Vitamin C attenuates memory loss induced by post-traumatic stress like behavior in a rat model

Journal Pre-proof Vitamin C Attenuates Memory Loss Induced by Post-Traumatic Stress like Behavior in a Rat Model Karem. H. Alzoubi, Alaa F. Shatnawi, Mohammad A. Al-Qudah, Mahmoud A. Alfaqih

PII:

S0166-4328(19)31247-1

DOI:

https://doi.org/10.1016/j.bbr.2019.112350

Reference:

BBR 112350

To appear in:

Behavioural Brain Research

Received Date:

12 August 2019

Revised Date:

5 November 2019

Accepted Date:

7 November 2019

Please cite this article as: Alzoubi KH, Shatnawi AF, Al-Qudah MA, Alfaqih MA, Vitamin C Attenuates Memory Loss Induced by Post-Traumatic Stress like Behavior in a Rat Model, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112350

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Vitamin C Attenuates Memory Loss Induced by Post-Traumatic Stress like Behavior in a Rat Model

Running title: Vitamin C reduces oxidative stress and memory impairment induced by PTSD-like

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behavior

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“Karem. H. Alzoubi1*, Alaa F. Shatnawi2, Mohammad A. Al-Qudah3 and Mahmoud A. Alfaqih2 Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology,

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Irbid-22110, Jordan 2

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Department of Physiology and Biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid-22110, Jordan

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Department of Pathology and Microbiology, Faculty of Medicine, Jordan University of Science and

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Technology, Irbid-22110, Jordan

Corresponding Author:

Karem Alzoubi, PhD

Professor of Pharmacology

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Department of Clinical Pharmacy Faculty of Pharmacy

Jordan University of Science and Technology Irbid, Jordan 22110” Tel.: +962-2-7201000 Ext.: (23525)

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Abstract Oxidative stress is associated with neuronal damage in many brain regions including

the

hippocampus;

an

area

in

the

brain

responsible

of

memory

processing. Oxidative stress is also linked with many psychiatric conditions including post-traumatic stress disorder (PTSD). PTSD is triggered by traumatic and

many

PTSD

patients

show

signs

of

memory

impairment.

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experience

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Vitamin C is a water-soluble vitamin with antioxidant properties. Herein, we hypothesized that memory impairment observed during PTSD could be a result

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of oxidative stress in hippocampal tissues and that prophylactic vitamin C administration may reduce oxidative stress in the hippocampus and prevent

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memory impairment. The above hypothesis was tested in a rat model where

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PTSD-like behavior was induced through single prolonged stress (SPS). Short and long-term memory was tested using a radial arm water maze (RAWM). We

of

the

decrease

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found that SPS induced a significant increase in the oxidized glutathione levels hippocampus. in

This

glutathione

reduction

peroxidase

was and

accompanied catalase

with

enzyme

a

significant

activity,

and

a

significant increase in lipid peroxidation. Intriguingly, vitamin C administration

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successfully attenuated memory impairment and all of the changes observed in oxidative stress markers. Our findings demonstrate that vitamin C could prevent oxidative stress and memory impairment induced by SPS model of PTSD-like behavior in rat. Key words: Vitamin C, PTSD, Single Prolonged Stress, memory, maze, oxidative stress.

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Introduction

Reactive oxygen species (ROS) are regularly produced by the cellular systems of aerobic organisms (Nohl et al., 2003). These species are released inadvertently during the process of cellular respiration and are also generated by inflammatory cells to combat invading

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microorganisms (Spooner and Yilmaz, 2011). Despite their involvement in normal physiological

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functions, ROS can potentially damage cellular systems via oxidation of DNA, proteins and lipids (Radak et al., 2011). Indeed, in view of these damaging effects, aerobic organisms have developed

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multiple defense mechanisms that inactivate ROS and prevent them from destroying cells. The

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superoxide dismutase (SOD), glutathione and catalase systems are examples of the above (Diaz-

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Vivancos et al., 2015; Valko et al., 2016). Considering the potentially damaging effects of ROS, it is not surprising that several chronic disease conditions are linked with the increased generation

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of ROS above threshold levels that supersede the above defense systems; a cellular state commonly described as “oxidative stress” (Lushchak, 2014). Examples of these diseases include diabetes, Alzheimer, atherosclerosis, traumatic brain injury and post-traumatic stress disorder (PTSD)

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(Smith et al., 2000; Jay et al., 2006; Singh and Jialal, 2006; Alzoubi et al., 2018c; Alquraan et al., 2019). Further evidence on the central role of ROS in mediating the etiology and pathogenesis of the above disorders comes from studies performed on preclinical and clinical models which showed that inactivating ROS or limiting their production is linked with improved treatment outcome and/or prevention of many of the above disorders (Fenster et al., 2003). 3

