Restraint stress exacerbates apoptosis in a 6-OHDA animal model of Parkinson disease

Restraint stress exacerbates apoptosis in a 6-OHDA animal model of Parkinson disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Restraint stress exacerbates apoptosis in a 6-OHDA animal model of Parkinson disease Sara El Idrissi, Nada Fath, Hind Ibork, Khalid Taghzouti, Meryem Alamy, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2196389/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jan, 2023 Read the published version in Neurotoxicity Research → Version 1 posted 7 You are reading this latest preprint version Abstract Activation of the apoptotic pathway has been associated with promoting neuronal cell death in the pathophysiology of Parkinson disease (PD). Nonetheless, the mechanisms by which it may occurs remain unclear. It has been suggested that stress-induced oxidation and potentially apoptosis may play a major role in the progression of PD. Thus, in this study, we aimed to investigate the effect of subchronic restraint stress on striatal dopaminergic activity, iron, p53, caspase-3, and plasmatic acetylcholinesterase (AChE) levels in an animal model of PD induced by administration of 6-hydroxydopamine(6-OHDA) in the medial forebrain bundle (MFB). The obtained results showed that restraint stress exacerbates motor coordination deficits and anxiety in animals treated with 6-OHDA in comparison to animals receiving saline, and it had no effect on object recognition memory. On another hand, 6-OHDA decreased dopamine(DA) levels, increased iron accumulation, and induced overexpression of the pro-apoptotic factors caspase-3, p53, and AChE. More interestingly, post-lesion restraint stress exacerbated the expression of caspase-3 and AChE without affecting p53 expression. These findings suggest that subchronic stress may accentuate apoptosis and may contribute to DA neuronal loss in the striatal regions and possibly exacerbate the progression of PD. Parkinson disease restraint stress medial forebrain bundle dopamine apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction PD is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) [1, 2]. PD is the second most common neurodegenerative disorder with a prevalence of 2–3% within the population above 65 years of age [3]. Several studies have shown males are more susceptible to PD, with a male-female ratio that varies between 1.37 to 3.7 [4, 5]. PD presents a serious burden on the patient and caregivers [6]. Yet, its aetiology is not completely understood. PD is characterized by several motor symptoms such as akinesia, bradykinesia, tremor and rigidity. Additionally, PD patients show other motor deficits, including gait disturbance, impaired handwriting, posture instability, grip force and speech deficits (Kouli et al., 2018, Balestrino and Schapira, 2020). The neuropathological hallmark of PD includes neurodegeneration, neuro-inflammation, altered dopaminergic activity and increased oxidative stress in the nigrostriatal pathway [7, 8]. Many risk factors were proposed to be involved in the pathogenesis and the progression of PD including environmental stress-induced increased cortisol levels [9]. It has been shown that acute and chronic stress affects the dopaminergic nigrostriatal pathway alongside dopaminergic neurons in the mesocorticolimbic pathways, leading to impaired locomotor activity, suggesting an interaction between stress and the onset of the motor symptoms of PD [10–12]. Furthermore, motor deficits associated with PD, on their own, may present a stressful event that contributes to the aggravation of PD's neuropathology [13]. Clinical studies have shown that stress-increased cortisol is associated with the worst functional scores for motor, and cognitive symptoms of PD [14]. Several animal models were developed to understand the physiopathology of PD. Among these models, the 6-OHDA animal model [15]. It was demonstrated that unilateral injection of 6-OHDA into the substantia nigra pars compacta (SNpc), leads to a rapid onset of neuronal loss [16]. Whereas, injecting 6-OHDA in the medial forebrain bundle (MFB) induces the lesion of the nigrostriatal pathway within 3–5 weeks resulting in the initiation of a deleterious cascade of events leading to neuronal loss [17–19]. It has been shown that 6-OHDA neuronal loss is mediated, in at least one part, by oxidative stress resulting from increased iron release from Fe-S cluster proteins of the mitochondrial respiratory chain and from other iron-storing cell compartments which subsequently exacerbate reactive oxygen species (ROS) production via the Fenton’s reaction [20, 21]. Iron is a transition metal, mandatory for the normal functioning of cells and the metabolism of neurotransmitters including DA, but when iron exceeds the homeostatic levels, it becomes toxic, leading to oxidative damage and eventually cell death [22]. The deleterious effects of 6-OHDA can be aggravated by stress which was suggested to play a role in the onset of PD [23, 24]. It has been shown that chronic restraint stress triggers dopaminergic and noradrenergic neurodegeneration via apoptosis by activating caspase enzymes, priory expressed as inactive precursors lacking protease activity [24, 25]. Nevertheless, apoptosis, which is a mode of programmed cell death (PCD), crucial for mammalian development as it controls cell numbers, tissue development, and clears damaged structures [26, 27]. The apoptotic processes depend on multiple factors such as the activation of the p53 transcription factor, known as tumour protein 53, and considered one of the primary measures in the evaluation of neural responses to stressful events [8, 28] as it controls DNA damage, senescence, ROS formation, as well as activating pro-apoptotic and suppressing anti-apoptotic proteins [8, 29]. Apoptosis also depends on caspase 3 expression and activity. Additionally, Caspase-3 has been identified as a key mediator of apoptosis and plays an important role in PD pathogenesis [30]. Several studies highlighted a positive correlation between the degree of neurodegeneration of dopaminergic neurons and the levels of caspase-3 expression, indicating that caspase-3 is a vulnerability factor and final effector in the apoptotic death of dopaminergic neurons in PD [31]. Interestingly, it has been shown that caspase-3 may induce DNA fragmentation and protein cleavage, including the Parkin protein implicated in the biochemical pathways underlying the aetiology of sporadic PD [32–34]. Apoptosis also depends on AChE integrity as it plays a major role in the termination of signal transmission in the cholinergic system and is implicated in the pathogenesis of neurodegenerative diseases by affecting inflammatory responses, oxidative stress, and the aggregation of pathological proteins [35, 36]. It has been shown that decreased expression of AChE reduces apoptotic markers in different cell lines whereas augmented AChE activity increases apoptosis [37]. Based on these studies, we hypothesised that restraint stress may aggravate the degeneration of the nigrostriatal pathway in the 6-OHDA PD animal model by inducing oxidative stress, through induction of ROS release and iron build-up, and/or by upregulating apoptotic factors such as caspase 3, p53 and AChE. To our best knowledge, no study investigated the expression of iron, p53, caspase-3, and AChE in animal models of PD exposed to stress. Therefore, we aimed to investigate the behavioural impairments and related neurochemical changes following post-lesion restraint stress in adult male rats injected with 6-OHDA in the MFB. 2. Methodology 2.1. Animals Thirty-two male Wistar rats (7 to 8 weeks old) obtained from the animal facility of the Faculty of Sciences at Mohammed V University in Morocco were used in this randomised blinded study. The animals were housed under standard laboratory conditions; a 12-hour light/dark cycle with lights on at 6 am and they had free access to standard chow and tap water. The animals were divided into four groups; (1) A control group received an injection of vehicle (saline solution containing 0.2% ascorbic acid) into the left MFB; (2) 6-OHDA group; (3) vehicle + stress group, injected with vehicle and subjected to 7 days’ restraint stress and the (4) 6-OHDA + stress group. Restraint stress was applied one week following 6-OHDA or vehicle administration into the MFB. The experimental paradigm was summarised in Fig. 1 . All experiments were conducted in compliance with the ARRIVE guidelines and they were approved by the Animal Ethics Sub-committee of Mohammed V University. 2.2. Stereotaxic surgery and restraint stress On PND 70–72, a solution of 6-OHDA was freshly prepared, kept on ice, and protected from light exposure with tin foil. The solution consisted of 5 µg 6-OHDA hydrobromide (Sigma-Aldrich) dissolved in 4 µl of sterile saline containing 0.2% ascorbic acid. The surgical site and instruments were sterilised before surgery. Rats (240–310 g), were injected intraperitoneally (i.p) with desipramine at 15 mg/kg (Sigma-Aldrich) 30 minutes before the injection of 6-OHDA to prevent the degeneration of the noradrenergic neurons. Prior to the stereotaxic surgery, the rats were anaesthetized with sodium pentobarbital (60 mg/kg, i,p). To avoid the confounding effect of direct mechanical damage to the striatum or substantia nigra [11], 6-OHDA or vehicle were injected into the left MFB at a rate of 1 µl per 2 minutes using a Hamilton syringe. The coordinates of injection were 4.7 mm anterior to the interaural line, 1.7 mm lateral to the midline and 8.6 mm ventral to the dura based on the Stereotaxic Atlas of Rat Brain by Paxinos and Watson [11, 38]. The rate of infusion was set at 1µl / 2 minutes. To facilitate optimal diffusion of the solution, the needle was left in place for an additional 5 minutes following the infusion of 6-OHDA. Subsequently, the needle was slowly retracted from the brain and the incision was sutured using a clinisilk suture gauge 5/0 needle. Following surgery, the rats were placed on a heating pad for 30 minutes to prevent hypothermia and then the animals were returned to their home cages for recovery. Six days following the surgery, rats in the two stressed groups were moved to an isolated behavioural room away from the non-stressed animals and placed in rodent restrainers (114 mm long x 28 mm diameter) for 3 hours per day from 9h00 to 12h00 for 7 consecutive days. At the end of each session, the animals were taken back to their home cages. 