In this study, we employed a between-group experimental design to examine the effects of moderate exercise on motor and brain function in a PD rat model. Behavioral analyses, ROI selection, and c-Fos/TH quantification were performed without blinding to group allocation.

Fifteen-week-old male unilateral PD rats with medial forebrain bundle (MFB) lesions (MFB model) were used. Following confirmation of MFB lesions by the apomorphine-induced rotation test, ten rats were randomly assigned to either the exercise group (EX; n = 5) or the sedentary control group (SED; n = 5).

The animals were housed individually in cages under a 12-h light/dark cycle. Water was provided ad libitum. Food intake was restricted to maintain body weight at 90% - 95% of the initial body weight to enhance learning and task performance during the behavioral experiments.

Unilateral 6-OHDA-lesioned Wistar rats were purchased from Japan SLC Inc. (Shizuoka, Japan). The lesions had been produced by stereotaxic injection of 4 µL of 6-OHDA (2.25 mg/mL) into the right medial forebrain bundle after intraperitoneal administration of desipramine (25 mg/kg), a neuroprotective noradrenergic agent. The stereotaxic coordinates were 4.4 mm posterior to bregma, 1.5 mm lateral to the midline, and 7.8 mm ventral to the skull surface.

In all animals, the right hemisphere was designated as the lesioned side. Apomorphine-induced rotation tests were performed at 15 weeks of age. Rats exhibiting ≥7 contralateral rotations per minute were considered to have successful MFB lesions, and only these rats were included in the study.

Rats in the EX group performed treadmill exercise on a small-animal treadmill (KN-37; Natsume Seisakusho Co., Ltd., Tokyo, Japan) five times per week for four consecutive weeks (each session lasted 30 min). The exercise intensity gradually increased as follows: 5 - 10 m/min in week 1, 10 - 15 m/min in weeks 2 - 3, and 15 m/min in week 4. Rats in the SED group were placed on a treadmill for the same duration, with the belt speed set at 0 m/min.

The treadmill speed of 15 m/min was selected based on previous studies demonstrating that this workload is below the lactate threshold in rats and is generally considered to represent moderate-intensity exercise [11].

2.5.1. General Conditions

To assess exercise-induced excessive workload and stress, changes in body weight were calculated by subtracting the body weight measured on the day before the intervention from that measured on the final day of the intervention.

2.5.2. Behavioral Assessment Methods

1)Beam-WalkingTest

Beam-walking tests were conducted as previously described for PD rat models [8]. Briefly, behavioral performance during the task was recorded simultaneously in four directions (frontal, left lateral, right lateral, and top) using a web camera. Video recordings were acquired at 720 p resolution and 60 frames/s. Measurements were obtained before the exercise intervention and on the day after its completion, and were analyzed for differences between post- and pre-intervention values.

a) Number of Footslips

The number of slips of the fore and hind limbs within the measurement zone was visually counted from the lateral-view recordings.

b) Task Completion Time.

The time required to traverse the 500-mm measurement zone was measured using lateral-view recordings.

c) Image analysis.

i) Frontal View

Head inclination angle was calculated using the line connecting both eyes (1) and a reference line connecting two points on the beam projected on the screen (2) (Figure 1(a)). Mean and maximum head inclination angles within the measurement zone were recorded.

ii) Lateral View (Lesioned Side)

Head inclination angle was calculated using the line connecting the nose and ear canal (1) and a reference line connecting two points on the tape attached to the beam (2) (Figure 1(b)). The stride length was calculated based on forelimb heel coordinates (3). The mean, maximum, and minimum head inclination angles were analyzed, and the stride length was calculated as the average of two consecutive steps (Figure 1(c)).

iii) Top View

Head inclination angle was calculated using the line connecting the midpoint between the nose and both ears (1) and the beam reference line (3) (Figure 1(d)). Trunk inclination angle was calculated using the line connecting the midpoint between both ears and the base of the tail (2) together with the same beam reference line (3). The mean and maximum values of the head and trunk inclination angles were used in the analysis.

Figure 1.Analysis of gait and posture during beam walking. (a) Frontal view analysis: Head inclination angle was calculated using the line connecting both eyes (1) and a reference line connecting two points on the beam projected on the screen (2). The travel time within the measurement zone was determined using the clock displayed in the frontal and right-lateral images. ((b), (c)) Lateral view: Head inclination angle was calculated using the line connecting the nose and ear canal (1) and a reference line connecting two points on the tape attached to the beam (2) (b). Stride length was calculated based on the DeepLabCut coordinates of the forelimb heel (3) (c). (d) Top view: Head inclination angle was calculated using the line connecting the midpoint between the nose and ears (1) and the beam reference line (3). Trunk inclination angle was calculated using the line connecting the midpoint between ears and the base of the tail (2) together with the same beam reference line (3). Adapted from Ohno and Chigira [8], Open Veterinary Journal, 2025.

