Role of Cyclooxygenase Inhibitors in Diminution of Dissimilar Stress-induced Depressive Behavior and Memory Impairment in Rats

Accepted Manuscript Role of cyclooxygenase inhibitors in diminution of dissimilar stress-induced depressive behaviour and memory impairment in rats Tahira Perveen, Shaista Emad, Saida Haider, Sana Sadaf, Sara Qadeer, Zehra Batool, Yousra Sarfaraz, Sheeza Sheikh PII: DOI: Reference:

S0306-4522(17)30801-1 https://doi.org/10.1016/j.neuroscience.2017.11.014 NSC 18129

To appear in:

Neuroscience

Received Date: Accepted Date:

3 February 2017 6 November 2017

Please cite this article as: T. Perveen, S. Emad, S. Haider, S. Sadaf, S. Qadeer, Z. Batool, Y. Sarfaraz, S. Sheikh, Role of cyclooxygenase inhibitors in diminution of dissimilar stress-induced depressive behaviour and memory impairment in rats, Neuroscience (2017), doi: https://doi.org/10.1016/j.neuroscience.2017.11.014

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

1

TITLE:

Role of cyclooxygenase inhibitors in diminution of dissimilar stress-induced depressive behaviour and memory impairment in rats

AUTHORS: Tahira Perveen*, Shaista Emad, Saida Haider, Sana Sadaf, Sara Qadeer, Zehra Batool, Yousra Sarfaraz, Sheeza Sheikh AFFILIATION: Neurochemistry and Biochemical Neuropharmacology Research Unit, Department of Biochemistry, University of Karachi. CORRESPONDING AUTHOR* Dr. Tahira Perveen Professor Neurochemistry and Biochemical Neuropharmacology Research Unit, Department of Biochemistry, University of Karachi, Pakistan E-mail: [email protected]

2

Abstract Memory functions can be considerably affected by various life events and stress has shown to be a chief regulator. Different stress patterns have distinct effects on the overall functioning of the brain. Stress provokes inflammation not only in the periphery but also in the brain. Neuroinflammation causes alterations in neuronal structure and function, which eventually progress to the development of neurodegenerative diseases. Inflammatory reactions are modulated through communication between the nervous, endocrine and immune systems. An excessive release of stress hormones and changes in the neurotransmission system may cause cognitive impairments. The present study investigated dissimilar stress-related memory deficits and their diminution by non-steroidal anti-inflammatory drugs (NSAIDs). Treatment with cyclooxygenase inhibitors, which inhibit prostaglandin synthesis, has enhanced memory functions in a number of neuroinflammatory states. In this study, rats were exposed to a series of dissimilar stressors and behavioural parameters for depression and memory functions were examined. Corticosterone, serotonin (5-HT) and dopamine (DA) levels were also estimated. Results from the forced swim test, elevated plus maze test and Morris water maze test showed significant effects of NSAIDs. A significant decrease in plasma corticosterone and increased DA and 5-HT levels were observed in NSAIDs-treated dissimilar-stressed rats. This study demonstrates the therapeutic potential of NSAIDs for dissimilar stress-induced impaired memory functions and related

hormonal and

neurochemical changes in the rat brain. Keywords: depression, dopamine, memory, NSAIDs, serotonin, stress INTRODUCTION Stress is a potent modulator of the hypothalamic pituitary adrenal (HPA) axis. Stress-induced stimulation of the HPA axis involves a series of neuroendocrine changes that are regarded as

3

the stress response. This response to stress is identified by the release of stress mediators, such as glucocorticoids (GCs) and catecholamines. High levels of corticotropin-releasing factor that are present in the systemic circulation and pituitary gland also mediate neuroendocrine effects (Charmandari et al., 2005; Smith and Vale, 2006). GCs play essential functions in regulating the extent and period of activation of the HPA axis (Sapolsky et al., 2000). GCs can penetrate the brain quickly and can disrupt neurological functions. GCs stimulate acute neuroendocrine system in response to stress, which is advantageous and helps the body to cope with challenging situations. However, if this response persists for an extended period of time, the body is left in a state of constant stress, and this state leads to different diseases, such as immune system dysfunction, ischaemia, stroke, heart attack, depression, anxiety, dementia and Alzheimer’s disease (McEwen, 1998, 2004; Glaser and Kiecolt-Glaser, 2005; Chrousos, 2009). The effect of stress on the development of these ailments depends upon the type, duration and intensity of the stressors (Mercier et al., 2003). It is well recognized that neuropsychiatric illnesses are generally manifested by chronic exposure to adverse stimuli (Mercier et al., 2003). Studies on experimental animals that are exposed to chronic stress showed that animals develop behavioural changes, including decreased locomotion, increased anxiety states, depressive symptoms and memory impairments (Kim and Diamond, 2002). Studies also report that over-stimulation of the HPA axis and increased release of GCs are linked to memory impairments (De Kloet et al., 2005). Chronic stress exposure responses are not merely restricted to changes in behaviours but are also the cause of disturbances in immune responses. Immune system activation is a natural defence mechanism of the body against any infection or tissue injury. Immune dysfunction is generally observed in individuals who are repeatedly exposed to stress conditions and have a higher susceptibility to pathological conditions (Glaser and Kiecolt-Glaser, 2005; Reiche et al., 2005). Inflammation is due to an increased release of proinflammatory cytokines and a decreased release of anti-

