by Tukey's test).

      3.7 Effect of rutin and its modification with NO modulators on brain AChE levels

      Head trauma control animals showed a significant increase in the levels of AChE enzyme in both cerebral cortex and hippocampus as compared with the sham group (P < 0.01). Rutin (40 and 80 mg/kg) treatment significantly attenuated the increased AChE activity as compared with control animals (P < 0.01). L-NAME (10 mg/kg) pretreatment with subeffective dose of rutin (40 mg/kg) potentiated its effects, which was significant as compared with their effects alone. However, L-arginine (100 mg/kg) reversed the protective effect of rutin (40 mg/kg), which was significant as compared with rutin (40 mg/kg) alone. Besides, the per se effects of L-NAME (10 mg/kg) and L-arginine (100 mg/kg) on AChE levels did not show any significant effect as compared with control (F [9, 44] = 146.21 [P < 0.01]; Fig. 5).
      Figure thumbnail gr5
      Fig. 5Effects of rutin and its modification with NO modulators on brain AChE levels. Data expressed as mean ± standard error of mean. aP < 0.05 as compared with sham; bP < 0.05 as compared with control; cP < 0.05 as compared with R (40); dP < 0.05 as compared with L-NAME (10) (One-way ANOVA followed by Tukey's test). Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg).

      3.8 Effect of rutin and its modification with NO modulators on brain TNF-α and apoptotic factor (caspase-3) activity

      Brain injury significantly increased the activity of TNF-α and caspase-3 in both cerebral cortex and hippocampus of control animals as compared with sham group (P < 0.01). Treatment with rutin (40 and 80 mg/kg) significantly attenuated the increased activity of TNF-α and caspase-3 as compared with control (P < 0.01). However, rutin (20 mg/kg) did not show any significant improvement as compared with sham group. Furthermore, L-NAME (10 mg/kg) pretreatment with subeffective dose of rutin (40 mg/kg) potentiated its protective effect (lowered TNF-α and caspase-3 levels), which was significant as compared with their effects alone; however, pretreatment with L-arginine (100 mg/kg) reversed the protective effect of rutin (40 mg/kg) alone. However, effects of L-NAME (10 mg/kg) and L-arginine (100 mg/kg) per se treatments did not produce any significant effect on TNF-α (F [9, 44] = 74.21 [P < 0.01]; Fig. 6) and caspase-3 levels (F [9, 44] = 54.23 [P < 0.01]; Fig. 7) as compared with sham group.
      Figure thumbnail gr6
      Fig. 6Effects of rutin and its modification with NO modulators on TNF-α activity. Data expressed as mean ± standard error of mean. aP < 0.05 as compared with sham; bP < 0.05 as compared with control; cP < 0.05 as compared with R (40); dP < 0.05 as compared with L-NAME (10) (One-way ANOVA followed by Tukey's test). Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg).
      Figure thumbnail gr7
      Fig. 7Effects of rutin and its modification with NO modulators on caspase-3 activity. Data expressed as mean ± standard error of mean. aP < 0.05 as compared with sham; bP < 0.05 as compared with control; cP < 0.05 as compared with R (40); dP < 0.05 as compared with L-NAME (10) (One-way ANOVA followed by Tukey's test). Control: head injury control, R (20, 40, and 80): rutin (20, 40, and 80 mg/kg), L-ARG (100): L-Arginine (100 mg/kg).