2)Apomorphine-InducedRotationTest

Apomorphine was diluted to 1.0 mg/mL saline and administered intraperitoneally at 1.0 mg/kg. The rotation test was conducted for 30 min in a cylindrical container (28 cm diameter).

The total number of rotations toward the intact hemisphere was counted for 20 min, excluding the first 5 min after rotation onset and the final 5 min of recording. Measurements were conducted the day after the intervention.

2.6.1. Tissue Collection and Processing

Kovács reported that Fos protein typically peaks 90 - 120 min after stimulation [12]. Therefore, tissues were collected 90 min after treadmill exercise at 15 m/min, corresponding to the exercise intensity used in week 4, on day 2 after the completion of the exercise intervention in the EX group. Rats in the SED group did not perform treadmill exercise before perfusion. To minimize the influence of circadian variation on c-Fos expression, the SED group was perfused at the same time of day as the EX group on the following day.

The rats were deeply anesthetized with a mixture of ketamine and xylazine and transcardially perfused with heparinized saline at room temperature, followed by perfusion fixation with 4% paraformaldehyde in phosphate-buffered saline. Brains were immediately removed and fixed with 4% paraformaldehyde.

2.6.2. Tyrosine Hydroxylase (TH) and c-Fos Immunohistochemistry

Immunohistochemical staining for TH and c-Fos was performed on paraffin-embedded coronal sections. Brain tissue was collected at the striatal level corresponding to bregma +0.2 mm, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 4 µm.

Sections were deparaffinized in xylene, rehydrated using a graded ethanol series, and washed with Tris-buffered saline (TBS). Antigen retrieval was performed by autoclaving at 120˚C for 10 min. Endogenous peroxidase activity was blocked with 0.3% H₂O₂ in methanol, and nonspecific binding was blocked using skim milk in TBS.

Sections were incubated overnight at 4˚C with rabbit monoclonal anti-TH (E2L6M) or anti-c-Fos (E2I7R) XP primary antibodies (1:100; Cell Signaling Technology, Danvers, MA, USA), washed with TBS, and incubated with SignalStain Boost IHC Detection Reagent (HRP, rabbit; Cell Signaling Technology, Danvers, MA, USA). Immunoreactivity was visualized using 3,3’-diaminobenzidine, and nuclei were counterstained with Mayer’s hematoxylin. The sections were dehydrated in a graded ethanol series, cleared in xylene, and coverslipped.

2.6.3. Immunohistochemical Image Analyses

1)THImmunostaining

For TH staining, we calculated the percentage of TH-positive areas relative to the total area within the region of interest (ROI) in lesioned and intact hemispheres (Figure 2(a)). The ROIs were defined using the midline, dorsal, ventral, and lateral striatal boundaries as anatomical landmarks.

Image analysis was performed using the Fiji platform of the open-source software ImageJ (v.2.3.0) [13]. Images were captured under a digital microscope (LPE-06BK; Sanwa Supply Inc., Osaka, Japan).

2)c-FosImmunostaining

For c-Fos staining, the striatum in each hemisphere was divided into the dorsomedial, dorsolateral, ventromedial, and ventrolateral subregions (Figure 2(b)). In each subregion, c-Fos-positive cells were visually counted within a rectangular ROI (100 µm in height × 300 µm in width) positioned near the center of the region, and the total number of positive cells was calculated. Images were acquired using a biological microscope (BA81; Shimadzu Rika Corporation).

Figure 2. Tyrosine hydroxylase (TH) and c-Fos immunostaining in the striatum. (a) Representative images of TH immunostaining showing coronal brain sections at the striatal level corresponding to bregma +0.2 mm. Regions of interest (ROIs) were defined in lesioned and intact hemispheres, and the percentage of TH-positive areas was calculated relative to the total ROI area. Scale bar = 5 mm. (b) Representative c-Fos immunostaining images are shown. The striatum was divided into dorsomedial, dorsolateral, ventromedial, and ventrolateral subregions. The number of c-Fos-positive cells was counted within a rectangular ROI (100 µm in height × 300 µm in width) placed at the center of each subregion, and the total number of positive cells was calculated. Arrows indicate c-Fos-positive cells. Scale bar = 100 µm.