4

inflammatory cytokines (Averbeck and Reeh, 2001; Reiche et al., 2004; Wojdasiewicz et al., 2014). GCs have been shown to enhance memory following exposure to acute stress; in contrast, the excessive release of GCs during chronic stress is reported to impair memory functions (Sorrells et al., 2009). Previous studies reported that high GC levels produce an augmented inflammatory response and induce excitotoxicity in neurons. The inflammatory response of GCs is due to increased production of inflammatory mediators, which further worsen inflammation both in the peripheral and central nervous systems (Denes et al., 2010; Sorrells et al., 2014). Prostaglandins are also involved in stress-induced adrenocorticotropic hormone release via adrenergic stimulation of both α-1 and α-2 receptors (Watanabe et al., 1991; Gadek-Michalska et al., 2002). Inflammation of the CNS causes neuronal injury and modulates synaptic plasticity. Synaptic plasticity is an essential process in the development of memories (Calabrese et al., 2009). Depression and anxiety are the most prevalent health issues around the globe. Depression and memory dysfunction may share a closely related inflammatory aetiology. Patients suffering from major depressive disorder have impaired memory, lack of attention and concentration and usually fail to achieve routine tasks (Allison and Ditor, 2014). Serotonergic and dopaminergic systems are altered following exposure to stressful stimuli. The rate of synthesis and release of biogenic amines under stress conditions may determine the functioning of the brain and the associated risk factors. Several studies reported the involvement of biogenic amines in learning and cognitive functions (Baker and Reynolds, 1989; Saleem et al., 2014). In general, neuroendocrine, immune and neurotransmitter systems are interlinked with each other, and changes in any of these systems may trigger changes in other systems (Leonard, 2010). A practical understanding of the relationship between these systems is necessary to select suitable therapeutic targets. The use of non-steroidal antiinflammatory drugs (NSAIDs) is a recent, novel therapeutic strategy for protecting against

5

stress-induced depressive behaviours and memory loss (Leonard, 2010). Stress models, such as chronic unpredictable stress, produce more effective and prolonged stress responses and are widely accepted stress protocols for investigating depression in rats as well as the underlying mechanisms of stress (Gouirand and Matuszewich, 2005; Monteiro et al., 2015). These stress procedures appear to be stronger and cause marked changes in immunological, physiological, behavioural, biochemical and metabolic functions. Anti-inflammatory approaches for the management of dissimilar stress-induced depressive behaviours have shown beneficial outcomes for maintaining a healthy lifestyle (Guo et al., 2009). Therefore, the current study was designed to investigate dissimilar stress-induced behavioural despair and memory loss and the potential of NSAIDs to attenuate these behavioural deficits. The present study was further extended to determine the biochemical and neurochemical changes that follow the administration of NSAIDs in dissimilar stress-exposed rats. EXPERIMENTAL PROCEDURES Animals and chemicals Male Sprague-Dawley rats (weight: 150-200 g) were purchased from Aga Khan University Hospital, Karachi, Pakistan. Animals were housed individually under a 12:12 h light: dark cycle, at 25±2°C with free access to food and water. All experimental protocols were approved by the institutional ethics and animal care committee and carried out in strict accordance with the National Institute of Health’s Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised1985). NSAIDs (indomethacin and diclofenac sodium), dichloromethane, sulfuric acid-ethanol reagent, octyl sodium sulfate (OSS), methanol ethylenediaminetetraacetic acid (EDTA) and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, USA).

6

Restraining procedure Restraint stress is a commonly used, authenticated model of stress in animals, and it allows for minimal movement, including in the tail, and causes no pain in animals. The animals were restrained in ventilated, closed plastic tubes that permitted restricted lateral movement of rats in the restrainer tube (Delaney et al., 2012). Noise exposure Noise stress was given to rats using generator noise, which was recorded and amplified by loud speakers in an isolated room. The speakers were placed approximately 30 cm above the animal cages. The level of recorded noise was set at 100 dB and intensity was quantified by a sound level meter DS102 (Naqvi et al., 2012). Shaker stress Shaker stress was given to rats by exposing them to horizontal shakes at a high speed of 200rpm for approximately 10 minutes (Orsetti et al., 2007). Experimental protocol In this study, thirty-six rats were randomly divided into unstressed (n=18) and dissimilarstressed (n=18) groups. Each group was subdivided into control (n=6), indomethacin (n=6) and diclofenac sodium (n=6) groups. Rats in the control group were injected with saline (0.9%), rats in the indomethacin group received a dose of 7.5 mg/kg (Pappius and Wolfe, 1983; Emad et al., 2017), and rats in the diclofenac sodium group received a dose of 5.0 mg/kg (Novaes et al., 2014; Emad et al., 2017) of respective drug. All animals were injected intraperitoneally for seven days. Rats from the dissimilar-stressed group were exposed to repeated dissimilar stress, such as restraint stress, noise stress and shaker stress, after saline

7

and respective drug treatments. The rats from the unstressed group remained in their home cages throughout this period. No stressor was given repetitively for more than two days, and during the 7 days of exposure, each stressor was given two or three times. Twenty-four hours after the last stress session, the following behavioural tests were administered in unstressed and repeatedly dissimilar-stressed rats: the forced swim test (FST) to assess depression, the elevated plus-maze test (EPM) to assess short-term memory and the Morris water maze test (MWM) to assess long-term memory. All tests were conducted in the light period between 9:00 AM to 5:00 PM. Rats were then decapitated using the guillotine method (Emad et al., 2017), and their plasma and brain samples were collected. Their brains were detached from the skull immediately, within 30 s after decapitation. All samples were kept at -70 ᵒC until being investigated for biochemical and neurochemical assays. Measurement of depressive behaviour by forced swim test (FST) The FST was used to monitor the antidepressant effects of the drugs. The test was carried out in a similar way to that explained earlier (Detke et al., 1997). The test involved the scoring of struggling (active) or immobility (passive) behaviours of rats when they were forced to swim in a cylinder filled with water that had no exit (Slattery and Cryan, 2012). Rats were placed separately in a tank (53, 19, 28 cm). The water in the tank was filled to a height of 18 cm. The height of the water was maintained such that the animal was supposed to swim freely. The animal was held in the container with a cut-off time of 5 minutes. The behavioural scoring of the FST was carried out by noting the immobility time of the rats. After completion of the test, the rats were dried completely with a towel and placed in their home cages. Measurement of short-term memory by elevated plus maze test (EPM)