In humans, vitamin C (ascorbic acid) is an essential water-soluble nutrient. Vitamin C is a cofactor of enzymes involved in the hydroxylation of proline and lysine residues of collagen; a process mandatory for the folding, maturation and strength of the collagen triple-helix (Traber and Stevens, 2011). Furthermore, vitamin C is involved in the synthesis of catecholamines, carnitine,

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cholesterol, some peptide hormones and amino acids (Padayatty and Levine, 2016). In addition to its role in the above biosynthetic reactions, vitamin C is considered an antioxidant (Irshad and

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Chaudhuri, 2002). The exact mechanism by which vitamin C plays this role is unknown, however,

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it is thought that vitamin C protects the cellular DNA, proteins and lipids from the oxidative

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damage of ROS by being oxidized itself. Indeed, the ascorbic acid form of vitamin C can donate

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up to two electrons to various ROS thereby inactivating them. The antioxidant properties of vitamin C could explain the preventive effects of diets rich in this vitamin (i.e. fruits and

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vegetables) on the risk of cardiovascular disease and cancer; conditions strongly linked with oxidative stress (Padayatty et al., 2003; Mandl et al., 2009). Furthermore, vitamin C implemented several beneficial functions such as immunostimulant and anti-inflammatory manners (Spoelstra-

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de Man et al., 2018; Zhang et al., 2018b). Intriguingly, previous study on mice given LPS and pretreated with vitamin C has shown to prevent pro-inflammatory cytokines involving tumor necrosis factor-alpha and normalizing the signal pathway and malondialdehyde level in the hippocampus of the LPS treated group (Zhang et al., 2018a).

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Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition. Development of PTSD is observed in war-veterans; however, the symptoms of PTSD could be precipitated through exposure to any life-threatening event such as sexual assault, home displacement and road traffic accidents (Kirkpatrick and Heller, 2014). Individuals affected with PTSD develop a number of

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symptoms including re-experiencing symptoms in which they relive the precipitating trauma and avoidance symptoms in which they modify their behavior in an attempt to avoid any event which

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may remind them of the trauma, and hyperarousal (American Psychiatric Association, 2013).

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Moreover, PTSD is associated with cognitive decline and memory loss that was shown in both

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human (Ritchie et al., 2019) and animal studies (Richter-Levin, 1998; Cohen et al., 2007; Patki et

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al., 2014a; Alzoubi et al., 2017; Alzoubi et al., 2018d; Alzoubi et al., 2018c; El-Elimat et al., 2019). Memory loss in PTSD patients could be severe enough to affect day to day activities of affected

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individuals (Ritchie et al., 2019).

The exact reason behind the cognitive decline in PTSD patients is under investigation. Several reports indicated that it may be linked with the neurodegeneration of the hippocampus, an

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area in the brain responsible for processing and storing memory (Miller and Sadeh, 2014; Alzoubi et al., 2017; Miller et al., 2018; Alzoubi et al., 2019). Indeed, destruction of the neuronal tissues of the hippocampus is observed in several psychiatric conditions with overlapping symptoms of PTSD such as depression and Alzheimer and is observed on MRI images of the brains of PTSD patients. Preventing or delaying the onset of memory loss in PTSD requires a mechanistic 5

understanding of its pathophysiology at the molecular level. Although still under extensive investigation, several reports indicated that the above memory loss could be related to increased oxidative stress in the hippocampus eventually leading to its neurodegeneration (Soleimani et al., 2016; Rahimzadegan and Soodi, 2018). Concomitantly, recent study has observed neurotoxicity

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in mice model and deterioration of antioxidant defense system in brain tissue, vitamin C has mitigated these actions by increasing hippocampus neurogenesis and potentiating memory (Nam

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et al., 2019). This hypothesis is supported by several studies which reported a decrease in memory

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loss in animal models of PTSD-like behavior upon the use of pharmacological reagents that

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normally scavenge ROS (Alzoubi et al., 2018b; Alzoubi et al., 2018a). In this report, we

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investigated the effect of vitamin C administration on the memory loss in an animal model of

Methods

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PTSD-like behavior including the levels of several oxidative markers in the hippocampus.