Between-group differences in change scores (post-intervention minus pre-intervention values) were analyzed to compare the effects of the exercise intervention between the two groups. The Shapiro-Wilk test was used to assess normality. Levene’s test was used to evaluate the homogeneity of variance in normally distributed data. Student’s t-test and Welch’s t-test were used for normally distributed data with equal and unequal variances, respectively. The Mann-Whitney U test was used for data that were not normally distributed. Statistical significance was set at p < 0.05. Data are presented as mean ± standard error.

Portions of the manuscript were translated from Japanese to English using ChatGPT (OpenAI, San Francisco, CA, USA). The generated content was carefully reviewed and edited by the authors to ensure accuracy, clarity, and consistency. The authors take full responsibility for the final content of the manuscript.

Body weight changes did not differ significantly between the groups. The change in body weight was 13.60 ± 4.41 and 16.50 ± 1.08 g in the EX and SED groups, respectively (Student’s t-test).

3.2.1. Number of Footslips

No footslips were observed in either group within the measurement zone during the beam-walking test.

3.2.2. Task Completion Time

The change in task completion time was −0.15 ± 0.22 s in the EX group and −0.85 ± 0.73 s in the SED group, with no significant differences between them (Student’s t-test).

3.2.3. DeepLabCut-Based Gait and Posture Analyses

1)FrontalView(HeadInclinationAngle)

The mean head inclination angle was −1.34˚ ± 0.11˚ in the EX group and −1.97˚ ± 0.48˚ in the SED group, with no significant difference between groups (Student’s t-test).

The maximum head inclination angle was −0.83˚ ± 0.48˚ in the EX group and −0.88˚ ± 1.10˚ in the SED group, with no significant difference between groups (Student’s t-test).

2)LateralView

a) Head-inclination angles

The mean head inclination angle was 6.72˚ ± 4.19˚ in the EX group and 9.79˚ ± 2.47˚ in the SED group, with no significant difference between groups (Student’s t-test).

The maximum head inclination angle was 5.92˚ ± 3.93˚ in the EX group and 5.07˚ ± 3.47˚ in the SED group, with no significant difference between groups (Student’s t-test).

The minimum head inclination angle was 12.61˚ ± 5.71˚ in the EX group and 18.70˚ ± 6.36˚ in the SED group, with no significant difference between groups (Student’s t-test).

b) Stride Length

Stride length was 27.92 ± 6.84 mm in the EX group and 37.02 ± 9.21 mm in the SED group, with no significant difference between groups (Student’s t-test).

3)TopView

a) Head-inclination angles

The mean head inclination angle was −5.66˚ ± 1.79˚ in the EX group and −6.79˚ ± 1.58˚ in the SED group, with no significant difference between groups (Student’s t-test).

The maximum head inclination angle was −8.14˚ ± 2.05˚ in the EX group and −3.82˚ ± 5.80˚ in the SED group, with no significant difference between groups (Student’s t-test).

b) Trunk Inclination Angle

The mean trunk inclination angle was 2.66˚ ± 1.06˚ in the EX group and 3.79˚ ± 1.11˚ in the SED group, with no significant difference between groups (Student’s t-test).

The maximum trunk inclination angle was 2.40˚ ± 3.44˚ in the EX group and 10.23˚ ± 3.99˚ in the SED group, with no significant difference between groups (Student’s t-test).

3.2.4. Apomorphine-Induced Rotation Test

The total number of rotations toward the intact side was 286.20 ± 27.44 in the EX group and 224.20 ± 43.17 in the SED group, with no significant difference between groups (Student’s t-test).

3.3.1. TH Immunostaining

In the intact hemisphere, the percentage of TH-positive area relative to the total ROI area was 13.74% ± 0.39% in the EX group and 14.77% ± 2.83% in the SED group, with no significant difference between groups (Welch’s t-test).

In the lesioned hemisphere, the percentage of TH-positive area was 0.83% ± 0.22% in the EX group and 1.32% ± 0.35% in the SED group, with no significant difference between groups (Student’s t-test).

3.3.2. c-Fos Immunostaining

In the intact hemisphere, the total number of c-Fos-positive cells was 0.80 ± 0.37 in the EX group and 0.00 ± 0.00 in the SED group, with no significant difference between groups (Mann-Whitney U test).

In the lesioned hemisphere, the total number of c-Fos-positive cells was significantly higher in the EX group (2.40 ± 0.67) than in the SED group (0.40 ± 0.40) (p < 0.05, Mann-Whitney U test).