8

The EPM was used to monitor the short-term memory capability of the rats. The apparatus was made of Perspex plastic, with 4 arms in a 50 × 10 cm area. The height of two of the enclosed arms of the EPM was 40 cm. The two open and closed arms of the EPM were joined by a central square (10 × 10 cm), which gave the apparatus an appearance of a plus sign (+). The whole apparatus was elevated 60 cm above the floor. The placement of the apparatus in the laboratory remained the same during the entire experiment, allowing the rats to use extra maze cues to facilitate learning. The method and technique followed was the same as reported earlier (Haider et al., 2011). During the training session, rats were separately positioned at one end of the open arm in such a way that they could not face the central platform. The transfer latency (time taken in seconds for the rat to enter into one end of the closed arms with all four paws) was recorded. The training session during which the rat could explore the maze was limited to 90 seconds. To monitor the memory retention of the rats, a test session was performed after 1 hour. During the test session, the transfer latency was examined. The improved memory function of the rats was based on a significant reduction of the transfer latency in EPM. Measurement of long-term memory by Morris water maze (MWM) The MWM is a well-recognized, conventional cognitive testing apparatus that involves an animal’s use of spatial learning and memory to both find a hidden platform just beneath the surface of a circular pool of water and to memorize the location of the platform from earlier trials (Morris, 1981). The MWM apparatus consisted of a circular pool of water with a diameter of 45 cm, height of 37 cm and water depth of 12 cm. The pool was made with metal that was painted white on the inside surface, and the escape platform was also made of metal with a flat metallic top with a surface diameter of 8 cm, which was 2 cm below the surface of the water during water maze training. The MWM tank was filled with water at a temperature

9

of 23±2°C.The water was made opaque with milk to hide the platform and to permit proficient pathways for rats to swim. The long-term memory of rats was examined in terms of latency to find the hidden platform. On the first day, four training trials were performed to monitor the escape latency. The rats were placed in the tank with a cut-off time of 120 seconds to locate and mount the hidden platform. If the rats were positioned on the platform successfully, then they were allowed to stay there for 10 seconds (Haider et al., 2007).The test session was performed 24 hours after the training session in order to monitor the longterm memory of rats. Estimation of plasma corticosterone levels A fluorimetric assay was used to estimate the concentration of plasma corticosterone, as described previously (Mattingly, 1962). The method of estimation is based on the particular fluorescence of 11-hydroxy-corticoids in concentrated sulfuric acid (conc. H2SO4). Plasma samples were separated from whole blood after centrifugation at 4°C in heparinized centrifuge tubes. The extraction technique for plasma corticosterone was principally the same as reported earlier (Peterson and Pierce, 1960). First, 200 µl of plasma was collected in glass stoppered tubes. Then, 1500 µl of dichloromethane was added, followed by vigorous shaking and centrifugation. The upper layer of the mixture was aspirated and 1 ml aliquots of the dichloromethane layer, which contained steroids, were transferred to another glass tube. To this tube, 1 ml of fluorogenic reagent containing a sulfuric acid-ethanol reagent (7: 3 v/v) was added and mixed using a vortex mixer for 30 seconds. The contents in the tubes were left for 60-90 minutes. The upper organic phase was removed by aspiration and the lower layer of acid-alcohol reagent was transferred to a small cuvette for fluorescence to be read at 460 nm excitation and 570 nm emission wavelengths. Estimation of biogenic amines

10

Reversed-phase high performance liquid chromatography with an electrochemical detector (HPLC-ECD) was used to analyse the levels of biogenic amines (Haider et al., 2015). Brain samples were homogenized in 5-10 volumes of perchloric acid to extract biogenic amines and their metabolites. The mobile phase used for this method was comprised of octyl sodium sulfate (0.023%) in 0.1 M phosphate buffer, with a pH value of 2.9. The solid phase was a 5µ Shim-pack ODS column with an internal diameter of 4.0 mm and a length of 150 mm. The electrochemical detector was operated at a potential of +0.8 V, and the pressure of the pump was 2000-3000 psi. Statistical analysis Results are represented as the mean±SD (n=6 per group). Analyses were performed by twoway analysis of variance (ANOVA) to determine the effects of the various factors involved. Individual comparisons were performed by Tukey’s test and p>0.05 was considered nonsignificant. RESULTS

Effect of NSAIDs on depression following repeated dissimilar stress

Fig.1 shows the effects of NSAIDs on the FST in unstressed and repeatedly dissimilarstressed male rats. Data analysis revealed a significant effect of stress (df=1, 30; F=1109.82; p<0.01), a significant effect of drugs (df=2, 30; F=925.383; p<0.01), and a significant interaction between the two factors (df=1, 30; F=119.41; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly decreased (p<0.01) struggling time in the FST. Administration of indomethacin and diclofenac sodium significantly increased (p<0.01) struggling time in unstressed and repeatedly dissimilar-

11

stressed rats. However, indomethacin and diclofenac sodium significantly decreased struggling time in repeatedly dissimilar-stressed rats compared to respective unstressed rats. Effect of NSAIDs on short-term memory following repeated dissimilar stress