Animals and treatment

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Male Wistar rats used in this study (150-200 grams in weight) were purchased from the animal care facility of Jordan University of Science and Technology (JUST). Rats were kept cages made of plastic (3-4 animals/cage) in a climate-controlled room (24 ± 1oC), they had an ad libitum access to food and water, and 12 hours light/dark cycle (light on 7:00 am) with the experimental procedure being performed during the light cycle. Tail labeling was used to distinguish rats of the different treatment group. The study protocol was approved by the Institutional Animal Care and 6

Use Committee (IACUC) of JUST. All lab and animal house personnel who handled rats followed the Institutional Animal Care and Use Committee Guidebook, 2nd edition of 2002. Four groups (n=15/group) of animals were randomly allocated: control, vitamin C, single prolonged stress (SPS) group, and vitamin C plus SPS group (SPS + Vitamin C). Vitamin C was administered via oral gavage at a dose of 100mg/kg/day, six days a week for a total duration of four weeks. Vitamin C was also administered on the day when the behavioral test was performed. The

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above vitamin C dose was previously shown to preserve cognition in conditions other than PTSD

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(Loukavenko et al., 2016; Pourmemar et al., 2017; Stepanichev et al., 2017) and was thus chosen for this study. The SPS and vitamin C -SPS groups were subjected to SPS procedure, an established

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model of PTSD-like behavior in animals (Patki et al., 2014a; Solanki et al., 2015; Alzoubi et al.,

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2017; Liu et al., 2017; Alzoubi et al., 2018b; Chen et al., 2018; Wang et al., 2018; El-Elimat et al., 2019; Lin et al., 2019), one week following the initiation of vitamin C administration (Fig 1). Water

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alone was administered to animals of the control and SPS groups once daily on the same days the other two groups received vitamin C.

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Single Prolonged Stress (SPS) procedure

All animals of the SPS or SPS + Vitamin C groups received SPS Single prolonged stress (SPS) to introduce PTSD behavior in animals. The SPS was carried out in three stages as described previously (Patki et al., 2014a; Solanki et al., 2015; Alzoubi et al., 2017; Alzoubi et al., 2018c). In

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the first stage, a rodent restrainer (Part # 82, IITC Life Science, Woodland Hills, CA, USA) was used to restrain the animal for two hours. The above procedure guaranteed full immobilization. The second stage, which followed immediately, was a twenty-minute forced swimming performed in a transparent cylinder (height: 50 cm; diameter: 35 cm; water depth: 30 cm; water temperature: 24˚C). Right after that, animals were allowed to recuperate in for 15 minutes in pre-prepared plastic

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cages. Finally, 1-2 minutes ether anesthesia until loss of consciousness was applied (Yamamoto et al., 2009; Li et al., 2010; Patki et al., 2014b; Alzoubi et al., 2017; Alzoubi et al., 2018c). The radial arm water maze (RAWM)

It is used to test spatial learning and memory. Details of the RAWM were previously described (Alzoubi et al., 2015; Mhaidat et al., 2015a; Alzoubi et al., 2016). The RAWM has 6 stainless steel

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arms and a central black circular water-filled tube with a hidden platform located on the target arm, which was always the same arm for each rat during both learning and memory phases. The starting

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arm was different in each of the learning trials or memory tests. All experiments were carried out in

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dimly-light room with visual cues that were fixed at the wall of the room during experiment forming a spatial reference for the animal during the learning and memory phase. Water

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temperature was maintained at 23±1C°. The learning phase was performed prior to the short term

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and long term memory tests. The learning phase consisted of twelve trials; six trials were performed consecutively followed by a 5 minutes rest before performing the remaining six trials. Short-term memory testing was performed 30 minutes after the end of the learning phase, whereas

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long-term memory testing was performed 5 hours and 24 hours following the end of the last trial of the learning phase. During the learning phase, each rat was given a one min to swim freely to the target arm but it was guided to the target arm after having one minute without finding the target

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arm. Each animal was left on the target arm for 15 seconds to explore their location according to the visual cues. In memory test, each rat was given one minute to locate the hidden platform (with no 15 seconds chance on the target arm). An error was recorded when the rat entered to an arm other than the target arm.

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2.4. Dissection of the Hippocampus

Animals were euthanized and brain was dissected out and put on top of a normal saline soaked filter paper. The filter paper was placed over a crushed ice filled cold glass plate (Alzoubi et al., 2013). Both left and right lopes of the hippocampus were separated from the remaining cerebral tissues and directly placed in Eppendorf tube that were a pre-labeled for that purpose.

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The tube was then placed in liquid nitrogen before being stored at -30C° until further analysis.