      4. Discussion

      Traumatic head injury is a serious health concern, which results into physical, cognitive, and psychosocial disturbances, seen in both civilian and military population. Cognitive dysfunction is a normal consequence of head trauma, which is seen in various age groups particularly among young population [
      • Ozdemir D.
      • Baykara B.
      • Aksu I.
      • et al.
      Relationship between circulating IGF-1 levels and traumatic brain injury-induced hippocampal damage and cognitive dysfunction in immature rats.
      ]. In the present study, we revealed that rutin, a common dietary flavonoid, could reverse the impaired cognition and oxidative damage along with attenuation of neuroinflammation and cell death cascade associated with rat model of head trauma. On the other hand, L-NAME pretreatment potentiated the beneficial effects of rutin, whereas L-arginine appeared to reverse its protective effects. These results clearly suggest that the beneficial effects of rutin against head injury are dependent on modulation of NO pathway.
      In the present study, memory performances were evaluated using MWM and EPM. These two tests are often used as complementary to each other. Along with the measurement of anxiety behavior, EPM has also been used as a model for assessment of learning and memory in our laboratory [
      • Sharma A.C.
      • Kulkarni S.K.
      Evaluation of learning and memory mechanisms employing elevated plus-maze in rats and mice.
      ,
      • Kumar A.
      • Naidu P.S.
      • Seghal N.
      • Padi S.S.
      Effect of curcumin on intracerebroventricular colchicine-induced cognitive impairment and oxidative stress in rats.
      ]. In MWM test, ELT and path length to reach the hidden platform was significantly increased; however, TSTQ (retrieval trial) was significantly decreased in head trauma rats as compared with sham group suggesting impairment of learning and memory. The results from EPM further substantiated the findings with MWM test as ITL and RTL in EPM was significantly increased in head-injury rats. These findings are in conformity with the recent report from Uysal et al. [
      • Uysal N.
      • Baykara B.
      • Kiray M.
      • et al.
      Combined treatment with progesterone and magnesium sulfate positively affects traumatic brain injury in immature rats.
      ], which showed memory impairment associated with head trauma. In the present study, chronic treatment with rutin significantly improved the deficits in cognitive behavior observed in head trauma rats in both MWM and EPM. The memory restorative potentials of rutin are in line with the previous findings [
      • Gupta R.
      • Singh M.
      • Sharma A.
      Neuroprotective effect of antioxidants on ischaemia and reperfusion-induced cerebral injury.
      ,
      • Javed H.
      • Khan M.M.
      • Ahmad A.
      • et al.
      Rutin prevents cognitive impairments by ameliorating oxidative stress and neuroinflammation in rat model of sporadic dementia of Alzheimer type.
      ].
      Oxidative stress is a state of imbalance between free radical generation and antioxidant defense enzymes of the body. Increased production of free radicals causes discrepancy between oxidative species and antioxidant enzymes leading to generation of neurodegenerative diseases [
      • Schulz J.B.
      • Lindenau J.
      • Seyfried J.
      • Dichgans J.
      Glutathione, oxidative stress and neurodegeneration.
      ]. Oxidative stress is known to be one of the leading factors in the pathogenesis of traumatic head injury [
      • Bayir H.
      • Kochanek P.M.
      • Clark R.S.
      Traumatic brain injury in infants and children: mechanisms of secondary damage and treatment in the intensive care unit.
      ]. Generation of free radicals, such as superoxide and hydroxyl ions, after head trauma are the major contributors in the pathogenesis of secondary injury cascade [
      • Ji X.
      • Tian Y.
      • Xie K.
      • Liu W.
      • Qu Y.
      • Fei Z.
      Protective effects of hydrogen-rich saline in a rat model of traumatic brain injury via reducing oxidative stress.
      ]. A recent report also suggests a strong induction of oxidative/nitrosative stress markers in mild traumatic brain injury model of rat [
      • Abdul-Muneer P.M.
      • Schuetz H.
      • Wang F.
      • et al.
      Induction of oxidative and nitrosative damage leads to cerebrovascular inflammation in an animal model of mild traumatic brain injury induced by primary blast.