Fig. 2 shows the effect of NSAIDs on the EPM test in unstressed and repeatedly dissimilarstressed male rats. Data analysis revealed a significant effect of stress (df=1, 30; F=101.491; p<0.01), a significant effect of drugs (df=2,30; F=411.631; p<0.01), and a significant interaction between the two factors (df=1,30; F=17.891; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly increased (p<0.01) time taken to enter the closed arm in the EPM test. Administration of indomethacin and diclofenac sodium significantly decreased (p<0.01) time taken to enter the closed arm in unstressed and repeatedly dissimilar-stressed rats. However, indomethacin and diclofenac sodium significantly increased (p<0.01) time taken to enter the closed arm in repeatedly dissimilarstressed rats compared to respective unstressed rats. Effect of NSAIDs on long-term memory following repeated dissimilar stress

Fig.3 shows the effect of NSAIDs on the MWM test in unstressed and repeatedly dissimilarstressed male rats. Data analysis revealed a significant effect of stress (df=1, 30; F=381.88; p<0.01), a significant effect of drugs (df=2,30; F=304.28; p<0.01), and a significant interaction between the two factors (df=1,30; F=4.94; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly increased (p<0.01) time taken to reach the hidden platform in the MWM test. Administration of indomethacin and diclofenac sodium significantly decreased (p<0.01) time taken to reach the hidden platform in unstressed and repeatedly dissimilar-stressed rats. However, indomethacin and diclofenac sodium

12

significantly increased (p<0.01) time taken to reach the hidden platform in repeatedly dissimilar-stressed rats compared to respective unstressed rats. Effect of NSAIDs on plasma corticosterone levels following repeated dissimilar stress Fig.4 shows the effect of NSAIDs on plasma corticosterone levels in unstressed and repeatedly dissimilar-stressed male rats. Data analysis revealed a significant effect of dissimilar stress (df=1, 30; F=1045.95; p<0.01), a significant effect of drugs (df=2, 30; F=880.07; p<0.01), and a significant interaction between the two factors (df=1, 30; F=724.90; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly

increased

(p<0.01)

plasma

corticosterone

levels.

Administration

of

indomethacin and diclofenac sodium significantly decreased (p<0.01) corticosterone levels in dissimilar-stressed rats compared to unstressed rats. However, significantly increased (p<0.05) corticosterone levels were observed by indomethacin in dissimilar-stressed rats compared to respective unstressed rats. Effect of NSAIDs on brain monoamine levels following repeated dissimilar stress

Monoamines and their metabolites were estimated using the HPLC-ECD method. Fig.5a shows the effect of NSAIDs on dopamine (DA) levels in repeatedly dissimilar-stressed male rats. The results of the DA estimation indicated a non-significant effect of repeated dissimilar stress (df=1,30; F=1.222; p>0.05), a significant effect of NSAIDs (df=2,30; F=79.047; p<0.01), and a significant interaction between the two factors (df=2,30; F=15.932; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly decreased (p<0.01) brain DA levels. Administration of indomethacin and diclofenac sodium significantly increased (p<0.01) DA levels in unstressed and repeatedly dissimilar-stressed rats.

13

Fig.5b shows the effect of NSAIDs on DOPAC levels in repeatedly dissimilarstressed male rats. The results indicated a significant effect of repeated dissimilar stress (df=1, 30; F=21.478; p<0.01), a significant effect of NSAIDs (df=2, 30; F=129.438; p<0.01) and a significant interaction between the two factors (df=2, 30; F=13.160; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly increased (p<0.01) DOPAC levels. Administration of indomethacin and diclofenac sodium significantly decreased (p<0.01) DOPAC levels in unstressed and repeatedly dissimilar-stressed rats. Fig.5c shows the effect of NSAIDs on DA turnover in repeatedly dissimilar-stressed male rats. The results indicated a significant effect of repeated dissimilar stress (df=1,30; F=57.970; p<0.01), a significant effect of NSAIDs (df=2,30; F=194.930; p<0.01), and a significant interaction between the two factors (df=2,30; F=50.269; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly increased (p<0.01) DA turnover. Administration of indomethacin and diclofenac sodium significantly decreased (p<0.01) DA turnover in unstressed and repeatedly dissimilar-stressed rats. Fig.6a shows the effect of NSAIDs on serotonin (5-HT) levels in repeatedly dissimilar-stressed male rats. The results indicated a significant effect of repeated dissimilar stress (df=1,30; F=73.142; p<0.01), a significant effect of NSAIDs (df=2,30; F=31.408; p<0.01), and a non-significant interaction between the two factors (df=2,30; F=0.271; p>0.05). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly decreased (p<0.01) brain 5-HT levels. Administration of indomethacin significantly increased (p<0.01) 5-HT levels in unstressed and repeatedly dissimilar-stressed rats. However, indomethacin and diclofenac sodium significantly decreased 5-HT levels in dissimilarstressed rats compared with respective unstressed rats.

14

Fig.6b shows the effect of NSAIDs on 5-HIAA levels in repeatedly dissimilarstressed male rats. The results indicated a significant effect of repeated dissimilar stress (df=1, 30; F=13.628; p<0.01), a significant effect of NSAIDs (df=2, 30; F=125.228; p<0.01), and a significant interaction between the two factors (df=2, 30; F=12.565; p<0.05). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly increased (p<0.01) brain 5-HIAA levels. Administration of indomethacin and diclofenac sodium significantly decreased (p<0.01) 5-HIAA levels in unstressed and repeatedly dissimilarstressed rats. Fig.6c shows the effect of NSAIDs on 5-HT turnover in repeatedly dissimilar-stressed male rats. The results indicated a significant effect of repeated dissimilar stress (df=1,30; F=127.814; p<0.01), a significant effect of NSAIDs (df=2,30; F=188.536; p<0.01), and a significant interaction between the two factors (df=2,30; F=41.514; p<0.01). Post hoc analysis by Tukey’s test showed that repeated dissimilar stress significantly increased (p<0.01) 5-HT turnover. Administration of indomethacin and diclofenac sodium significantly decreased (p<0.01) 5-HT turnover in unstressed and repeatedly dissimilar-stressed rats. However, diclofenac sodium increased 5-HT turnover in repeatedly dissimilar-stressed rats compared with respective unstressed rats. DISCUSSION Stress-related psychiatric illnesses manifest as a dysfunction of the neuroimmune-endocrine system. Inflammation plays an essential function in the progression of stress-induced depression and cognitive decline in humans and animals (Pehrson et al., 2015). Uncontrollable stress situations modulate behavioural, biochemical and neurochemical effects in the brain (Elenkov et al., 1999). The present study showed that after exposure to repeated situations of dissimilar stress, rats displayed behavioural deficits as evidenced by