2.3. Open field test The open field test was conducted in a rectangular box (100 cm length x 100 cm width x 40 cm height) made of plexiglass. The floor of the open field apparatus was virtually divided into 25 equal squares, brightly illuminated by a white light lamp (100 W) and suspended 2 meters above the open field arenas. Each animal was placed in the bottom right corner of the apparatus and video recorded for 5 min. For each rat, the total distance travelled, the average speed (as an indicator of locomotor activity), the number of entries to the central zone and the time spent in the central zone (as an indicator of anxiety-like behaviour) were measured using Anymaze software (Stoelting. Co). The apparatus was cleaned with 70% alcohol after each trial. 2.4. Novel object recognition test The novel object recognition test was conducted in the same open-field apparatus. This task evaluates the ability of the animal to discriminate between novel and familiar objects. It relies on the rat's innate preference for novelty, if the rat recognizes a familiar object; it will spend most of its time at the novel object. The duration of the test is 3 days; during the first day, the animals are familiarised with the apparatus for 5 min; on the 2nd day or the habituation phase, two identical objects are presented to the animal and placed 15 cm from the sidewall in diagonal corners opposite each other. Each rat was allowed to explore the objects for 5 min; on the 3rd day (test day) one of the training objects is replaced with a novel object. The exploration index was calculated during the habituation phase as a ratio of the total time spent by the animal exploring the objects and the trial duration (5 min), while the discrimination index (DI) was calculated during the test day as fellow; DI =[(time spent exploring the novel object - time spent exploring the familiar object)/total exploration time] [39]. 2.5. The beam-walking test The beam-walking test consisted of a training phase and a test session. During the training phase, the rats were subjected to three trials during which they were encouraged to cross a wooden beam (2 cm in diameter,100 cm in length and elevated 70 cm above the floor) to reach a black box placed on the other extremity of the beam. 24 hours later animals were tested on the same beam. gait and time to cross the beam were counted as indicators of motor coordination [40, 41]. 2.6. Rotarod test The apparatus consisted of 7-cm-diameter plastic drums machined with grooves to improve grip (Panlab Harvard Apparatus, Barcelona, Spain). It could be set on accelerating speed (4, 10, 12, 15, 19, 22, 26, 29 34 and 40 rpm, 30 s at each speed). Before testing, the rats were trained for 2 days. During the first day, rats were trained for 3 min with an unlimited number of trials on the rotarod. Followed by four trials of a maximum of 60 s with 30 s intervals. On the second day, rats were placed on the rotarod at accelerating speed for a maximum of 300 s. on the testing day, each rat was individually placed on the rotarod at accelerating speed for a maximum of 300 s, and the latency to fall off the rotarod and the maximum speed reached within this time were recorded. Immediately after each session, the apparatus was thoroughly cleaned with cotton pads wetted with 70% ethanol; water solution and dried. Rats were allowed to habituate to the experimental room for 60 min before both training and testing. Training and testing were performed between 10:00 AM and 1:00 PM [42]. 2.7. Samples preparation 24 hours after that last behavioural test, the animals were decapitated using a guillotine, the striatal brain structures were dissected out using plastic forceps (Sigma-Aldrich, USA) to minimize trace metal ion contamination, washed with phosphate buffer solution (1 x PBS, pH = 7.4), weighted and stored at -80°C until the day of analysis. Trunk blood was collected in heparinized tubes and centrifuged at 3000 g for 10 minutes. The plasma was separated in the supernatant and kept at − 80°C until further use. 2.8. Striatal Dopamine and DOPAC determination A high-performance liquid chromatography (HPLC) system (Agilent technologies HP 1100 series) with electrochemical detection (Agilent Hewlett Packard 1049A) set at 750 mV was used to analyse DA and its metabolites 3,4-Dihydroxyphenylacetic acid (DOPAC). The striatal homogenate was thawed and centrifuged at 4°C for 2 min at 12,000 g. Ten microliters of each supernatant per sample were injected onto a LiChrospher 100 cartridge column, RP-18,5 µm, 3 × 125 mm (Agilent) maintained at 4°C. The mobile phase consisted of 0.1 M Na2HPO4 (pH 3.3), 0.15 mM EDTA and 25% methanol. The flow rate was 0.8 ml/min. The analytes (DA and DOPAC) were identified by their retention times compared with their corresponding standards. Their concentration was estimated by comparison of the area under the curve using the straight-line equation y = mx + c and presented as ng/mg protein [43]. 2.9. Determination of striatal iron levels For iron content analysis, Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to measure iron levels in the striatum following the procedure previously explained by [44]. Briefly, a mixture of 0.5 ml of concentrated hydrochloric acid (HCL) 2 N, and 200 mg of striatal sample was sonicated and homogenized utilizing a Misonix Sonicator XL2000-010 (Newtown CT, USA) until a homogenate was acquired. 70% perchloric acid (0.1 ml) was added to treat the samples following which the samples were incubated in a water bath at 50°C for 24–36 hours. The samples were centrifuged at 600 g for 1 h and subsequently filter-syringed through a 0.45 µm pore filter. Standard iron solution (50 mg/l) was diluted with nitric 70% perchloric acid 100, 50, 25 and 12.5 times and used to draw the standard curved line. To analyze the standards and samples, Perkin Elmer Optima 5300 DV Optimal Emission Spectrometer (Waltham MA, USA) was used at a detection wavelength of 259.94 nm. 2.10. Assay protocol for Caspase3, AChE, and p53 The levels of caspase-3, p53, and AChE were quantified using commercially available sandwich-ELISA assay kits (Elabscience, USA). The striatal tissue was sonicated and homogenized in a buffered solution containing 400 mM NaCl, 2.0 mM EDTA, 2.0 mM benzamidine, 0.1% Triton-X, 0.5% BSA, 0.1 mM PMSF, Aprotinin (9.7 TIU/ml), 0.1 mM benzethonium chloride and 0.1 M phosphate buffer (pH = 7.4). All procedures were accomplished according to the instructions of the manufacturer. The sensitivity of the kits was reported as 46.875pg/mL for p53, 0.188ng/mL for caspase-3 and 0.47ng/mL for AChE. The coefficient of variation for the assays was < 10%. Recombinant preparations were used for the establishment of the standard curves for p53, caspase-3, and AChE analysis. 2.11. Statistical analysis We tested normality using the Shapiro-Wilk test. Data that assumed gaussian distribution were analyzed using a two-way analysis of variance (two-way ANOVA) followed by Bonferroni’s post hoc test. All statistical analyses were performed using the GraphPad Prism software (Version 8.0). Data were expressed as mean ± SEM and significance were set at p < 0.05. 3. Results 3.1. 6-OHDA and restraint stress evoke anxiety-like behaviour To measure locomotor activity and anxiety-like behaviour, we used the open-field test. The obtained results revealed a significant effect of 6-OHDA on locomotor activity expressed as a significant decrease in the total distance travelled (F (1,28) = 14.22, p = 0.0008, Fig. 2 A), and decreased average speed (F (1, 28) = 6.001, p = 0.0208, Fig. 2 B) as has been revealed by the two-way ANOVA analysis. 6-OHDA induced an anxiogenic effect expressed by a decreased number of entries (F (1, 28) = 4.411, p = 0.0448) (Fig. 2 C), and reduced time spent in the central zone of the open field (F (1,28) = 7.171, p = 0.0123, Fig. 2 D). However, restraint stress had no effect on the total distance travelled (F (1, 28) = 0.03434, p = 0.8543, Fig. 2 A) neither on the average speed (F (1, 28) = 1.149, p = 0.2930, Fig. 2 B). Furthermore, restraint stress induced anxiety as it significantly decreased the time spent in the central zone (F (1, 28) = 9.534; P = 0. 0045, Fig. 2 D) without affecting the numbers of entries to the central zone ( F (1, 28) = 2.810, P = 0.1048; Fig. 2 C). Moreover, data analysis revealed no significant interaction between 6-OHDA injection and restraint stress in all open field measured parameters; neither on the total distance travelled (F (1, 28) = 1.413; P = 0.2445), the average speed (F (1, 28 ) = 0.7091, p = 0. 4069); the number of entries to the central zone (F (1, 28) = 0.5082, p = 0.4818), or the time spent in the central zone (F (1, 28) = 0.1792, p = 0.6753,Fig. 2 ). 3.2. 6-OHDA and restraint stress did not affect the episodic-like memory To evaluate the effect of 6-OHDA injection and exposure to restraint stress on episodic memory we used the novel object recognition test. Data analysis using two-way ANOVA test indicated that 6-OHDA treatment had no effect on the index of exploration (F (1, 28) = 2.531, p = 0. 1228, Fig. 3 A) neither on the index of discrimination (F (1, 28) = 1.341, p = 0.2566, Fig. 3 B). Similarly, restraint stress had no effect on the index of exploration (F (1, 28) = 0.09427, p = 0.7611) neither on the index of discrimination (F (1, 28) = 1.470, p = 0.2355). 3.3. 6-OHDA alone or combined with restraint stress-induced gait impairments The hind limb implication in the gait impairment was analysed using the beam-walking test. Our obtained results showed a main effect of the 6-OHDA on gait-related behaviour as expressed by increased latency to start crossing (F (1, 28) = 54.91, p < 0.0001, Fig. 4 A), and increased time spent to cross the beam (F (1, 28) = 16.41, p = 0.0004, Fig. 4 B). However, restraint stress alone had no significant effect on gait-related behaviour, neither on the latency to start crossing (F (1, 28) = 1.257, p = 0.2717, Fig. 4 A) or the total time spent to cross the beam (F (1, 28) = 0.3570, p = 0.5550, 4B). Moreover, two-way ANOVA indicated no interaction between 6-OHDA and restraint stress when analysing the latency to start crossing (F (1, 28) = 1.257, p = 0.2717; Fig. 4 A) but it revealed a significant interaction between 6-OHDA and restraint stress when analysing the total time spent crossing the beam (F (1, 28) = 5.673, p = 0.0243, Fig. 4 B). Bonferroni’s posthoc comparisons test showed that 6-OHDA combined with exposure to restraint stress increased significantly the total time spent crossing the beam compared to saline/no stress group (p = 0.0164), and saline/restraint stress group (p = 0.0006) (Fig. 4 B). 3.4. 6-OHDA impaired motor coordination The impact of 6-OHDA and stress exposure on motor coordination and balance was evaluated by the rotarod test. Our results revealed a significant effect of 6-OHDA (F (1, 28) = 16. 62, p = 0. 