Adult male Sprague Dawley rats (250-360 g) from Harlan Laboratories (Indianapolis, IN) were used (n=8-10/group). Animals were treated in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee. Rats were randomly divided into four weight-matched groups: two groups exposed to CVS and the other two serving as non-stressed control groups. Each of the two groups was divided again with half receiving the catecholamine-specific neurotoxin 6-OHDA and the other half receiving vehicle injections (control/vehicle, control/6-OHDA, CVS/vehicle, CVS/6-OHDA). Animals were anesthetized (87 mg/kg ketamine, 13 mg/kg xylazine; i.p.), placed into a stereotaxic apparatus, and received two unilateral injections of 6-OHDA (10 µg each in 2 µl saline + 0.2% ascorbic acid) or 6-OHDA vehicle (saline + ascorbic acid) placed into the right striatum [AP:+1.6mm, ML:-2.4, DV:-4.2 and AP:+0.2, ML:-2.6, DV:-7.0; coordinates according to the rat brain atlas of Paxinos and Watson (1)]. The striatal injection of 6-OHDA results in a relatively progressive degeneration of the dopaminergic nigrostriatal pathway (2,3).
The CVS regimen used has been characterized extensively (4,5). The following eight stressors composed the present CVS regimen: cold exposure (1h in a 4°C room), restraint (1h in plastic restraint tubes), vibration (1h on shaker), hypoxia (9% oxygen, 30 min), cold swim (10 min in 16-18°C water), warm swim (20 min in 31-33°C water), crowding (6 rats/cage, overnight), isolation (1 rat/cage, overnight). After surgeries, stressors requiring mobility (i.e. the swim stressors) were not administered. Stressors were administered in a random, unpredictable fashion to prevent habituation because habituating stressors do not produce depressive-like symptoms and do not result in nigral dopamine cell loss (6). Animals in the CVS groups were exposed to one stressor in the morning and one in the afternoon, with the overnight stressors administered intermitantly throughout the regimen. Stress-naïve controls consisted of rats that were handled briefly each day.
Forelimb asymmetry test
Behavioral data were collected using the forelimb asymmetry test (cylinder test) to assess differences in motor impairment as previously described (7). The cylinder test was administered during the dark cycle under low-light conditions, two and four weeks post-lesion for all experiments. Each rat was placed individually into a transparent Plexiglas cylinder and allowed to naturally explore the walls by rearing up and using their forelimbs for weight support. A rat was considered to have completed the test when it had performed 20-25 touches. A percent limb usage score was calculated from the behavioral data using the formula [(contralateral side + ½ both) / (ipsilateral side + contralateral side + both)] x 100. A score of 50% indicates equal use of both forelimbs. Statistical analyses included one-way ANOVA followed by Neuman-Kuels post-hoc test. Data are presented as mean ± SEM. Significance is considered at P < 0.05.
One day after the final behavior test, the animals were sacrificed and processed for tyrosine hydroxylase (TH; catecholamine biosynthetic enzyme and marker for midbrain dopamine cells) and NeuN (a general neuronal marker) immunohistochemistry as previously described (8). In each group, cells immunostained for TH and NeuN were counted in the SNpc on evenly spaced sections throughout the rostrocaudal extent of the ventral mesencephalon. Cell estimates of both the lesioned and unlesioned sides of the SNpc were determined using Stereo Investigator 5.05 (MicroBrighfield, Williston, VT) utilizing unbiased stereological techniques (9). The sections were viewed on an Olympus BX-60 microscope (Melville, NY) using a CCD video camera (HV-C20, Hitachi, San Jose, CA). Contours were determined at 2.5X magnification and cell counting was performed at 60X using the optical fractionator. Random sample sites were determined by the software on a grid size of 170x100 for TH immunostaining and 220x220 for NeuN immunostaining. Each sample section had a guard of 2 µm placed. The Gundersen correction was used to calculate the coefficient of error for each animal and was 0.10 or lower. The extent of cell loss was determined by comparing the lesioned side to the contralateral unlesioned side of the SNpc. Statistical analyses included one-way ANOVA followed by Neuman-Kuels post-hoc test. Data are presented as mean ± SEM. Significance is considered at P < 0.05.