15

depressive behaviours measured in the FST and memory impairments measured in the EPM and MWM tests. Depression and cognitive decline are significant features of stress-induced neuroinflammation (DeLegge and Smoke, 2008; Allison and Ditor, 2014). In the current study, it was observed that rats exposed to dissimilar stress exhibited decreased struggling in the FST. It is believed that peripheral cytokines may affect various functions of the CNS, as they can penetrate the CNS via transporters into the brain (Yarlagadda et al., 2009). Increased levels of cytokines may exacerbate sickness behaviours (Anisman and Merali, 1999) with concomitant development of symptoms of depression (Dantzer et al., 2008). The depressive behaviour in dissimilar-stressed rats might be due to the stress-provoked release of proinflammatory mediators (Zhang and An, 2007). Inflammation is regarded as one of the important biological factors that lead to the emergence of major depressive disorders. The activation of inflammatory events following physical or psychological stress conditions, along with environmental factors, trauma or injury produce cytokines and other inflammatory mediators, including prostaglandins and nitric oxide, which progressively lead to cognitive impairments as seen in various neurodegenerative diseases (Cunningham et al., 2009; GądekMichalska et al., 2013). Therefore, the depression-like symptoms and concomitant cognitive decline observed in the present study may be attributed to increased inflammatory responses in rats exposed to dissimilar stress situations. Administration of the NSAIDs indomethacin and diclofenac sodium showed positive outcomes in attenuation of depressive behaviour in dissimilar-stressed rats when compared with those of respective controls. Inflammatory mediators, such as PGs produced by cyclooxygenase enzymes, stimulate the HPA axis (Gadek-Michalska et al., 2002). Upon exposure to repeated dissimilar stress situations, inflammatory pathways are over-stimulated; and the continuous release of PGs in turn leads to activation of the HPA axis causing further release of GCs. The continuous release of corticosterone in response to dissimilar stress exerts deleterious effects on the CNS and

16

dampens memory functions. In the present study, transfer latency in EPM and escape latency in MWM increased in rats exposed to dissimilar stress. Dissimilar stress provokes the release of inflammatory mediators (Glaser and Kiecolt-Glaser, 2005), which may activate the HPA axis to release corticosterone resulting in altered memory functions (DeLegge and Smoke, 2008). Administration of indomethacin and diclofenac sodium attenuated both short- and long-term memory impairments in dissimilar-stressed rats compared to respective controls. In the present study, fourfold rise in plasma corticosterone levels was observed in dissimilarstressed rats compared to unstressed controls. It is suggested that over-stimulation of the HPA axis and the subsequent increase in corticosterone levels might be due to cytokine and prostaglandin production leading to depressive behaviour and cognitive deficits. It has also been previously reported that elevated corticosterone levels alter synaptic plasticity and memory functions and decrease neurogenesis (McEwen and Magarinos, 2001). Under stressful situations, PGs induce hyperactivity of the HPA-axis through activation of adrenergic receptors (Gądek-Michalska et al., 2013). Previously, it was observed that administration of indomethacin reduces hyperactivity of the HPA axis in rats (GadekMichalska et al., 2002). It is therefore possible that in the present study, indomethacin and diclofenac sodium may have blocked cyclooxygenase-mediated PG biosynthesis, which may have reduced hyperactivity of the HPA-axis via the adrenergic system and hence normalized the stress response in rats. Pretreatment with NSAIDs could be beneficial in alleviating the symptoms of depression and cognitive deficits in rats exposed to dissimilar stress. In the current study, effects of dissimilar stress on the neurotransmission system were also observed. The dopaminergic and serotonergic systems are implicated in learning and memory functions (Haider et al., 2006). Alterations in dopaminergic and serotonergic neurotransmission are reported in cognitive deficits and depressive behaviour (Graeff et al., 1996; Savitz et al., 2009). In the present study, dissimilar stress affected DA and 5-HT

17

metabolism. Rats exposed to dissimilar stress exhibited increased DA and 5-HT turnover. Previous studies also reported enhanced 5-HT turnover and reduced 5-HT levels in response to variable stress (Drossopoulou et al., 2004). Proinflammatory cytokines also enhance noradrenergic, dopaminergic and serotonergic metabolism in the brain (Wilson et al., 2002). Potential alterations in DA and 5-HT turnover have been observed in both humans and animals when exposed to different stress paradigms (Cox et al., 2011). Studies reveal that cytokines reduce the availability of tryptophan by activating the enzyme indoleamine 2,3dioxygenase (Song et al., 2009). It can be suggested that elevated proinflammatory mediator cytokines released in dissimilar stress-exposed rats may reduce the availability of tryptophan and hence the biosynthesis of 5-HT (Felger and Lotrich, 2013). It was also reported previously that NSAIDs inhibit the activity of indoleamine 2,3-dioxygenase (Dairam et al., 2006). Therefore, the increased 5-HT levels following the administration of indomethacin and diclofenac sodium that were observed in the present study may be due to the inhibition of the indoleamine 2,3-dioxygenase enzyme via increased tryptophan availability. In the current study, it was observed that dissimilar stress situations favour the catabolism of biogenic amines, which reduces the availability of brain monoamines (Ahmad et al., 2010). Indomethacin and diclofenac sodium normalized the HPA axis and serotonergic and dopaminergic activity in rats exposed to dissimilar stress. In conclusion, the present study states that indomethacin and diclofenac sodium improve memory function and concomitantly increase brain 5-HT and DA levels, suggesting that these NSAIDs may be beneficial for patients suffering from stress-associated neurological disorders. Conflicts of interest The authors declare no conflicts of interest. Acknowledgement

18

The authors wish to acknowledge the Higher Education Commission of Pakistan for providing the necessary funds for this study.