0003), no effect of restraint stress (F (1, 28) = 1.975, p = 0. 1709) and no interaction between these two parameters on the latency to fall (F (1, 28) = 0.7761, p = 0.3858, Fig. 5 ). 3.5. 6-OHDA evoked DA depletion in the striatum To investigate the effect of 6-OHDA injection and restraint stress on the nigrostriatal pathway, we measured the levels of DA and DOPAC as well as DA turnover in the striatum. Two-way ANOVA analysis revealed that 6-OHDA decreased significantly DA (F (1, 28) = 59.49, p < 0.0001, Fig. 6 A), and DOPAC (F (1, 28) = 44.73, p < 0.0001, Fig. 6 B) and had no effect on DA turnover (F (1, 28) = 2.344, p = 0.1370, Fig. 6 C). Whereas, restraint stress had no effect on DA (F (1, 28) = 3.875, p = 0.0590, Fig. 6 A), neither on DOPAC (F (1. 28) = 0 03673, p = 0.8494, Fig. 6 B) or DA turnover (F (1, 28) = 0.1287, p = 0.7225, Fig. 6 C). Additionally data analysis revealed no significant interaction between 6-OHDA and restraint stress when analysing DA, DOPAC or DA turnover levels in the striatum (F (1, 28) = 0.1220, p = 0.7295; F (1, 28) = 1.250, p = 0.2730; F (1, 28) = 0.2165, p = 0.6453; respectively) (Fig. 6 ). 3.6. 6-OHDA increased iron build-up in the striatum It was reported that iron may play an important role in the pathophysiology of PD. Thus, we investigated iron levels in the striatal structures following 6-OHDA and stress exposure. Both, 6-OHDA and restraint stress had a significant effect on striatal iron levels (F (1, 28) = 44.76, p < 0.0001; F (1, 28) = 6.213, p = 0.0189, respectively). However, there was no significant interaction between these two factors (F (1, 28) = 0.07972, p = 0.7798, Fig. 7 ). 3.7. Restraint stress aggravates apoptosis in 6-OHDA-treated animals In order to study the role of apoptosis in 6-OHDA and restraint stress-induced deficits, we evaluated the expression of p53 and caspase-3 in the striatum. As depicted in Fig. 8 , the obtained results revealed a significant effect of 6-OHDA treatment on p53 levels in the striatum (F (1, 28) = 43.59, p < 0.0001) while restraint stress had no effect (F (1, 28) = 1.461, p = 0.2368) and no interaction between these two factors was observed (F (1, 28) = 0.1646, p = 0.6881). However, both, 6-OHDA and restraint stress (F (1, 28) = 52.01, p < 0.0001; F (1, 28) = 10. 70, p = 0.0028, respectively) significantly increased caspase 3 levels in the striatum (Fig. 9 ). Additionally, these two parameters had a significant interaction (F (1, 28) = 5.919, p = 0, 0216, Fig. 9 ). Bonferroni’s multiple comparison test revealed a significant increase in caspase-3 levels in 6-OHDA animals in comparison to the saline/no stress group (p = 0.0129), while 6-OHDA post-lesion restraint stress exacerbated caspase 3 expressions in comparison to non-stressed animals treated with 6-OHDA and to the animals that were subjected to restraint stress only (p < 0.0001, p < 0.0001, respectively, Fig. 9 ). 3.8. 6-OHDA and restraint stress increased AChE plasmatic levels The obtained results, showed that 6-OHDA and restraint stress had a significant effect on AChE levels in the plasma (F (1, 28) = 31.16, p < 0.0001; F (1, 28) = 8.318, p = 0.0075, respectively) and a significant interaction between them (F (1, 28) = 6.700, p = 0.0151). Bonferroni’s post comparisons test showed that 6-OHDA combined with restraint stress notably increased the concentration of AChE in the plasma in comparison to saline/no stress, saline/restraint stress and restraint stress only groups (p < 0.0001, p < 0.0001, p = 0.0036 respectively). 4. Discussion The main objective of our study was to investigate the effect of 6-OHDA injection in the MFB of male rats' motor, emotionality and cognitive behaviour as well as oxidative stress-related indicators such as iron; and apoptotic markers including caspase-3 and p53 in the striatum, plasmatic AChE level; striatal DA level and metabolism. We have also evaluated whether exposure to post-operative restraint stress would exaggerate the adverse consequences induced by 6-OHDA. First, we have confirmed that the injection of 6-OHDA into the MFB of male rats mimics the motor coordination deficits observed in PD. Our results showed that 6-OHDA injection in the MFB causes a significant impairment of motor coordination, particularly of the hind limb, and relatively decreased locomotor activity and velocity. 6-OHDA – induced coordination deficits were exacerbated by post-lesion restraint stress. This result was corroborated by an earlier study by Zhou et al., (2015), who investigated the effect of 6-OHDA injection in three different sites; SNpc, striatum, and MFB on gait deficits. Their findings showed that the MFB group has an apparent and stable gait impairment compared to SNpc and striatum groups [45]. Also, it has been previously shown that 6-OHDA lesions combined with restraint stress may alter motor coordination through a synergistic effect. Alongside, our result revealed severe impairments in the hind limb use while Ngema and Mabandla ., (2017) showed an altered forelimb use in gaiting and postural stability. Additionally, the deleterious effect of restraint stress in 6-OHDA-treated animals might be explained by its effect on the hypothalamic-pituitary-adrenal axis (HPA). It has been shown that stress, including restraint stress, may induce hyper-activation of the HPA and increase glucocorticoid release which may exaggerate the motor coordination deficits induced by 6-OHDA [23, 46]. 6-OHDA injection in the MFB also induced anxiety-like behaviour that was further aggravated by restraint stress. In line with our results, it has been shown that bilateral injection of 6-OHDA in the SNpc, induces anxiety-like behaviour, reduces DA and noradrenaline release in the prefrontal cortex(PFC), striatum and amygdala and increases serotonin levels in the amygdala [47]. Accordingly, restraint stress may exacerbate anxiety-like behaviour in the 6-OHDA treated animals by altering the brain monoamine systems, in particular the DA, as it has been shown that restraint stress substantially reduces SNpc DA and noradrenergic neuronal cell numbers in rats locus coeruleus [24]. On another hand, 6-OHDA lesions alone or combined with restraint stress had no effect on the episodic memory which suggests that the neural substrates underlying cognitive function might be less sensitive to the deleterious effects of 6-OHDA injection into the MFB. A similar observation was made by Marshall et al., (2019) who suggested that MFB unilateral lesion alone is insufficient to recapitulate the recognition memory deficits seen in PD patients [48]. However, a previous study using a more challenging cognitive and mnemonic task such as the Morris water maze, demonstrated that unilateral 6-OHDA lesion in MFB induces cognitive dysfunction in rats [49]. Moreover, it has been shown that an extended restraint stress duration such as 4h/day during 14 consecutive days may alter motor, emotional and cognitive functions in rats [50, 51] suggesting that 7-day exposure to restraint stress used in our study might be not sufficient to impair the neuronal circuits involved in the episodic-like memory process. The motor coordination impairments observed in animals injected with 6-OHDA and subjected to restraint stress were associated with a significant decrease in striatal DA and DOPAC levels, no effect on DA turnover, elevated iron build-up, and increased apoptotic factors expression caspase3, p53, and elevated plasmatic AChE level. The unchanged DA/DOPAC ratio between control, 6-OHDA and 6-OHDA + restraint stress groups suggest that decreased levels of DA and DOPAC in the striatum might be due to neurodegeneration of dopaminergic neurons of the SNpc and not an altered DA metabolism. These results are in line with previous studies demonstrating that 6-OHDA injection in the MFB may induce a progressive degeneration of dopaminergic neurons in the nigrostriatal pathway [18, 52]. Moreover, it was shown that 6-OHDA injection in the striatum dysregulates mitochondria by inhibiting complex I and IV of the respiratory chain, dysregulates the expression of divalent metal transporter 1(DMT1), and ferroportin 1 (FPN1) by activating IRP1, inhibits hepcidin release. These alterations consequently lead to abnormal accumulation of iron [53, 54] as has been demonstrated in our animal model. In line with these studies, our results showed that 6-OHDA injection in the MFB increased the levels of caspase 3, p53, and acetylcholinesterase, which might be subsequent to a mitochondrial dysfunction inducing ROS and cytochrome C release into the cytoplasm that mediates apoptosis [55]. Furthermore, autophagy may also be implicated in 6-OHDA-induced neurodegeneration, as it has been shown that 6-OHDA dysregulates autophagy by oligomerizing pro-apoptotic proteins; in particular; BCL2 Associated X(BAX) that leads to increased mitochondrial cytochrome C levels in the cytosol, and caspases activation [56]. In addition to p53 and caspase 3 overexpression, our results showed a significant increase of AChE in the plasma after 6-OHDA injection. Taken into account that the functional integrity of the basal ganglia relies on a balanced interaction between dopaminergic, cholinergic, GABAergic and glutamatergic systems, and the fact that overexpression of AChE contributes to cell apoptosis by altering apoptotic protease-activating-factor-1(Apaf-1) and cytochrome C [57, 58], we suggest that increased levels of AChE in the plasma of 6-OHDA animals may be due to dopaminergic cell loss in the nigrostriatal pathway. Increased levels of plasmatic AChE can be attributed to a compensatory response to the loss of functional cholinergic neurons [59, 60] or an increase in caspase-mediated cleavage of cytosolic AChE and subsequent a translocation of cleaved and full-length AChE into the cell nucleus [61]. On the other hand, exposure to subchronic restraint stress alone had no effect on iron accumulation, neither on DA turnover, p53, caspase 3 levels in the striatum or AChE levels in the plasma. A recent study showed that exposure to subchronic restraint stress 150 min /day for five consecutive days may induce apoptotic responses by increasing BAX/Bcl-2 ratio and elevating caspase 3 and caspase 9 levels in the prefrontal cortex and the hippocampus [62] which indicate that the nigrostriatal pathway might be less vulnerable to the deleterious effects of subchronic stress. However, when combined with 6-OHDA, restraint stress exacerbated the levels of caspase 3 and AChE which suggests that subchronic stress may reinforce the expression of pro-apoptotic factors and aggravate the loss of dopaminergic neurons in the nigrostriatal pathway. It was demonstrated that exposure to restraint stress for 7 days after 6-OHDA injection decreased the concentration of neurotrophic factors such as glial cell-line derived neurotrophic factor and Neurotrophin-3 in the nigrostriatal pathway which may consequently contribute to neurodegeneration [24, 63]. Taken together, these results give further support to 6-OHDA MFB lesion as a suitable model for understanding the neuronal substrates implicated in the pathological process of PD, suggesting a synergistic effect between restraint stress and 6-OHDA treatment, and indicating that stress may accelerate the progression of PD by triggering dopaminergic neurodegeneration and increasing oxidative stress and apoptotic processes. In conclusion, the present study provides evidence that post-lesion stress may aggravate apoptosis through increasing caspase-3 and AChE activities and may alter motor coordination and exacerbate anxiety behaviour in the 6-OHDA animal model of PD. Further studies using a multi-environmental stress approach and complex cognitive tasks reflecting the human condition are needed to understand the mechanisms implicated in the aetiology and/or the progression of PD. Declarations Ethical Approval: All experiments were conducted in compliance with the ARRIVE guidelines and they were approved by the Animal Ethics Sub-committee of Mohammed V University in Rabat, Morocco. Competing interests : The authors declare no competing interests. Authors' contributions: S.E., K.T., M.A. and O.A. designed the study; S.E., N.F., H.I., and O.A. performed experiments and data analysis; S.E., N.F., M. 