      ]. Similarly, the present study also shows a significant increase in lipid peroxidation, nitrite levels, and a marked reduction in reduced GSH and catalase enzyme levels in both cerebral cortex and hippocampus regions of head trauma rat. However, chronic treatment with rutin significantly mitigated head trauma–mediated alterations in the levels of biological antioxidant enzymes. This effect could be attributed to rutin's free radical scavenging and neuroprotective properties [
      • Javed H.
      • Khan M.M.
      • Ahmad A.
      • et al.
      Rutin prevents cognitive impairments by ameliorating oxidative stress and neuroinflammation in rat model of sporadic dementia of Alzheimer type.
      ]. In another studies, rutin enhanced the activity of antioxidant enzymes, such as GSH peroxidase and reductase [
      • Kamalakkannan N.
      • Prince P.S.M.
      Rutin improves the antioxidant status in streptozotocin-induced diabetic rat tissues.
      ], and showed protection against lipid peroxidation [
      • Lopez-Revuelta A.
      • Sanchez-Gallego J.I.
      • Hernandez-Hernandez Z.
      • Sanchez-Yague J.
      • Llanillo M.
      Membrane cholesterol contents influence the protective effects of quercetin and rutin in erythrocytes damaged by oxidative stress.
      ], which relates to its antioxidant effects.
      Cholinergic function is important for the process of learning and memory, and its alteration plays a key role in the development of cognitive impairment. Hippocampus has abundant inputs from the basal forebrain cholinergic system and acetylcholine (ACh) is known to have a major role in the course of learning and memory [
      • Prado V.F.
      • Martins-Silva C.
      • de Castro B.M.
      • et al.
      Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition.
      ]. ACh is degraded by the enzyme AChE, thus terminating the physiological action of neurotransmitter in brain. Loss of cholinergic neurons and alteration of the ACh neurotransmission has been shown in experimental model of brain trauma [
      • Scremin O.U.
      • Li M.G.
      • Roch M.
      • Booth R.
      • Jenden D.J.
      Acetylcholine and choline dynamics provide early and late markers of traumatic brain injury.
      ]. Similarly, in our current findings, we found a significant increase in the levels of AChE in different brain regions of head trauma rats, which was further attenuated on chronic treatment with rutin.
      Traumatic head injury causes damage to the neuronal tissues through the process of primary and secondary injury cascades. Although primary injury leads to edema formation and tissue loss within the brain, secondary injury involves several mechanisms, including the initiation of oxidative stress and release of many inflammatory cytokines [
      • Lenzlinger P.M.
      • Hans V.H.
      • Jöller-Jemelka H.I.
      • Trentz O.
      • Morganti-Kossmann M.C.
      • Kossmann T.
      Markers for cell-mediated immune response are elevated in cerebrospinal fluid and serum after severe traumatic brain injury in humans.
      ]. Neuronal inflammation is a well-known process involved in the onset of several neurodegenerative disorders, including Alzheimer disease. Furthermore, head injury–induced mitochondrial damage is known to initiate the process of apoptotic cell death by the release and activation of proapoptotic factors, such as caspases [
      • Mazzeo A.T.
      • Beat A.
      • Singh A.
      • Bullock M.R.
      The role of mitochondrial transition pore, and its modulation, in traumatic brain injury and delayed neurodegeneration after TBI.
      ]. In the present study, we observed a significant elevation in the levels of proinflammatory cytokines (TNF-α) and apoptotic factors (caspase-3) in both cerebral cortex and hippocampal regions, which is indicative of enhanced neuroinflammation and cell death in two main brain regions involved in memory. These findings are in concurrence with those reported from other laboratories [
      • Mao S.S.
      • Hua R.
      • Zhao X.P.
      • et al.
      Exogenous administration of PACAP alleviates traumatic brain injury in rats through a mechanism involving the TLR4/MyD88/NF-kappaB pathway.
      ,
      • Raghupathi R.
      • Conti A.C.
      • Graham D.I.
      • et al.
      Mild traumatic brain injury induces apoptotic cell death in the cortex that is preceded by decreases in cellular Bcl-2 immunoreactivity.
      ]. On the other hand, treatment with rutin significantly reduced the levels of TNF-α and caspase-3 in both the brain regions of traumatic injury rats. The results are further supported by reports from recent studies [
      • Javed H.