REFERENCES

Ahmad A, Rasheed N, Banu N, Palit G (2010) Alterations in monoamine levels and oxidative systems in frontal cortex, striatum, and hippocampus of the rat brain during chronic unpredictable stress. Stress 13:356-365. Allison DJ, Ditor DS (2014) The common inflammatory etiology of depression and cognitive impairment: a therapeutic target. J Neuroinflammation 11:1. Anisman H, Merali Z (1999) Anhedonic and anxiogenic effects of cytokine exposure. In: Cytokines, stress, and depression, pp 199-233: Springer US. Averbeck B, Reeh P (2001) Interactions of inflammatory mediators stimulating release of calcitonin gene-related peptide, substance P and prostaglandin E 2 from isolated rat skin. Neuropharmacol 40:416-423. Baker GB, Reynolds GP (1989) Biogenic amines and their metabolites in Alzheimer's disease: noradrenaline, 5-hydroxytryptamine and 5-hydroxyindole-3-acetic acid depleted in hippocampus but not in substantia innominata. Neurosci Lett 100:335339. Calabrese F, Molteni R, Racagni G, Riva MA (2009) Neuronal plasticity: a link between stress and mood disorders. Psychoneuroendocrinology 34:S208-S216. Charmandari E, Tsigos C, Chrousos G (2005) Endocrinology of the stress response. Annu Rev Physiol 67:259-284. Chrousos GP (2009) Stress and disorders of the stress system. Nat Rev Endocrinol 5:374381.

19

Cox BM, Alsawah F, McNeill PC, Galloway MP, Perrine SA (2011) Neurochemical, hormonal, and behavioral effects of chronic unpredictable stress in the rat. Behav Brain Res 220:106-111. Cunningham C, Campion S, Lunnon K, Murray CL, Woods JF, Deacon RM, Rawlins JNP, Perry VH (2009) Systemic inflammation induces acute behavioral and cognitive changes and accelerates neurodegenerative disease. Biol Psychiatry 65:304-312. Dairam A, Antunes EM, Saravanan K, Daya S (2006) Non-steroidal anti-inflammatory agents, tolmetin and sulindac, inhibit liver tryptophan 2, 3-dioxygenase activity and alter brain neurotransmitter levels. Life Sci 79:2269-2274. Dantzer R, O'Connor JC, Freund GG, Johnson RW, Kelley KW (2008) From inflammation to sickness and depression: when the immune system subjugates the brain. Nature Rev Neurosci 9:46-56. De Kloet ER, Joels M, Holsboer F (2005) Stress and the brain: from adaptation to disease. Nature Rev Neurosci 6:463-475. Delaney G, Dawe K, Hogan R, Hunjan T, Roper J, Hazell G, Lolait S, Fulford A (2012) Role of nociceptin/orphanin FQ and NOP receptors in the response to acute and repeated restraint stress in rats. J Neuroendocrinol 24:1527-1541. DeLegge MH, Smoke A (2008) Neurodegeneration and inflammation. Nutr Clin Pract 23:35-41. Denes A, Thornton P, Rothwell N, Allan S (2010) Inflammation and brain injury: acute cerebral ischaemia, peripheral and central inflammation. Brain Behav Immun 24:708723. Detke MJ, Johnson J, Lucki I (1997) Acute and chronic antidepressant drug treatment in the rat forced swimming test model of depression. Exp Clin Psychopharm 5:107.

20

Drossopoulou G, Antoniou K, Kitraki E, Papathanasiou G, Papalexi E, Dalla C, Papadopoulou-Daifoti Z (2004) Sex differences in behavioral, neurochemical and neuroendocrine effects induced by the forced swim test in rats. Neuroscience 126:849-857. Elenkov IJ, Webster EL, Torpy DJ, Chrousos GP (1999) Stress, corticotropin‐releasing hormone, glucocorticoids, and the immune/inflammatory response: acute and chronic effects. Ann N Y Acad Sci 876:1-13. Emad S, Qadeer S, Sadaf S, Batool Z, Haider S, Perveen T (2017) Attenuation of stress induced memory deficits by nonsteroidal anti-inflammatory drugs (NSAIDs) in rats: Role of antioxidant enzymes. Pharmacol Rep 69:300-305. Felger JC, Lotrich FE (2013) Inflammatory cytokines in depression: neurobiological mechanisms and therapeutic implications. Neuroscience 246:199-229. Gadek-Michalska A, Bugajski J, Bugajski A, Glod R (2002) Effect of adrenergic antagonists and cyclooxygenase inhibitors on the nicotine-induced hypothalamic-pituitaryadrenocorticol activity. J Physiol Pharmacol 53:275-288. Gadek-Michalska A, Tadeusz J, Rachwalska P, Bugajski J (2013) Cytokines, prostaglandins and nitric oxide in the regulation of stress-response systems. Pharmacol Rep 65:16551662. Glaser R, Kiecolt-Glaser JK (2005) Stress-induced immune dysfunction: implications for health. Nat Rev Immunol 5:243-251. Gouirand AM, Matuszewich L (2005) The effects of chronic unpredictable stress on male rats in the water maze. Physiol Behav 86:21-31. Graeff FG, Guimarães FS, De Andrade TG, Deakin JF (1996) Role of 5-HT in stress, anxiety, and depression. Pharmacol Biochem Behav 54:129-141.