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Mallet, D., et al., Re-routing metabolism by the mitochondrial pyruvate carrier inhibitor MSDC-0160 attenuates neurodegeneration in a rat model of Parkinson’s disease. bioRxiv, 2022. Xu, M., et al., 6-Hydroxydopamine Induces Abnormal Iron Sequestration in BV2 Microglia by Activating Iron Regulatory Protein 1 and Inhibiting Hepcidin Release. Biomolecules, 2022. 12 (2): p. 266. Sodhi, R.K., et al., IDO-1 inhibition protects against neuroinflammation, oxidative stress and mitochondrial dysfunction in 6-OHDA induced murine model of Parkinson’s disease. Neurotoxicology, 2021. 84 : p. 184-197. Chung, Y., et al., Dysregulated autophagy is linked to BAX oligomerization and subsequent cytochrome c release in 6-hydroxydopmaine-treated neuronal cells. Biochemical and Biophysical Research Communications, 2021. 548 : p. 20-26. Knorr, D.Y., et al., Acetylcholinesterase promotes apoptosis in insect neurons. Apoptosis, 2020. 25 (9): p. 730-746. Junn, E. and M.M. 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Cite Share Download PDF Status: Published Journal Publication published 12 Jan, 2023 Read the published version in Neurotoxicity Research → Version 1 posted Editorial decision: Major revision 30 Oct, 2022 Reviews received at journal 29 Oct, 2022 Reviewers agreed at journal 26 Oct, 2022 Reviewers invited by journal 25 Oct, 2022 Editor assigned by journal 25 Oct, 2022 Submission checks completed at journal 25 Oct, 2022 First submitted to journal 23 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2196389","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":146882237,"identity":"e4449610-7988-4c22-99a5-bd7c6aa3ea17","order_by":0,"name":"Sara El Idrissi","email":"","orcid":"","institution":"Mohammed V University in Rabat","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"El","lastName":"Idrissi","suffix":""},{"id":146882238,"identity":"2bc2d337-8e7b-4a3a-b3ed-ec97ba1fc7ca","order_by":1,"name":"Nada Fath","email":"","orcid":"","institution":"Mohammed V University in Rabat","correspondingAuthor":false,"prefix":"","firstName":"Nada","middleName":"","lastName":"Fath","suffix":""},{"id":146882239,"identity":"8f27a411-d16c-4531-93e9-07057d3f854b","order_by":2,"name":"Hind Ibork","email":"","orcid":"","institution":"Mohammed V University in Rabat","correspondingAuthor":false,"prefix":"","firstName":"Hind","middleName":"","lastName":"Ibork","suffix":""},{"id":146882240,"identity":"f366d03f-3405-4ea1-b143-4322d141c714","order_by":3,"name":"Khalid Taghzouti","email":"","orcid":"","institution":"Mohammed V University in Rabat","correspondingAuthor":false,"prefix":"","firstName":"Khalid","middleName":"","lastName":"Taghzouti","suffix":""},{"id":146882241,"identity":"6c516f7e-9634-41b2-a187-066cc08ec5ba","order_by":4,"name":"Meryem Alamy","email":"","orcid":"","institution":"Mohammed V University in Rabat","correspondingAuthor":false,"prefix":"","firstName":"Meryem","middleName":"","lastName":"Alamy","suffix":""},{"id":146882242,"identity":"3cac0063-abb1-4ec5-b044-15aaa3e44528","order_by":5,"name":"Oualid Abboussi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYHACNmYwxcx8gIHxD2la2BIYGBtI0sLAY0CcFn4G5mePCyoO28u383yTurnjHgN/+wHWDT/waJFsYDM3nnHmcOKGw7zbpHPPFDNInElgu9mDR4vBAQYzad62wwkGzEAtOUD/MNxgYLvBg1cL+zeQFnv5Zp5nYC3yQC038YWcwQEesC2MDYd52KRz2xIYDIBabuOzRbKZpxzol3SgX9iMrXPOJPAYnklsuy2DRws/e/s2YIhZ28v3H354O6ciQU7u+OFjN9/g0cLAjMYHOonYCB0Fo2AUjIJRgBMAAKnNRnKEh+udAAAAAElFTkSuQmCC","orcid":"","institution":"Mohammed V University in Rabat","correspondingAuthor":true,"prefix":"","firstName":"Oualid","middleName":"","lastName":"Abboussi","suffix":""}],"badges":[],"createdAt":"2022-10-23 18:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2196389/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2196389/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12640-022-00630-3","type":"published","date":"2023-01-12T18:18:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":28369916,"identity":"b503fb02-3a88-41da-8c94-51a88ac5e441","added_by":"auto","created_at":"2022-10-28 14:39:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79215,"visible":true,"origin":"","legend":"\u003cp\u003eTimeline of the experimental paradigm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/8a8206f627853f998749c5f8.png"},{"id":28368987,"identity":"f385d7b5-a140-4650-a68f-06b601a6d9c5","added_by":"auto","created_at":"2022-10-28 14:34:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30965,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA insult and restraint stress on locomotor activity and anxiety-like behaviour induced anxiety-like behaviour. Data are reported as mean ± SEM of total distance travelled (A), average speed (B), number of entries to the central zone, and time spent in the central zone (D) from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/285d1527bf1370cc8ab0332a.png"},{"id":28368989,"identity":"382eb0a9-72ec-462e-81d6-a9691b96acb5","added_by":"auto","created_at":"2022-10-28 14:34:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15191,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA and restraint stress on episodic memory. Data are reported as mean ± SEM of the index of exploration (A) and index of discrimination (B) from the 6-OHDA group (n=8) and saline group (n=8). Values were analyzed using Two-way ANOVA followed by a Bonferroni post-test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/b670ea3a72c06249373d81cc.png"},{"id":28369918,"identity":"be09a4d5-489e-410d-a5e9-43b5824f797c","added_by":"auto","created_at":"2022-10-28 14:39:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22063,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u0026nbsp;6-OHDA and restraint stress on gait impairment. Data are reported as mean ± SEM of latency to start crossing (A) and total time to cross the beam (B) from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.0005, ****p \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/ad0292e5feda6dd6dfb53662.png"},{"id":28369917,"identity":"ee497abb-b1c3-4b0c-92a7-b20a1a7f77b2","added_by":"auto","created_at":"2022-10-28 14:39:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14532,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA and restraint stress on motor coordination. Data are reported as mean ± SEM of the latency to fall from the rotatory bar from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/68e1dd6367c240af0deac492.png"},{"id":28368993,"identity":"d5b6bd04-4246-42c6-8133-a6a595cbc694","added_by":"auto","created_at":"2022-10-28 14:34:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19781,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA and restraint stress on DA and DOPAC levels in the striatum. As well as DA turnover. Data are reported as mean ± SEM of DA (A), DOPAC (B) and DOPAC/DA ratio (C) from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.0005, ****p \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/d3c0d971d7f5f9435914c100.png"},{"id":28368995,"identity":"db7f37c0-db6c-4d39-8813-1816595c996f","added_by":"auto","created_at":"2022-10-28 14:34:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16358,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA and restraint stress on iron accumulation in the striatum Data are reported as mean ± SEM from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.0005, ****p \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/9bfd05cab4b095762b4174a1.png"},{"id":28368991,"identity":"a6f23c88-c3a2-447e-9a7f-eff0a596470d","added_by":"auto","created_at":"2022-10-28 14:34:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":15430,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA and restraint stress on the striatal tumor suppressor protein p53. Data are reported as mean ± SEM from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.0005, ****p \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/7b676bc595551d82bcd4cc59.png"},{"id":28368996,"identity":"c1517067-7fe6-4020-bf48-5f676ba2c20e","added_by":"auto","created_at":"2022-10-28 14:34:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":17478,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA and restraint stress on the levels of caspase-3 in the striatum. In addition, post-lesion exposure to stress exacerbated caspase-3 levels. Data are reported as mean ± SEM from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.0005, ****p \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/a0791fb5c85ad40d8a648e41.png"},{"id":28368992,"identity":"2a195554-2bed-43ab-8907-d655cac38249","added_by":"auto","created_at":"2022-10-28 14:34:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":13267,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 6-OHDA and restraint stress on the levels of AChE in the plasma. Data are reported as mean ± SEM from the 6-OHDA group (n=8) and saline group (n=8). Values were analysed using Two-way ANOVA followed by Bonferroni post-test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.0005, ****p \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/94d3d4441c046a6e743ab699.png"},{"id":44716602,"identity":"00f01a08-1ca9-465a-ac85-8db5a2fd3581","added_by":"auto","created_at":"2023-10-16 