Restraint Stress Exacerbates Apoptosis in a 6-OHDA Animal Model of Parkinson Disease

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 occur remain unclear. It has been suggested that stress-induced oxidation and potential 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 male Wistar rat 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.


Introduction
PD is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) (Trist et al. 2019;Bae et al. 2021). PD is the second most common neurodegenerative disorder with a prevalence of 2-3% within the population above 65 years of age (Dorsey et al. 2018). Several studies have shown males are more susceptible to PD, with a male-female ratio that varies between 1.37 and 3.7 (Armstrong and Okun 2020; Moisan et al. 2016). PD presents a serious burden on the patient and caregivers (Rocca 2018). 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 andSchapira 2020). The neuropathological hallmark of PD includes neurodegeneration, neuroinflammation, altered dopaminergic activity, and increased oxidative stress in the nigrostriatal pathway (Kouli et al. 2018;Luo et al. 2022). Many risk factors were proposed to be involved in the pathogenesis and the progression of PD including environmental stress-induced increased cortisol levels (Belvisi et al. 2020). 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 (Dodiya et al. 2020;Howells et al. 2005;Mitsumoto and Mori 2018). Furthermore, motor deficits associated with PD, on their own, may present a stressful event that contributes to the aggravation of PD's neuropathology (Blakemore et al. 2018). Clinical studies have shown that stress-increased cortisol is associated with the worst functional scores for motor and cognitive symptoms of PD (Soares et al. 2019). Several animal models were developed to understand the physiopathology of PD. Among these models, the 6-OHDA animal model (Simola et al. 2007). It was demonstrated that unilateral injection of 6-OHDA into the substantia nigra pars compacta (SNpc) leads to a rapid onset of neuronal loss (Casarrubea et al. 2019), 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 (Yuan et al. 2005;Rentsch et al. 2019;Bagga et al. 2015). 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 (Dixon and Stockwell 2014;Onukwufor et al. 2022). 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 (Nnah and Wessling-Resnick 2018). The deleterious effects of 6-OHDA can be aggravated by stress which was suggested to play a role in the onset of PD (Grigoruţă et al. 2020;Sugama et al. 2016). It has been shown that chronic restraint stress triggers dopaminergic and noradrenergic neurodegeneration via apoptosis by activating caspase enzymes, priorly expressed as inactive precursors lacking protease activity (Sugama et al. 2016;Salvesen and Riedl 2008). Nevertheless, apoptosis, which is a mode of programmed cell death (PCD), is crucial for mammalian development as it controls cell numbers and tissue development and clears damaged structures (Grilo and Mantalaris 2019;Saleem 2021). 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 (Luo et al. 2022;Morrison and Kinoshita 2000) as it controls DNA damage, senescence, and ROS formation, as well as activating pro-apoptotic and suppressing antiapoptotic proteins (Luo et al. 2022;Miller et al. 2000). 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 (Hartmann et al. 2000). 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 (Erekat 2018). 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 (Davoli et al. 2002;Kitazawa et al. 2003;Kahns et al. 2002). 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 (Greenblatt et al. 2003;Walczak-Nowicka and Herbet 2021). It has been shown that decreased expression of AChE reduces apoptotic markers in different cell lines, whereas augmented AChE activity increases apoptosis (Price et al. 2020). Based on these studies, we hypothesized 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.

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 randomized blinded study. The animals were housed under standard laboratory conditions, a 12-h 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-day restraint stress; and the (4) 6-OHDA + stress group. Restraint stress was applied 1 week following 6-OHDA or vehicle administration into the MFB. The experimental paradigm is summarized 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.

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 sterilized before surgery. Rats (240-310 g) were injected intraperitoneally (i.p.) with desipramine at 15 mg/ kg (Sigma-Aldrich) 30 min 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 (Howells et al. 2005), 6-OHDA or vehicle was injected into the left MFB at a rate of 1 μl per 2 min 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 (Howells et al. 2005;Yang et al. 2018). The rate of infusion was set at 1μl / 2 min. To facilitate optimal diffusion of the solution, the needle was left in place for an additional 5 min 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 min 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 × 28 mm diameter) for 3 h 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.