21

Guo J-Y, Li C-Y, Ruan Y-P, Sun M, Qi X-L, Zhao B-S, Luo F (2009) Chronic treatment with celecoxib reverses chronic unpredictable stress-induced depressive-like behavior via reducing cyclooxygenase-2 expression in rat brain. Eur J Pharmacol 612:54-60. Haider S, Khaliq S, Haleem DJ (2007) Enhanced serotonergic neurotransmission in the hippocampus following tryptophan administration improves learning acquisition and memory consolidation in rats. Pharmacol Rep 59:53. Haider S, Khaliq S, Ahmed S, Haleem D (2006) Long-term tryptophan administration enhances cognitive performance and increases 5HT metabolism in the hippocampus of female rats. Amino Acids 31:421-425. Haider S, Batool Z, Tabassum S, Perveen T, Saleem S, Naqvi F, Javed H, Haleem DJ (2011) Effects of walnuts (Juglans regia) on learning and memory functions. Plant Foods Hum Nutr 66:335-340. Kim JJ, Diamond DM (2002) The stressed hippocampus, synaptic plasticity and lost memories. Nature Rev Neurosci 3:453-462. Leonard BE (2010) The concept of depression as a dysfunction of the immune system. In: Depression: From Psychopathology to Pharmacotherapy, pp 53-71: Karger Publishers. Mattingly D (1962) A simple fluorimetric method for the estimation of free 11 hydroxycorticoids in human plasma. J Clin Pathol 15:374-379. McEwen BS (1998) Stress, adaptation, and disease: Allostasis and allostatic load. Ann N Y Acad Sci 840:33-44. McEwen BS (2004) Protection and damage from acute and chronic stress: allostasis and allostatic overload and relevance to the pathophysiology of psychiatric disorders. Ann N Y Acad Sci 1032:1-7.

22

McEwen BS, Magarinos AM (2001) Stress and hippocampal plasticity: implications for the pathophysiology of affective disorders. Hum Psychopharmacol Clin Exp 16:S7-S19. Mercier S, Buguet A, Cespuglio R, Martin S, Bourdon L (2003) Behavioural changes after an acute stress: stressor and test types influences. Behav Brain Res 139:167-175. Monteiro S, Roque S, de Sa-Calcada D, Sousa N, Correia-Neves M, Cerqueira JJ (2015) An efficient chronic unpredictable stress protocol to induce stress-related responses in C57BL/6 mice. Front Psychiatry 6:6. Morris RG (1981) Spatial localization does not require the presence of local cues. Learn Motiv 12:239-260. Naqvi F, Haider S, Perveen T, Haleem DJ (2012) Sub-chronic exposure to noise affects locomotor activity and produces anxiogenic and depressive like behavior in rats. Pharmacol Rep 64:64-69. Novaes APR, Desidera AC, Nascimento GC, Leite-Panissi CR (2014) Effects of Sodium Diclofenac on the Distribution of Fos Protein in Central Amygdala and Lateral Hypothalamus during Experimental Tooth Movement in Rats. World J Neurosci 4:183. Orsetti M, Canonico PL, Dellarole A, Colella L, Di Brisco F, Ghi P (2007) Quetiapine prevents anhedonia induced by acute or chronic stress. Neuropsychopharmacology 32:1783-1790. Pappius HM, Wolfe LS (1983) Effects of indomethacin and ibuprofen on cerebral metabolism and blood flow in traumatized brain. J Cereb Blood Flow Metab 3:448459. Pehrson AL, Leiser SC, Gulinello M, Dale E, Li Y, Waller JA, Sanchez C (2015) Treatment of cognitive dysfunction in major depressive disorder—a review of the preclinical evidence for efficacy of selective serotonin reuptake inhibitors, serotonin–

23

norepinephrine reuptake inhibitors and the multimodal-acting antidepressant vortioxetine. Eur J Pharmacol 753:19-31. Peterson RE, Pierce CE (1960) The metabolism of corticosterone in man. Eur J Clin Invest 39:741. Reiche EMV, Nunes SOV, Morimoto HK (2004) Stress, depression, the immune system, and cancer. Lancet Oncol 5:617-625. Reiche EMV, Morimoto HK, Nunes SMV (2005) Stress and depression-induced immune dysfunction: implications for the development and progression of cancer. Int Rev Psychiatry 17:515-527. Saleem S, Tabassum S, Ahmed S, Perveen T, Haider S (2014) Senescence related alteration in hippocampal biogenic amines produces neuropsychological deficits in rats. Pak J Pharm Sci 27:837-845. Sapolsky RM, Romero LM, Munck AU (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55-89. Savitz J, Lucki I, Drevets WC (2009) 5-HT 1A receptor function in major depressive disorder. Prog Neurobiol 88:17-31. Slattery DA, Cryan JF (2012) Using the rat forced swim test to assess antidepressant-like activity in rodents. Nat Protoc 7:1009-1014. Smith SM, Vale WW (2006) The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress. Dialogues Clin Neurosci 8:383. Song C, Zhang XY, Manku M (2009) Increased phospholipase A2 activity and inflammatory response but decreased nerve growth factor expression in the olfactory bulbectomized rat model of depression: effects of chronic ethyl-eicosapentaenoate treatment. J Neurosci 29:14-22.