18:27:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":707018,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2196389/v1/89683919-b092-4288-abfc-ad088eacbcf2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Restraint stress exacerbates apoptosis in a 6-OHDA animal model of Parkinson disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePD is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) [1, 2]. PD is the second most common neurodegenerative disorder with a prevalence of 2\u0026ndash;3% within the population above 65 years of age [3]. Several studies have shown males are more susceptible to PD, with a male-female ratio that varies between 1.37 to 3.7 [4, 5]. PD presents a serious burden on the patient and caregivers [6]. Yet, its aetiology is not completely understood. PD is characterized by several motor symptoms such as akinesia, bradykinesia, tremor and rigidity. Additionally, PD patients show other motor deficits, including gait disturbance, impaired handwriting, posture instability, grip force and speech deficits (Kouli et al., 2018, Balestrino and Schapira, 2020). The neuropathological hallmark of PD includes neurodegeneration, neuro-inflammation, altered dopaminergic activity and increased oxidative stress in the nigrostriatal pathway [7, 8]. Many risk factors were proposed to be involved in the pathogenesis and the progression of PD including environmental stress-induced increased cortisol levels [9]. It has been shown that acute and chronic stress affects the dopaminergic nigrostriatal pathway alongside dopaminergic neurons in the mesocorticolimbic pathways, leading to impaired locomotor activity, suggesting an interaction between stress and the onset of the motor symptoms of PD [10\u0026ndash;12]. Furthermore, motor deficits associated with PD, on their own, may present a stressful event that contributes to the aggravation of PD's neuropathology [13]. Clinical studies have shown that stress-increased cortisol is associated with the worst functional scores for motor, and cognitive symptoms of PD [14]. Several animal models were developed to understand the physiopathology of PD. Among these models, the 6-OHDA animal model [15]. It was demonstrated that unilateral injection of 6-OHDA into the substantia nigra pars compacta (SNpc), leads to a rapid onset of neuronal loss [16]. Whereas, injecting 6-OHDA in the medial forebrain bundle (MFB) induces the lesion of the nigrostriatal pathway within 3\u0026ndash;5 weeks resulting in the initiation of a deleterious cascade of events leading to neuronal loss [17\u0026ndash;19]. It has been shown that 6-OHDA neuronal loss is mediated, in at least one part, by oxidative stress resulting from increased iron release from Fe-S cluster proteins of the mitochondrial respiratory chain and from other iron-storing cell compartments which subsequently exacerbate reactive oxygen species (ROS) production via the Fenton\u0026rsquo;s reaction [20, 21]. Iron is a transition metal, mandatory for the normal functioning of cells and the metabolism of neurotransmitters including DA, but when iron exceeds the homeostatic levels, it becomes toxic, leading to oxidative damage and eventually cell death [22]. The deleterious effects of 6-OHDA can be aggravated by stress which was suggested to play a role in the onset of PD [23, 24]. It has been shown that chronic restraint stress triggers dopaminergic and noradrenergic neurodegeneration via apoptosis by activating caspase enzymes, priory expressed as inactive precursors lacking protease activity [24, 25]. Nevertheless, apoptosis, which is a mode of programmed cell death (PCD), crucial for mammalian development as it controls cell numbers, tissue development, and clears damaged structures [26, 27]. The apoptotic processes depend on multiple factors such as the activation of the p53 transcription factor, known as tumour protein 53, and considered one of the primary measures in the evaluation of neural responses to stressful events [8, 28] as it controls DNA damage, senescence, ROS formation, as well as activating pro-apoptotic and suppressing anti-apoptotic proteins [8, 29]. Apoptosis also depends on caspase 3 expression and activity. Additionally, Caspase-3 has been identified as a key mediator of apoptosis and plays an important role in PD pathogenesis [30]. Several studies highlighted a positive correlation between the degree of neurodegeneration of dopaminergic neurons and the levels of caspase-3 expression, indicating that caspase-3 is a vulnerability factor and final effector in the apoptotic death of dopaminergic neurons in PD [31]. Interestingly, it has been shown that caspase-3 may induce DNA fragmentation and protein cleavage, including the Parkin protein implicated in the biochemical pathways underlying the aetiology of sporadic PD [32\u0026ndash;34]. Apoptosis also depends on AChE integrity as it plays a major role in the termination of signal transmission in the cholinergic system and is implicated in the pathogenesis of neurodegenerative diseases by affecting inflammatory responses, oxidative stress, and the aggregation of pathological proteins [35, 36]. It has been shown that decreased expression of AChE reduces apoptotic markers in different cell lines whereas augmented AChE activity increases apoptosis [37]. Based on these studies, we hypothesised that restraint stress may aggravate the degeneration of the nigrostriatal pathway in the 6-OHDA PD animal model by inducing oxidative stress, through induction of ROS release and iron build-up, and/or by upregulating apoptotic factors such as caspase 3, p53 and AChE. To our best knowledge, no study investigated the expression of iron, p53, caspase-3, and AChE in animal models of PD exposed to stress. Therefore, we aimed to investigate the behavioural impairments and related neurochemical changes following post-lesion restraint stress in adult male rats injected with 6-OHDA in the MFB.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Animals\u003c/h2\u003e\n \u003cp\u003eThirty-two male Wistar rats (7 to 8 weeks old) obtained from the animal facility of the Faculty of Sciences at Mohammed V University in Morocco were used in this randomised blinded study. The animals were housed under standard laboratory conditions; a 12-hour light/dark cycle with lights on at 6 am and they had free access to standard chow and tap water. The animals were divided into four groups; (1) A control group received an injection of vehicle (saline solution containing 0.2% ascorbic acid) into the left MFB; (2) 6-OHDA group; (3) vehicle\u0026thinsp;+\u0026thinsp;stress group, injected with vehicle and subjected to 7 days\u0026rsquo; restraint stress and the (4) 6-OHDA\u0026thinsp;+\u0026thinsp;stress group. Restraint stress was applied one week following 6-OHDA or vehicle administration into the MFB. The experimental paradigm was summarised in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. All experiments were conducted in compliance with the ARRIVE guidelines and they were approved by the Animal Ethics Sub-committee of Mohammed V University.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Stereotaxic surgery and restraint stress\u003c/h2\u003e\n \u003cp\u003eOn PND 70\u0026ndash;72, a solution of 6-OHDA was freshly prepared, kept on ice, and protected from light exposure with tin foil. The solution consisted of 5 \u0026micro;g 6-OHDA hydrobromide (Sigma-Aldrich) dissolved in 4 \u0026micro;l of sterile saline containing 0.2% ascorbic acid. The surgical site and instruments were sterilised before surgery. Rats (240\u0026ndash;310 g), were injected intraperitoneally (i.p) with desipramine at 15 mg/kg (Sigma-Aldrich) 30 minutes before the injection of 6-OHDA to prevent the degeneration of the noradrenergic neurons. Prior to the stereotaxic surgery, the rats were anaesthetized with sodium pentobarbital (60 mg/kg, i,p). To avoid the confounding effect of direct mechanical damage to the striatum or substantia nigra [11], 6-OHDA or vehicle were injected into the left MFB at a rate of 1 \u0026micro;l per 2 minutes using a Hamilton syringe. The coordinates of injection were 4.7 mm anterior to the interaural line, 1.7 mm lateral to the midline and 8.6 mm ventral to the dura based on the Stereotaxic Atlas of Rat Brain by Paxinos and Watson [11, 38]. The rate of infusion was set at 1\u0026micro;l / 2 minutes. To facilitate optimal diffusion of the solution, the needle was left in place for an additional 5 minutes following the infusion of 6-OHDA. Subsequently, the needle was slowly retracted from the brain and the incision was sutured using a clinisilk suture gauge 5/0 needle. Following surgery, the rats were placed on a heating pad for 30 minutes to prevent hypothermia and then the animals were returned to their home cages for recovery.\u003c/p\u003e\n \u003cp\u003eSix days following the surgery, rats in the two stressed groups were moved to an isolated behavioural room away from the non-stressed animals and placed in rodent restrainers (114 mm long x 28 mm diameter) for 3 hours per day from 9h00 to 12h00 for 7 consecutive days. At the end of each session, the animals were taken back to their home cages.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. Open field test\u003c/h2\u003e\n \u003cp\u003eThe open field test was conducted in a rectangular box (100 cm length x 100 cm width x 40 cm height) made of plexiglass. The floor of the open field apparatus was virtually divided into 25 equal squares, brightly illuminated by a white light lamp (100 W) and suspended 2 meters above the open field arenas. Each animal was placed in the bottom right corner of the apparatus and video recorded for 5 min. For each rat, the total distance travelled, the average speed (as an indicator of locomotor activity), the number of entries to the central zone and the time spent in the central zone (as an indicator of anxiety-like behaviour) were measured using Anymaze software (Stoelting. Co). The apparatus was cleaned with 70% alcohol after each trial.