Open-Field Test
The open-field test was conducted in a rectangular box (100 cm length × 100 cm width × 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 m 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 the Anymaze software (Stoelting, Co.). The apparatus was cleaned with 70% alcohol after each trial.

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 familiarized 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 follows: DI = [(time spent exploring the novel object − time spent exploring the familiar object)/ total exploration time] (Abboussi et al. 2016).

The Beam-Walking Test
The beam-walking test consisted of a training phase and a test session conducted in an experimentation room illuminated at 70 lx. 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. The black box contained nesting material from the animal's home cage. Twenty-four hours later, animals were tested on the

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 and 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 (Sadeghian et al. 2022).

Sample Preparation
Twenty-four hours after that last behavioural test, the animals were decapitated using a guillotine, and the striatal brain structures were dissected out using plastic forceps (Sigma-Aldrich, USA) to minimize trace metal ion contamination. Waxholm space rat brain atlas was used to determine the striatal brain structures. Briefly, the superior boundary of the striatum was defined by the corpus callosum, the lateral boundary by the external capsule, and the medial boundary by the lateral ventricle and the corpus callosum. The inferior boundary of the striatum lies between grey matter structures and was delineated by a line drawn from the corpus callosum at the level of the rhinal fissure, extending to the lower boundary of the lateral ventricle and excluding the nucleus accumbens (Papp et al. 2014). The dissected striatal region was washed with phosphate buffer solution (1 × 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 min. The plasma was separated in the supernatant and kept at -80 °C until further use.

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 was 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 (Abboussi et al. 2020).

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 Levy et al. (2001). 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. Seventy per cent of 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 h. The samples were centrifuged at 600 g for 1 h and subsequently filtersyringed 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 analyse 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.

Assay Protocol for Caspase-3, 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 46.875 pg/ml for p53, 0.188 ng/ml for caspase-3, and 0.47 ng/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.

Statistical Analysis
We tested normality using the Shapiro-Wilk test. Data that assumed Gaussian distribution were analysed using a twoway 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 was set at p < 0.05.

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. 3A) neither

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).

Restraint Stress Exacerbates the Increase of Striatal Caspase-3 Levels 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.

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 stressrelated 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-OHDAinduced coordination deficits were exacerbated by postlesion 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 (Zhou et al. 2015). Also, it has been previously shown that 6-OHDA Values were analysed using two-way ANOVA followed by Bonferroni post-test; *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001 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 (Grigoruţă et al. 2020;Smith et al. 2008). 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 (Vieira et al. 2019). Accordingly, restraint stress may exacerbate anxietylike 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 (Sugama et al. 2016). 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 (Marshall et al. 2019). 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 (Ma et al. 2014). Moreover, it has been shown that an extended restraint stress duration such as 4 h/day during 14 consecutive days may alter motor, emotional, and cognitive functions in rats (Peay et al. 2020;Olave et al. 2022) 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 caspase-3, p53, and elevated plasmatic AChE level. The unchanged DA/DOPAC ratio between control, 6-OHDA, and 6-OHDA + restraint stress groups suggests 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 (Rentsch et al. 2019;Poletti et al. 2021). 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, and inhibits hepcidin release. These alterations consequently lead to abnormal accumulation of iron (Mallet et al. 2022;Xu et al. 2022) 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 (Sodhi et al. 2021). Furthermore, autophagy may also be implicated in 6-OHDA-induced neurodegeneration, as it has been shown that 6-OHDA dysregulates autophagy by oligomerizing proapoptotic proteins; in particular; BCL2 associated X(BAX) that leads to increased mitochondrial cytochrome C levels in the cytosol and caspase activation (Chung et al. 2021). 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 proteaseactivating-factor-1 (Apaf-1) and cytochrome C (Knorr et al. 2020;Junn and Mouradian 2001), 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 (Ali et al. 2015;Rehfeldt et al. 2022) or an increase in caspase-mediated cleavage of cytosolic AChE and subsequent a translocation of cleaved and full-length AChE into the cell nucleus (Xie et al. 2011). 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, nor AChE levels in the plasma. A recent study showed that exposure to subchronic restraint stress 150 min/day for 5 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 (Salehpour et al. 2019) 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 (Sugama et al. 2016;Ngema and Mabandla 2017). 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.