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Molecular assays

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The tissues of the hippocampus were homogenized in phosphate buffer (200µl). using a plastic pestle, the homogenization buffer was prepared by the reconstitution of one phosphate buffered saline tablet

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(Sigma Chemical CO., Saint Louis, MO) and two tablets of protease inhibitor (Sigma Chemical CO., Saint Louis, MO) in distilled water (200 ml). Then, mixture of homogenized tissues and buffer were centrifuged

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at 15000xg for 10 minutes (4C°) in order to eliminate tissues that failed to dissolve in the buffer and thus remained insoluble. The supernatant from the previous step was recovered and stored for further analysis.

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Total protein concentration in the recovered supernatant was measured using a commercially available kit purchased from Bio-rad (Bio-Rad, USA). For oxidative stress biomarkers, total glutathione, GSSG, GPx, catalase and Thiobarbituric acid reactive substance (TBARs), commercially available kits were utilized as per kit’s manufacturer’s instructions (total glutathione and GSSG: Glutathione assay kit, CS0260, Sigma-

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Aldrich, MI, USA, GPx: CGP1, Sigma-Aldrich, MI, USA, Catalase: catalase assay kit, Cayman Chem, Ann Arbor, MI, USA, TBARs: Cayman Chem, item № 10009055, Ann arbor, MI. USA). Absorbance for each assay was measured spectrophotometrically using a Epoch Microplate Spectrophotometer at specified wavelength in each of the after-mentioned kits (Bio-tek instruments, Highland Park, Winooski, USA). Statistical analysis

The number of errors were compared via two-way ANOVA followed by posttest for multiple 9

comparison. The repeated measures factor was Time and interaction with SPS/no SPS and vitamin C/no vitamin C were independent group dimensions. For the biochemical assays, one-way ANOVA plus Tukey’s posttest was used to achieve comparisons. The statistical software used was GraphPad Prism version 4.0. Significance was set at P < 0.05. Values all over the study are reported

Results

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The effect of SPS and/or vitamin C on learning and memory

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as mean ± SEM.

The number of errors in the learning phase was high in the initial trials, however, this

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number decreased with subsequent trials (Figure 1). There were, however, no significant

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differences in the number of errors between different experimental groups whether in the early or late trials. We conclude that neither SPS nor vitamin C significantly affected learning in our model.

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However, upon the examination of the effect of SPS and/or vitamin C on short term and long term memory (after 5 or 24 hours) a different picture emerged. Here, we found that SPS significantly

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affected short term (Figure 2a) and long term memory when tested after 5 hours (Figure 2b) or 24 hours (Figure 2c). Intriguingly, our findings indicated that prophylactic administration of vitamin C attenuated short and long term memory loss induced by SPS although vitamin C by itself had no significant effect on short or long term memory compared to control rats (Figures 2b and 2c).

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The effect of SPS and/or vitamin C on the levels of oxidative stress biomarkers in the hippocampus

It was observed in prior studies that memory loss could result from oxidative stress, we

therefore measured the levels of several oxidative stress markers in hippocampal tissues recovered from the brains of the rats of each experimental group. These markers included GSH, GSSG and TBARs. Additionally, we measured the activities of two enzymes involved in combating ROS; 10

these included the catalase and glutathione peroxidase (GPx) enzymes. We found that although SPS did not significantly affect the levels of reduced glutathione (GSH) in hippocampal tissues (Figure 3a), SPS induced a significant increase in oxidized glutathione levels (GSSG) (Figure 3b) and consequently a significant decrease in GSH/GSSG ratio (Figure 3c). It was also observed that vitamin C attenuated the effects of SPS on GSSG and GSH/GSSH ratio. Interestingly, vitamin C administration by itself did not significantly change the levels of GSH, GSSG or GSH/GSSG ratio

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compared to vehicle treated rats.

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Since changes in the levels of the above biomarkers could be a result of changes in the levels of enzymes expressed in cellular system to combat oxidative stress, we also measured the

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activity of GPx and catalase enzymes in hippocampus tissues. In this analysis, we found that SPS

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caused a significant reduction in the levels of GPx (Figure 4a) as well as in catalase enzyme activity (Figure 4b). Moreover, analogous to the effects of vitamin C on GSSG and GSH/GSSG ratio,

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prophylactic treatment with vitamin C prevented the reduction in GPx and catalase activities induced by SPS (Figure 4a and 4b). Finally, we found that a SPS resulted in an increase in the

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levels of TBARs; a marker of lipid peroxidation (Figure 5). The above increase in TBARs was completely prevented by vitamin C (Figure 5).