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Novel object recognition test\u003c/h2\u003e\n \u003cp\u003eThe novel object recognition test was conducted in the same open-field apparatus. This task evaluates the ability of the animal to discriminate between novel and familiar objects. It relies on the rat\u0026apos;s innate preference for novelty, if the rat recognizes a familiar object; it will spend most of its time at the novel object. The duration of the test is 3 days; during the first day, the animals are familiarised with the apparatus for 5 min; on the 2nd day or the habituation phase, two identical objects are presented to the animal and placed 15 cm from the sidewall in diagonal corners opposite each other. Each rat was allowed to explore the objects for 5 min; on the 3rd day (test day) one of the training objects is replaced with a novel object. The exploration index was calculated during the habituation phase as a ratio of the total time spent by the animal exploring the objects and the trial duration (5 min), while the discrimination index (DI) was calculated during the test day as fellow; DI =[(time spent exploring the novel object - time spent exploring the familiar object)/total exploration time] [39].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. The beam-walking test\u003c/h2\u003e\n \u003cp\u003eThe beam-walking test consisted of a training phase and a test session. During the training phase, the rats were subjected to three trials during which they were encouraged to cross a wooden beam (2 cm in diameter,100 cm in length and elevated 70 cm above the floor) to reach a black box placed on the other extremity of the beam. 24 hours later animals were tested on the same beam. gait and time to cross the beam were counted as indicators of motor coordination [40, 41].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6. Rotarod test\u003c/h2\u003e\n \u003cp\u003eThe apparatus consisted of 7-cm-diameter plastic drums machined with grooves to improve grip (Panlab Harvard Apparatus, Barcelona, Spain). It could be set on accelerating speed (4, 10, 12, 15, 19, 22, 26, 29 34 and 40 rpm, 30 s at each speed). Before testing, the rats were trained for 2 days. During the first day, rats were trained for 3 min with an unlimited number of trials on the rotarod. Followed by four trials of a maximum of 60 s with 30 s intervals. On the second day, rats were placed on the rotarod at accelerating speed for a maximum of 300 s. on the testing day, each rat was individually placed on the rotarod at accelerating speed for a maximum of 300 s, and the latency to fall off the rotarod and the maximum speed reached within this time were recorded. Immediately after each session, the apparatus was thoroughly cleaned with cotton pads wetted with 70% ethanol; water solution and dried. Rats were allowed to habituate to the experimental room for 60 min before both training and testing. Training and testing were performed between 10:00 AM and 1:00 PM [42].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7. Samples preparation\u003c/h2\u003e\n \u003cp\u003e24 hours after that last behavioural test, the animals were decapitated using a guillotine, the striatal brain structures were dissected out using plastic forceps (Sigma-Aldrich, USA) to minimize trace metal ion contamination, washed with phosphate buffer solution (1 x PBS, pH\u0026thinsp;=\u0026thinsp;7.4), weighted and stored at -80\u0026deg;C until the day of analysis. Trunk blood was collected in heparinized tubes and centrifuged at 3000 g for 10 minutes. The plasma was separated in the supernatant and kept at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.8. Striatal Dopamine and DOPAC determination\u003c/h2\u003e\n \u003cp\u003eA high-performance liquid chromatography (HPLC) system (Agilent technologies HP 1100 series) with electrochemical detection (Agilent Hewlett Packard 1049A) set at 750 mV was used to analyse DA and its metabolites 3,4-Dihydroxyphenylacetic acid (DOPAC). The striatal homogenate was thawed and centrifuged at 4\u0026deg;C for 2 min at 12,000 g. Ten microliters of each supernatant per sample were injected onto a LiChrospher 100 cartridge column, RP-18,5 \u0026micro;m, 3 \u0026times; 125 mm (Agilent) maintained at 4\u0026deg;C. The mobile phase consisted of 0.1 M Na2HPO4 (pH 3.3), 0.15 mM EDTA and 25% methanol. The flow rate was 0.8 ml/min. The analytes (DA and DOPAC) were identified by their retention times compared with their corresponding standards. Their concentration was estimated by comparison of the area under the curve using the straight-line equation y\u0026thinsp;=\u0026thinsp;mx\u0026thinsp;+\u0026thinsp;c and presented as ng/mg protein [43].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e2.9. Determination of striatal iron levels\u003c/h2\u003e\n \u003cp\u003eFor iron content analysis, Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to measure iron levels in the striatum following the procedure previously explained by [44]. Briefly, a mixture of 0.5 ml of concentrated hydrochloric acid (HCL) 2 N, and 200 mg of striatal sample was sonicated and homogenized utilizing a Misonix Sonicator XL2000-010 (Newtown CT, USA) until a homogenate was acquired. 70% perchloric acid (0.1 ml) was added to treat the samples following which the samples were incubated in a water bath at 50\u0026deg;C for 24\u0026ndash;36 hours. The samples were centrifuged at 600 g for 1 h and subsequently filter-syringed through a 0.45 \u0026micro;m pore filter. Standard iron solution (50 mg/l) was diluted with nitric 70% perchloric acid 100, 50, 25 and 12.5 times and used to draw the standard curved line. To analyze the standards and samples, Perkin Elmer Optima 5300 DV Optimal Emission Spectrometer (Waltham MA, USA) was used at a detection wavelength of 259.94 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e2.10. Assay protocol for Caspase3, AChE, and p53\u003c/h2\u003e\n \u003cp\u003eThe levels of caspase-3, p53, and AChE were quantified using commercially available sandwich-ELISA assay kits (Elabscience, USA). The striatal tissue was sonicated and homogenized in a buffered solution containing 400 mM NaCl, 2.0 mM EDTA, 2.0 mM benzamidine, 0.1% Triton-X, 0.5% BSA, 0.1 mM PMSF, Aprotinin (9.7 TIU/ml), 0.1 mM benzethonium chloride and 0.1 M phosphate buffer (pH\u0026thinsp;=\u0026thinsp;7.4). All procedures were accomplished according to the instructions of the manufacturer. The sensitivity of the kits was reported as 46.875pg/mL for p53, 0.188ng/mL for caspase-3 and 0.47ng/mL for AChE. The coefficient of variation for the assays was \u0026lt;\u0026thinsp;10%. Recombinant preparations were used for the establishment of the standard curves for p53, caspase-3, and AChE analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eWe tested normality using the Shapiro-Wilk test. Data that assumed gaussian distribution were analyzed using a two-way analysis of variance (two-way ANOVA) followed by Bonferroni\u0026rsquo;s post hoc test. All statistical analyses were performed using the GraphPad Prism software (Version 8.0). Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and significance were set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.1. 6-OHDA and restraint stress evoke anxiety-like behaviour\u003c/h2\u003e\n \u003cp\u003eTo measure locomotor activity and anxiety-like behaviour, we used the open-field test.\u003c/p\u003e\n \u003cp\u003eThe obtained results revealed a significant effect of 6-OHDA on locomotor activity expressed as a significant decrease in the total distance travelled (F \u003csub\u003e(1,28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;14.22, p\u0026thinsp;=\u0026thinsp;0.0008, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), and decreased average speed (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.001, p\u0026thinsp;=\u0026thinsp;0.0208, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) as has been revealed by the two-way ANOVA analysis. 6-OHDA induced an anxiogenic effect expressed by a decreased number of entries (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.411, p\u0026thinsp;=\u0026thinsp;0.0448) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), and reduced time spent in the central zone of the open field (F \u003csub\u003e(1,28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.171, p\u0026thinsp;=\u0026thinsp;0.0123, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, restraint stress had no effect on the total distance travelled (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.03434, p\u0026thinsp;=\u0026thinsp;0.8543, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) neither on the average speed (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.149, p\u0026thinsp;=\u0026thinsp;0.2930, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, restraint stress induced anxiety as it significantly decreased the time spent in the central zone (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.534; P\u0026thinsp;=\u0026thinsp;0. 0045, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD) without affecting the numbers of entries to the central zone \u003cstrong\u003e(\u003c/strong\u003eF \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.810, P\u0026thinsp;=\u0026thinsp;0.1048; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Moreover, data analysis revealed no significant interaction between 6-OHDA injection and restraint stress in all open field measured parameters; neither on the total distance travelled (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.413; P\u0026thinsp;=\u0026thinsp;0.2445), the average speed (F \u003csub\u003e(1, 28\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;0.7091, p\u0026thinsp;=\u0026thinsp;0. 4069); the number of entries to the central zone (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5082, p\u0026thinsp;=\u0026thinsp;0.4818), or the time spent in the central zone (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1792, p\u0026thinsp;=\u0026thinsp;0.6753,Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003ch2\u003e3.2. 6-OHDA and restraint stress did not affect the episodic-like memory\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003cp\u003eTo evaluate the effect of 6-OHDA injection and exposure to restraint stress on episodic memory we used the novel object recognition test. Data analysis using two-way ANOVA test indicated that 6-OHDA treatment had no effect on the index of exploration (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.531, p\u0026thinsp;=\u0026thinsp;0. 1228, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA) neither on the index of discrimination (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.341, p\u0026thinsp;=\u0026thinsp;0.2566, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similarly, restraint stress had no effect on the index of exploration (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.09427, p\u0026thinsp;=\u0026thinsp;0.7611) neither on the index of discrimination (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.470, p\u0026thinsp;=\u0026thinsp;0.2355).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.3. 