      • Khan M.M.
      • Ahmad A.
      • et al.
      Rutin prevents cognitive impairments by ameliorating oxidative stress and neuroinflammation in rat model of sporadic dementia of Alzheimer type.
      ,
      • Yang Y.C.
      • Lin H.Y.
      • Su K.Y.
      • et al.
      Rutin, a flavonoid that is a main component of Saussurea involucrata, attenuates the senescence effect in d-Galactose aging mouse model.
      ].
      NO is an essential signaling molecule involved in various physiological functions within our body. Recent studies also show that NO pathway has an important role in brain structure and functions [
      • Kanbak G.
      • Kartkaya K.
      • Ozcelik E.
      • et al.
      The neuroprotective effect of acute moderate alcohol consumption on caspase-3 mediated neuroapoptosis in traumatic brain injury: the role of lysosomal cathepsin L and nitric oxide.
      ]. There are three major forms of NOS viz neuronal, endothelial, and inducible. NOS viz neuronal is the principal enzyme synthesizing NO in the central nervous system [
      • Zhou L.
      • Zhu D.Y.
      Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications.
      ]. Excess production of NO in body along with other free radicals can produce several neurotoxic effects. NO reacts with reactive oxygen species and acts as an oxidant agent, which can target mitochondrial respiratory enzymes and causes mitochondrial dysfunction [
      • Pall M.L.
      Elevated, sustained peroxynitrite levels as the cause of chronic fatigue syndrome.
      ]. NO can inhibit phosphorylation and glycolytic pathways by causing nitrosylation of different proteins [
      • Zhang J.
      • Snyder S.H.
      Nitric oxide in the nervous system.
      ]. Increased expression of NOS further initiates the process of neuroinflammation and oxidative damage [
      • Contestabile A.
      Roles of NMDA receptor activity and nitric oxide production in brain development.
      ]. Neuronal injury due to excess production of both NO and peroxynitrite has been documented in Alzheimer's brain [
      • Koppal T.
      • Drake J.
      • Yatin S.
      • et al.
      Peroxynitrite-induced alterations in synaptosomal membrane proteins: insight into oxidative stress in Alzheimer's disease.
      ]. All these different factors accounts for increase in NO production, leading to an enhanced free radical production and cell death. In the current investigation, L-NAME (a nonselective inhibitor of NOS) pretreatment with subeffective dose of rutin potentiated the protective effects of rutin. However, L-arginine (a NO donor) pretreatment attenuated its protective effect. These results are in line with the recent review report, which states that excess NO production by L-arginine can adversely affect brain functions leading to the development of neurodegenerative diseases such as Alzheimer disease [
      • Virarkar M.
      • Alappat L.
      • Bradford P.G.
      • et al.
      L-arginine and nitric oxide in CNS function and neurodegenerative diseases.
      ]. However, the individual effects of L-NAME and L-arginine treatment did not produce any significant effect as compared with head trauma control. Previous reports suggest the inhibitory effects of various flavonoids against NO production [
      • Marzouk M.S.
      • Soliman F.M.
      • Shehata I.A.
      • Rabee M.
      • Fawzy G.A.
      Flavonoids and biological activities of Jussiaea repens.
      ]. Failure of protection by L-arginine suggests that increased NO levels could have enhanced the process of nitrergic signaling causing behavioral and biochemical deficits. However, inhibition of nitrergic signaling by L-NAME could be one of the possible reasons for restoration of learning and memory process.

      5. Conclusions

      The findings of the present study propose that antioxidant and anti-inflammatory effects of rutin may have increased the endogenous defensive capacity of the brain via modulation of NO pathway to combat against oxidative stress mediated neuroinflammatory and cell death cascade, which may have resulted in protection against neurodegeneration of hippocampal and cerebral cortex neurons and cognitive loss in rat model of traumatic head injury.

      Acknowledgment

      The authors gratefully acknowledge the financial support of Council of Scientific and Industrial Research (CSIR), New Delhi for carrying out this work. Authors would like to thank Dr Thirunavukkarasu Angappan, Deputy General Manager, Panacea Biotec Ltd, Lalru for the supply of experimental animals.

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