Discussion

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Current study demonstrated that chronic vitamin C treatment prevented the deleterious effect of oxidative stress biomarkers within the hippocampus during memory impairment in the SPS animal model of PTSD-like behavior. To test our hypothesis, we applied SPS to create cognitive impairment, as well as we tested the spatial and learning memory using the radial arm water maze. Results revealed that SPS induced short- and long- term memory impairments, which were in

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consistence with previous studies that examined cognition, using the RAWM (Patki et al., 2014b; Alzoubi et al., 2017; Alzoubi et al., 2018c), and Morris water maze (Wang et al., 2010; Wen et al., 2015) in SPS exposed animals. Learning function was not altered due to SPS, which is consistent with previous reports (Alzoubi et al., 2017; Alzoubi et al., 2018c; Alquraan et al., 2019; El-Elimat et al., 2019).

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On the other hand, both short- and long- term memories, which were impaired by SPS model of PTSD-like behavior, were normal with vitamin C treatment. This protective impact of vitamin C

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on impaired memory shown in present study was in accordance with previous other studies that

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examined vitamin C protective effects on impairment of cognitive functions in conditions other than PTSD (Mhaidat et al., 2015b; Alqudah et al., 2018). Interestingly, vitamin C deficiency in

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guinea pigs was shown to evoke spatial memory deterioration that was proposed to be due to the impairment in neurotransmission and synaptic development, which was linked to increase in

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oxidative stress (Hansen et al., 2018).

The mechanism by which vitamin C alleviated memory was probably by potentiating the

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antioxidant defense system in the hippocampus (Mhaidat et al., 2015a; Alqudah et al., 2018). Oxidative stress was related to the pathophysiology of PTSD (Miller and Sadeh, 2014; Du et al., 2016). The hippocampal levels or activities of antioxidant molecules and oxidative capacity

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enzymes (GSH/GSH ratio, catalase, and GPx) were shown to be reduced in rats exposed to SPS. Moreover, the levels of GSSG and TBARS were elevated significantly due to SPS. This is in accordance with previously published studies (Alzoubi et al., 2017, 2018b; Britnell et al., 2017). Furthermore, the levels of TBARS and other oxidative stress biomarkers were reduced when vitamin C treatment was instituted on top of memory impairment induced by LPS treatment (Zhang et al., 2018a). These outcomes were put into context the potential effect of oxidative stress 12

in extending PTSD symptoms and related complications. On the contrary, vitamin C normalized GSSG, GPx, catalase, and TBARS levels during SPS. In accordance, vitamin C was shown to possess nephroprotective effects that oppose cisplatin induced nephrotoxicity in rats (Abdel-Daim et al., 2019). In conclusion, oxidative stress imbalance in the hippocampus, which induced memory impairment

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in the SPS model of PTSD-like behavior might be alleviated by chronic vitamin C treatment.

Conflicts of interest

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None declared. Acknowledgements

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This study was financially supported via grant number: 43/2017, from Deanship of Research at

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the Jordan University of Science and Technology to KA.

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Figure Legends:

Fig. 1: Effect of Vitamin C and/or SPS model of PTSD-like behavior on Learning performance in the Radial arm water maze (RAWM). Each animal was trained for six

phase).

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experimental groups. Values are mean±SEM. N=15/group.

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consecutive trials separated by 5 min rest, then another six consecutive trials (the

Fig. 2: Effect of Vitamin C and/or SPS model of PTSD-like behavior on memory function during the RAWM. A: Number of errors in short-term memory test (after 30min). B: Number of errors in long-term memory test (after 5hr). C: Number of errors in long-term memory test (after 24hr). *Indicates significant difference compared other groups at P<0.05. Values are mean±SEM. N=15/group.

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Fig. 3: Levels of GSH, GSSG and GSH/GSSG ratio in SPS and/or vitamin C treatment. A: GSH level: no change was observed in the levels of GSH among all experimental groups. , B: GSSG level: the GSSG levels were increased, whereas the GSH/GSSG ratio was decreased in the SPS group. These changes were prevented in the SPS + vitminc group. *Indicates significant difference compared with other groups

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(P<0.05). Values are mean±SEM. N=15/group.

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Fig. 4: Effect of vitamin C and/or SPS on activities of GPx and catalase. The GPx and catalase enzymes activities were reduced by the SPS, which was prevented by vitamin C. *Indicates significant difference compared with other groups (P<0.05). Values are

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mean±SEM. N=15/group.

Fig. 5: Effect of vitamin C and/or SPS on levels of TBARs. Significant increase in TBARS levels were induce by the SPS model of PTSD-like behavior. This effect was 21

prevented by administration of vitamin C. *Indicates significant difference compared

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with other groups (P<0.05). Values are mean±SEM. N=15/group.

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