6-OHDA alone or combined with restraint stress-induced gait impairments\u003c/h2\u003e\n \u003cp\u003eThe hind limb implication in the gait impairment was analysed using the beam-walking test. Our obtained results showed a main effect of the 6-OHDA on gait-related behaviour as expressed by increased latency to start crossing (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;54.91, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), and increased time spent to cross the beam (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16.41, p\u0026thinsp;=\u0026thinsp;0.0004, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, restraint stress alone had no significant effect on gait-related behaviour, neither on the latency to start crossing (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.257, p\u0026thinsp;=\u0026thinsp;0.2717, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) or the total time spent to cross the beam (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.3570, p\u0026thinsp;=\u0026thinsp;0.5550, 4B). Moreover, two-way ANOVA indicated no interaction between 6-OHDA and restraint stress when analysing the latency to start crossing (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.257, p\u0026thinsp;=\u0026thinsp;0.2717; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) but it revealed a significant interaction between 6-OHDA and restraint stress when analysing the total time spent crossing the beam (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.673, p\u0026thinsp;=\u0026thinsp;0.0243, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Bonferroni\u0026rsquo;s posthoc comparisons test showed that 6-OHDA combined with exposure to restraint stress increased significantly the total time spent crossing the beam compared to saline/no stress group (p\u0026thinsp;=\u0026thinsp;0.0164), and saline/restraint stress group (p\u0026thinsp;=\u0026thinsp;0.0006) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003e3.4. 6-OHDA impaired motor coordination\u003c/h2\u003e\n \u003cp\u003eThe impact of 6-OHDA and stress exposure on motor coordination and balance was evaluated by the rotarod test. Our results revealed a significant effect of 6-OHDA (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16. 62, p\u0026thinsp;=\u0026thinsp;0. 0003), no effect of restraint stress (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.975, p\u0026thinsp;=\u0026thinsp;0. 1709) and no interaction between these two parameters on the latency to fall (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.7761, p\u0026thinsp;=\u0026thinsp;0.3858, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003e3.5. 6-OHDA evoked DA depletion in the striatum\u003c/h2\u003e\n \u003cp\u003eTo investigate the effect of 6-OHDA injection and restraint stress on the nigrostriatal pathway, we measured the levels of DA and DOPAC as well as DA turnover in the striatum.\u003c/p\u003e\n \u003cp\u003eTwo-way ANOVA analysis revealed that 6-OHDA decreased significantly DA (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;59.49, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), and DOPAC (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;44.73, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB) and had no effect on DA turnover (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.344, p\u0026thinsp;=\u0026thinsp;0.1370, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Whereas, restraint stress had no effect on DA (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.875, p\u0026thinsp;=\u0026thinsp;0.0590, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA), neither on DOPAC (F \u003csub\u003e(1. 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0 03673, p\u0026thinsp;=\u0026thinsp;0.8494, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB) or DA turnover (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1287, p\u0026thinsp;=\u0026thinsp;0.7225, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Additionally data analysis revealed no significant interaction between 6-OHDA and restraint stress when analysing DA, DOPAC or DA turnover levels in the striatum (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1220, p\u0026thinsp;=\u0026thinsp;0.7295; F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.250, p\u0026thinsp;=\u0026thinsp;0.2730; F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2165, p\u0026thinsp;=\u0026thinsp;0.6453; respectively) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003e3.6. 6-OHDA increased iron build-up in the striatum\u003c/h2\u003e\n \u003cp\u003eIt was reported that iron may play an important role in the pathophysiology of PD. Thus, we investigated iron levels in the striatal structures following 6-OHDA and stress exposure.\u003c/p\u003e\n \u003cp\u003eBoth, 6-OHDA and restraint stress had a significant effect on striatal iron levels (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;44.76, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.213, p\u0026thinsp;=\u0026thinsp;0.0189, respectively). However, there was no significant interaction between these two factors (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.07972, p\u0026thinsp;=\u0026thinsp;0.7798, Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec21\"\u003e\n \u003ch2\u003e3.7. Restraint stress aggravates apoptosis in 6-OHDA-treated animals\u003c/h2\u003e\n \u003cp\u003eIn order to study the role of apoptosis in 6-OHDA and restraint stress-induced deficits, we evaluated the expression of p53 and caspase-3 in the striatum.\u003c/p\u003e\n \u003cp\u003eAs depicted in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, the obtained results revealed a significant effect of 6-OHDA treatment on p53 levels in the striatum (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;43.59, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) while restraint stress had no effect (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.461, p\u0026thinsp;=\u0026thinsp;0.2368) and no interaction between these two factors was observed (F\u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1646, p\u0026thinsp;=\u0026thinsp;0.6881). However, both, 6-OHDA and restraint stress (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;52.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10. 70, p\u0026thinsp;=\u0026thinsp;0.0028, respectively) significantly increased caspase 3 levels in the striatum (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Additionally, these two parameters had a significant interaction (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.919, p\u0026thinsp;=\u0026thinsp;0, 0216, Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Bonferroni\u0026rsquo;s multiple comparison test revealed a significant increase in caspase-3 levels in 6-OHDA animals in comparison to the saline/no stress group (p\u0026thinsp;=\u0026thinsp;0.0129), while 6-OHDA post-lesion restraint stress exacerbated caspase 3 expressions in comparison to non-stressed animals treated with 6-OHDA and to the animals that were subjected to restraint stress only (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively, Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec22\"\u003e\n \u003ch2\u003e3.8. 6-OHDA and restraint stress increased AChE plasmatic levels\u003c/h2\u003e\n \u003cp\u003eThe obtained results, showed that 6-OHDA and restraint stress had a significant effect on AChE levels in the plasma (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;31.16, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.318, p\u0026thinsp;=\u0026thinsp;0.0075, respectively) and a significant interaction between them (F \u003csub\u003e(1, 28)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.700, p\u0026thinsp;=\u0026thinsp;0.0151). Bonferroni\u0026rsquo;s post comparisons test showed that 6-OHDA combined with restraint stress notably increased the concentration of AChE in the plasma in comparison to saline/no stress, saline/restraint stress and restraint stress only groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, p\u0026thinsp;=\u0026thinsp;0.0036 respectively).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe main objective of our study was to investigate the effect of 6-OHDA injection in the MFB of male rats' motor, emotionality and cognitive behaviour as well as oxidative stress-related indicators such as iron; and apoptotic markers including caspase-3 and p53 in the striatum, plasmatic AChE level; striatal DA level and metabolism. We have also evaluated whether exposure to post-operative restraint stress would exaggerate the adverse consequences induced by 6-OHDA.\u003c/p\u003e \u003cp\u003eFirst, we have confirmed that the injection of 6-OHDA into the MFB of male rats mimics the motor coordination deficits observed in PD. Our results showed that 6-OHDA injection in the MFB causes a significant impairment of motor coordination, particularly of the hind limb, and relatively decreased locomotor activity and velocity. 6-OHDA \u0026ndash; induced coordination deficits were exacerbated by post-lesion restraint stress. This result was corroborated by an earlier study by Zhou et al., (2015), who investigated the effect of 6-OHDA injection in three different sites; SNpc, striatum, and MFB on gait deficits. Their findings showed that the MFB group has an apparent and stable gait impairment compared to SNpc and striatum groups [45]. Also, it has been previously shown that 6-OHDA lesions combined with restraint stress may alter motor coordination through a synergistic effect. Alongside, our result revealed severe impairments in the hind limb use while Ngema and Mabandla ., (2017) showed an altered forelimb use in gaiting and postural stability. Additionally, the deleterious effect of restraint stress in 6-OHDA-treated animals might be explained by its effect on the hypothalamic-pituitary-adrenal axis (HPA). It has been shown that stress, including restraint stress, may induce hyper-activation of the HPA and increase glucocorticoid release which may exaggerate the motor coordination deficits induced by 6-OHDA [23, 46]. 