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Early life stress, such as maternal separation, causes adaptive changes in neural mechanisms that have adverse effects on the neuroplasticity of the brain in adulthood. As a consequence, children who are exposed to stress during developmentmay be predisposed to neurodegenerative disorders in adulthood. A possible mechanism for increased vulnerability to neurodegeneration may be dysfunctional mitochondria. Protection from neurotoxins, such as 6- hydroxydopamine (6-OHDA), has been observed following voluntary exercise. The mechanism of this neuroprotection is not understood and mitochondria may play a role. The purpose of this study was to determine the effects of maternal separation and exercise on mitochondrial function in a rat model of Parkinson’s disease. Maternally separated (pups separated from the dam for 3 h per day from postnatal day (P) 2-14) and non-separated rats were placed in individual cages with or without attached running wheels for 1 week prior to unilateral infusion of 6-OHDA (5 μg/4 μl, 0.5 μl/min) into the leftmedial forebrain bundle at P60. After 2 h recovery, rats were returned to their cages and wheel revolutions recorded for a further 2 weeks. On P72, the rats’ motor function was assessed using the forelimb akinesia test. On P74, rats were sacrificed for measurement of mitochondrial function. Exercise increased the respiratory control index (RCI) in the non-lesioned hemisphere of 6-OHDA-lesioned rats. This effect was evident in the striatum of non-separated rats and the prefrontal cortex of maternally separated rats. These results suggest that early life stress may reduce the adaptive response to exercise in the striatum, a major target of dopamine neurons, but not the prefrontal cortex in this model of Parkinson’s disease. © The Author(s) 2012.
Thirty-nine male Sprague–Dawley rats (Rattus Norvegicus) weighing between 180 g and 280 g were used in the study. Rats were housed in a temperature-controlled room (21–24°C) in the Satellite Animal Facility at the University of Cape Town. Animals had access to standard rat chow and water ad libitum. The housing facility was maintained on a 12-hour light/dark cycle (lights on at 06 h00). The study was approved by the Faculty of Health Sciences Animal Research Ethics Committee of the University of Cape Town and adhered to international guidelines. On postnatal day 1 (P1), rats were sexed and culled to 8 males per litter. If the litter had less than 8 males, female littermates were added to standardize litter size and to allow for equal suckling from the dam. Maternal separation was performed as described by Daniels et al. (2004). For the experimental group the mother was physically removed from the home cage and placed in a separate clean plexiglass cage with clean bedding from P2 to P14. A small amount of bedding from the home cage was placed in the holding cage to decrease the stress the dam might be exposed to due to the separation. The pups were taken to a different room (separation room) to prevent communication with the dam (by means of ultrasound vocalization) for a period of 3 h from 09 h00 to 12 h00 daily. The temperature in the separation room was maintained between 31 and 34°C to prevent hypothermia. After the 3-hour separation period the pups were returned to the Satellite Animal Facility and reunited with their dams. The home cage was cleaned every fourth day and great care was taken not to physically disturb the pups and to minimize other factors (smell, sound) that could cause stress to the rat pups. During the cleaning of the home cages, the dam was removed and about half of the bedding which was not occupied by the pups was changed to ensure that the dam recognised the odour of her cage and her litter when returned to her home cage. The non-separated pups were kept with their dams in their home cages, under normal housing conditions in the Satellite Animal Facility. After P14 the litters were left with their dams until P21 when the litters were weaned. From P21 to P48 the rats were reared under normal housing conditions (standard rat chow and water ad libitum). On P48 rats were transferred to a room with a 23 h00-11 h00 light/dark cycle in order to facilitate recording and observation of their activity. After 6 days of adaptation to the new light/dark cycle, on P54, 20 maternally separated rats and 19 non-maternally separated rats were weighed and further divided into exercised (runners) and non-exercised (non-runners) groups. Ten maternally separated (MS) rats, and 9 non-separated (NS) rats were placed in cages with attached running wheels with a counter device that recorded the number of revolutions. One complete revolution measured one meter in distance. The remaining maternally separated rats (MS non-runners, n = 10) and non-separated rats (NS non-runners, n = 10) were placed in plexiglass cages that allowed 2 rats to be housed separately using a divider. The number of revolutions the rats made in the running wheels was recorded daily between 10 h00 and 11 h00 prior to the onset of their dark cycle. Stereotaxic surgery was performed according to Mabandla et al. (2004). On P60, the rats were weighed and taken to the surgery room. A norepinephrine transporter blocker, desipramine (15 mg/kg, Sigma St. Louis, MO, U.S.A), was injected intraperitoneally 30 min before surgery to prevent uptake of the neurotoxin, 6-OHDA, by noradrenergic neurons. Rats were deeply anaesthetised with a mixture of halothane and oxygen administered by means of a calibrated Blease Vaporiser (DATUM). All rats received 6-OHDA.HCl (5 μg/4 μl saline, Sigma, St. Louis, MO, U.S.A) infusion unilaterally (0.4 μl/min) into the left medial forebrain bundle at the following stereotaxic co-ordinates: 4.7 mm anterior to lambda, 1.6 mm lateral to midline and 8.4 mm ventral to dura, according to the rat brain atlas of Paxinos and Watson (1986). The infusion needle remained in the medial forebrain bundle for a period of 4 min post infusion to allow the neurotoxin to disperse into the tissue. Following gentle retraction of the needle, the burr-hole was sealed with sterile bone-wax and the scalp incision was swabbed with betadine/alcohol solution and sutured. The rats were allowed to recover from anaesthesia before they were returned to their respective cages. The number of revolutions made by the rats in cages with attached running wheels was recorded for a further 14 days after the surgery. On P72 (twelve days post lesion), the rats were assessed for motor function deficit. The rats were taken from the Satellite Facility at least 1 h before testing so as to enable the rats to acclimatize to the new conditions in the behavioural testing room. The light intensity in the behavioural testing room was 48 lux. The step test provides a robust measure of impairment in movement initiation, and thus measures the severity of the effect of the 6-OHDA lesion on limb function (Tillerson et al. 2001). The rat was supported by its torso, such that the hindquarters and the forelimb not being tested were held above the testing surface, allowing the rat to transfer its bodyweight onto the forelimb being tested. With the use of the thumb and index finger, the forelimb not being tested and head were held firmly to minimize head movement and to simultaneously gently propel the rat forward. The length of step of each forelimb was recorded in 3 successive trials and the mean step length was calculated. On P74, rats were sacrificed and their brains rapidly removed from the skull for measurement of mitochondrial function using an Oxygraph oxygen electrode (Hansatech Instruments, UK). Left and right prefrontal cortex and striatum were rapidly dissected from the brain, on an ice-cold glass plate, weighed and homogenized in 20 mM phosphate buffer (20 mM potassium phosphate, 20 mM potassium chloride, 1.6 mM EDTA, 5 mM magnesium chloride, 1 mM sodium malate, 10 mM sodium pyruvate 123 mM sucrose, 2 mM Tris, pH 7.4) and oxygen consumption measured in an Oxygraph, as an index of mitochondrial function. Two ml of homogenate was placed in the Oxygraph chamber and allowed to equilibrate to 39°C. Basal respiration was recorded for 5–10 s prior to addition of 1 mM adenosine diphosphate (ADP) to the chamber and oxygen tension was recorded until depletion. Respiratory control index (RCI) which indicates the tightness of coupling between respiration and phosphorylation, was calculated from the rate of oxygen consumption during ADP oxidation to ATP relative to the rate of oxygen consumption after ADP depletion. The phosphate:oxygen ratio (P:O) was also calculated from the oxygen consumption data. The P:O ratio provides a measure of the relationship between ATP synthesised and oxygen consumed. The validity of the technique was demonstrated by addition of NADH to the brain tissue homogenate prior to the measurement of oxygen consumption which did not alter the rate of respiration, thus confirming that the mitochondria were intact. The rate of oxygen consumption was not influenced by aeration of the homogenate, indicating that sufficient oxygen was present in the sample to measure mitochondrial respiration. The mean RCI was 10.8 which compares favourably with reported values of 2.2–4.9 for isolated brain mitochondria (Sugiyama and Fujita 1985; Xiong et al. 1999; Costa and Colleoni 2000). Protein concentration was determined according to the method described by Bradford (1976). Briefly, the Bradford protein assay is a colorimetric assay. A series of protein standards were prepared (0–1500 μg BSA/ml). Serial dilutions of the sample to be measured were also prepared. Each sample (standard and unknown) was pipetted into a spectrophotometer tube before adding a dye for the color reaction. The absorbance was measured using a spectrophotometer. The absorbance of the standards versus their concentration was plotted on graph paper following which the concentration of the unknown sample was extrapolated. Statistica (Statsoft Inc., Oklahoma, USA) was used for statistical analysis of the data. The t-test for dependent variables was used to determine differences between lesioned and non-lesioned hemispheres. Two-way analysis of variance (ANOVA) was performed to detect significant differences between treatment groups. Significant difference was accepted at p < 0.05. When the ANOVA showed a significant difference, the Newman-Keuls post-hoc test was used. Results are reported as mean ± standard error of the mean (mean ± SEM).
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