24

Sorrells SF, Caso JR, Munhoz

CD, Sapolsky RM (2009) The stressed CNS: when

glucocorticoids aggravate inflammation. Neuron 64:33-39. Sorrells SF, Munhoz CD, Manley NC, Yen S, Sapolsky RM (2014) Glucocorticoids increase excitotoxic injury and inflammation in the hippocampus of adult male rats. Neuroendocrinology 100:129-140. Watanabe T, Morimoto A, Morimoto K, Nakamori T, Murakami N (1991) ACTH release induced in rats by noradrenaline is mediated by prostaglandin E2. J Physiol 443:431. Wilson CJ, Finch CE, Cohen HJ (2002) Cytokines and cognition—the case for a head‐to‐toe inflammatory paradigm. J Am Geriatr Soc 50:2041-2056. Wojdasiewicz P, Poniatowski LA, Szukiewicz D (2014) The role of inflammatory and antiinflammatory cytokines in the pathogenesis of osteoarthritis. Mediators Inflamm 2014:561459. Yarlagadda A, Alfson E, Clayton AH (2009) The blood brain barrier and the role of cytokines in neuropsychiatry. Psychiatry (1550-5952) 6. Zhang J-M, An J (2007) Cytokines, inflammation and pain. Int Anesthesiol Clin 45:27.

25

Figure Legends Fig. 1 Effect of NSAIDs on forced swim test in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective saline-treated controls, ++p<0.01 from the respective unrestrained controls Fig. 2. Effect of NSAIDs on elevated plus maze test in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective salinetreated controls, ++p<0.01 from the respective unrestrained controls. Fig. 3. Effect of NSAIDs on Morris water maze test in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective salinetreated controls, ++p<0.01 from the respective unrestrained controls. Fig. 4. Effect of NSAIDs on plasma corticosterone levels in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 and *p<0.05 from the respective saline-treated controls, ++p<0.01,+p<0.05 from the respective unrestrained controls. Fig. 5a. Effect of NSAIDs on DA levels in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective saline-treated controls, ++p<0.01 from the respective unrestrained controls. Fig. 5b. Effect of NSAIDs on DOPAC levels in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective saline-treated controls, ++p<0.01 from the respective unrestrained controls.

26

Fig. 5c. Effect of NSAIDs on DA turnover in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective saline-treated controls, ++p<0.01 from the respective unrestrained controls. Fig. 6a. Effect of NSAIDs on 5-HT levels in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective saline-treated controls, ++p<0.01 from the respective unrestrained controls. Fig. 6b. Effect of NSAIDs on 5-HIAA levels in dissimilar-stressed rats. Values are mean±SD (n=6).Significant differences by Tukey’s test **p<0.01 from the respective saline-treated controls, ++p<0.01 from the respective unrestrained controls. Fig. 6c. Effect of NSAIDs on 5-HT turnover in dissimilar-stressed rats. Values are mean±SD (n=6). Significant differences by Tukey’s test **p<0.01 from the respective saline-treated controls, ++p<0.01 and +p<0.05 from the respective unrestrained controls.

27

Highlights •

Depressive behaviour and memory impairment were observed following exposure to dissimilar stress in rats.

•

Non-steroidal anti-inflammatory drugs attenuated the stress-induced behavioural deficits in rats.

•

Reduction in neuroinflammation by NSAIDs could normalize the activity of HPA axis and subsequent release of corticosterone.

•

Improvement in behavioural deficits by NSAIDs could be due to increased neurotransmitter levels in dissimilar stressed rats.

•

Both indomethacin and diclofenac sodium may play significant role in neurodegenerative diseases

28

29

Forced swim test

Saline Indomethacin Diclofenac sodium

Fig. 1

350

Struggling time (sec)

300

** **

**++

250

**++ 200

++ 150 100 50 0

Unstressed

Dissimilar stressed

30

Elevated plus maze

Saline Indomethacin Diclofenac Sodium

Fig. 2

Transfer latency (sec)

80

++

70

**++

60 50

**++

40 30

**

20 10

**

0

Unstressed

Dissimilar stressed

31

Morris water maze

Saline Indomethacin Diclofenac Sodium

Fig. 3

100

++

Escape latency (sec)

90 80

**++

70 60 50

**

40 30

**++

**

20 10 0

Unstressed

Dissimilar stressed

c

32

Plasma corticosterone level 450

Concentration (µg/ml)

400 350

Saline Indomethacin Diclofenac Sodium

Fig. 4 ++

300 250 200 150

*

**+ **

100 50 0

Unstressed

Dissimilar stressed

33

Brain DA levels 160

Concentration (ng/g)

140 120

Fig. 5a

Saline Indomethacin Diclofenac sodium

**

** **

100

**

80 60

++ 40 20 0

Unstressed

Dissimilar stressed

34

Brain DOPAC levels 450

Concentration(ng/g)

400 350

Fig. 5b

saline indomethacin Diclofenac sodium

++

300 250 200 150

**

**

** **

100 50 0

Unstressed

Dissimilar stressed

35

Brain DA turnover 12

DOPAC/DA ratio

10

Fig. 5c

Saline Indomethacin Diclofenac sodium

++

8

6

4

2

** **

** **

0

Unstressed

Dissimilar stressed

36

Brain 5-HT levels 400

Fig. 6a Saline Indomethacin Diclofenac sodium

**

Concentration (ng/g)

350 300

++

250

**++

200

++

150 100 50 0

Unstressed

Dissimilar stressed

37

Brain 5-HIAA levels 350

Concentration (ng/g)

300

Fig. 6b

Saline Indomethacin Diclofenac sodium

++

250 200

** 150

**

** **

100 50 0

Unstressed

Dissimilar stressed

38

Brain 5-HT turnover

5-HIAA/5-HT ratio

2.5

2

Fig. 6c

Saline Indomethacin Diclofenac sodium

++

1.5

**+

1

** 0.5

**

**

Unstressed

Dissimilar stressed

0

39