6-OHDA injection in the MFB also induced anxiety-like behaviour that was further aggravated by restraint stress. In line with our results, it has been shown that bilateral injection of 6-OHDA in the SNpc, induces anxiety-like behaviour, reduces DA and noradrenaline release in the prefrontal cortex(PFC), striatum and amygdala and increases serotonin levels in the amygdala [47]. Accordingly, restraint stress may exacerbate anxiety-like behaviour in the 6-OHDA treated animals by altering the brain monoamine systems, in particular the DA, as it has been shown that restraint stress substantially reduces SNpc DA and noradrenergic neuronal cell numbers in rats locus coeruleus [24]. On another hand, 6-OHDA lesions alone or combined with restraint stress had no effect on the episodic memory which suggests that the neural substrates underlying cognitive function might be less sensitive to the deleterious effects of 6-OHDA injection into the MFB. A similar observation was made by Marshall et al., (2019) who suggested that MFB unilateral lesion alone is insufficient to recapitulate the recognition memory deficits seen in PD patients [48]. However, a previous study using a more challenging cognitive and mnemonic task such as the Morris water maze, demonstrated that unilateral 6-OHDA lesion in MFB induces cognitive dysfunction in rats [49]. Moreover, it has been shown that an extended restraint stress duration such as 4h/day during 14 consecutive days may alter motor, emotional and cognitive functions in rats [50, 51] suggesting that 7-day exposure to restraint stress used in our study might be not sufficient to impair the neuronal circuits involved in the episodic-like memory process.\u003c/p\u003e \u003cp\u003eThe motor coordination impairments observed in animals injected with 6-OHDA and subjected to restraint stress were associated with a significant decrease in striatal DA and DOPAC levels, no effect on DA turnover, elevated iron build-up, and increased apoptotic factors expression caspase3, p53, and elevated plasmatic AChE level. The unchanged DA/DOPAC ratio between control, 6-OHDA and 6-OHDA\u0026thinsp;+\u0026thinsp;restraint stress groups suggest that decreased levels of DA and DOPAC in the striatum might be due to neurodegeneration of dopaminergic neurons of the SNpc and not an altered DA metabolism. These results are in line with previous studies demonstrating that 6-OHDA injection in the MFB may induce a progressive degeneration of dopaminergic neurons in the nigrostriatal pathway [18, 52]. Moreover, it was shown that 6-OHDA injection in the striatum dysregulates mitochondria by inhibiting complex I and IV of the respiratory chain, dysregulates the expression of divalent metal transporter 1(DMT1), and ferroportin 1 (FPN1) by activating IRP1, inhibits hepcidin release. These alterations consequently lead to abnormal accumulation of iron [53, 54] as has been demonstrated in our animal model. In line with these studies, our results showed that 6-OHDA injection in the MFB increased the levels of caspase 3, p53, and acetylcholinesterase, which might be subsequent to a mitochondrial dysfunction inducing ROS and cytochrome C release into the cytoplasm that mediates apoptosis [55]. Furthermore, autophagy may also be implicated in 6-OHDA-induced neurodegeneration, as it has been shown that 6-OHDA dysregulates autophagy by oligomerizing pro-apoptotic proteins; in particular; BCL2 Associated X(BAX) that leads to increased mitochondrial cytochrome C levels in the cytosol, and caspases activation [56]. In addition to p53 and caspase 3 overexpression, our results showed a significant increase of AChE in the plasma after 6-OHDA injection. Taken into account that the functional integrity of the basal ganglia relies on a balanced interaction between dopaminergic, cholinergic, GABAergic and glutamatergic systems, and the fact that overexpression of AChE contributes to cell apoptosis by altering apoptotic protease-activating-factor-1(Apaf-1) and cytochrome C [57, 58], we suggest that increased levels of AChE in the plasma of 6-OHDA animals may be due to dopaminergic cell loss in the nigrostriatal pathway. Increased levels of plasmatic AChE can be attributed to a compensatory response to the loss of functional cholinergic neurons [59, 60] or an increase in caspase-mediated cleavage of cytosolic AChE and subsequent a translocation of cleaved and full-length AChE into the cell nucleus [61]. On the other hand, exposure to subchronic restraint stress alone had no effect on iron accumulation, neither on DA turnover, p53, caspase 3 levels in the striatum or AChE levels in the plasma. A recent study showed that exposure to subchronic restraint stress 150 min /day for five consecutive days may induce apoptotic responses by increasing BAX/Bcl-2 ratio and elevating caspase 3 and caspase 9 levels in the prefrontal cortex and the hippocampus [62] which indicate that the nigrostriatal pathway might be less vulnerable to the deleterious effects of subchronic stress. However, when combined with 6-OHDA, restraint stress exacerbated the levels of caspase 3 and AChE which suggests that subchronic stress may reinforce the expression of pro-apoptotic factors and aggravate the loss of dopaminergic neurons in the nigrostriatal pathway. It was demonstrated that exposure to restraint stress for 7 days after 6-OHDA injection decreased the concentration of neurotrophic factors such as glial cell-line derived neurotrophic factor and Neurotrophin-3 in the nigrostriatal pathway which may consequently contribute to neurodegeneration [24, 63]. Taken together, these results give further support to 6-OHDA MFB lesion as a suitable model for understanding the neuronal substrates implicated in the pathological process of PD, suggesting a synergistic effect between restraint stress and 6-OHDA treatment, and indicating that stress may accelerate the progression of PD by triggering dopaminergic neurodegeneration and increasing oxidative stress and apoptotic processes.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study provides evidence that post-lesion stress may aggravate apoptosis through increasing caspase-3 and AChE activities and may alter motor coordination and exacerbate anxiety behaviour in the 6-OHDA animal model of PD. Further studies using a multi-environmental stress approach and complex cognitive tasks reflecting the human condition are needed to understand the mechanisms implicated in the aetiology and/or the progression of PD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eAll experiments were conducted in compliance with the ARRIVE guidelines and they were approved by the Animal Ethics Sub-committee of Mohammed V University in Rabat, Morocco.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e:\u0026nbsp;The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u0026nbsp;\u003c/strong\u003eS.E., K.T., M.A. and O.A. designed the study; S.E., N.F., H.I., and O.A. performed experiments and data analysis; S.E., N.F., M. A. and O.A. wrote the main manuscript text and prepared all figures; K.T., M.A. and O.A. supervised this study and all authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTrist, B.G., D.J. Hare, and K.L. 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Mabandla, \u003cem\u003ePost 6-OHDA lesion exposure to stress affects neurotrophic factor expression and aggravates motor impairment.\u003c/em\u003e Metabolic Brain Disease, 2017. \u003cstrong\u003e32\u003c/strong\u003e(4): p. 1061-1067.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurotoxicity-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ntre","sideBox":"Learn more about [Neurotoxicity Research](http://bacandrology.biomedcentral.com/)","snPcode":"12640","submissionUrl":"https://submission.nature.com/new-submission/12640/3","title":"Neurotoxicity Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Parkinson disease, restraint stress, medial forebrain bundle, dopamine, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-2196389/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2196389/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eActivation of the apoptotic pathway has been associated with promoting neuronal cell death in the pathophysiology of Parkinson disease (PD). Nonetheless, the mechanisms by which it may occurs remain unclear. It has been suggested that stress-induced oxidation and potentially apoptosis may play a major role in the progression of PD. Thus, in this study, we aimed to investigate the effect of subchronic restraint stress on striatal dopaminergic activity, iron, p53, caspase-3, and plasmatic acetylcholinesterase (AChE) levels in an animal model of PD induced by administration of 6-hydroxydopamine(6-OHDA) in the medial forebrain bundle (MFB). The obtained results showed that restraint stress exacerbates motor coordination deficits and anxiety in animals treated with 6-OHDA in comparison to animals receiving saline, and it had no effect on object recognition memory.\u003c/p\u003e \u003cp\u003eOn another hand, 6-OHDA decreased dopamine(DA) levels, increased iron accumulation, and induced overexpression of the pro-apoptotic factors caspase-3, p53, and AChE. More interestingly, post-lesion restraint stress exacerbated the expression of caspase-3 and AChE without affecting p53 expression. These findings suggest that subchronic stress may accentuate apoptosis and may contribute to DA neuronal loss in the striatal regions and possibly exacerbate the progression of PD.\u003c/p\u003e","manuscriptTitle":"Restraint stress exacerbates apoptosis in a 6-OHDA animal model of Parkinson disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-28 14:34:39","doi":"10.21203/rs.3.rs-2196389/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-10-30T11:13:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-10-29T10:52:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8a414bc5-2201-4ce5-95d9-3ebc3dd95d45","date":"2022-10-26T04:34:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-25T12:47:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-25T11:12:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-10-25T11:12:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurotoxicity Research","date":"2022-10-23T18:52:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurotoxicity-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ntre","sideBox":"Learn more about [Neurotoxicity Research](http://bacandrology.biomedcentral.com/)","snPcode":"12640","submissionUrl":"https://submission.nature.com/new-submission/12640/3","title":"Neurotoxicity Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"52f584d4-83a5-40ba-b940-e3eec13c16ae","owner":[],"postedDate":"October 28th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T18:24:18+00:00","versionOfRecord":{"articleIdentity":"rs-2196389","link":"https://doi.org/10.1007/s12640-022-00630-3","journal":{"identity":"neurotoxicity-research","isVorOnly":false,"title":"Neurotoxicity Research"},"publishedOn":"2023-01-12 18:18:16","publishedOnDateReadable":"January 12th, 2023"},"versionCreatedAt":"2022-10-28 14:34:39","video":"","vorDoi":"10.1007/s12640-022-00630-3","vorDoiUrl":"https://doi.org/10.1007/s12640-022-00630-3","workflowStages":[]},"version":"v1","identity":"rs-2196389","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2196389","identity":"rs-2196389","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}