Benefits of progressive resistance training on motor performance and muscular hypertrophy in rats with Parkinson’s disease

ABSTRACT Parkinson’s disease (PD) is a progressive neurodegenerative condition defined by the presence of primary debilitating motor symptoms. This study aims to investigate the benefits of high-intensity progressive resistance training on muscle tissue and motor performance before and after the induction of PD in rats. A total of 80 male Wistar rats (Rattus norvegicus, var. albinus) aged 40 days and weighing between 250 and 450g were used. A total of 40 animals were subjected to PD surgery to induce electrolytic injury and were randomly assigned to the following subgroups: animals trained before PD induction (PA-Exa); animals trained after PD induction (PA-Exd); animals trained before and after PD induction (PA-Exad); and sedentary animals with PD induction (PA-Sed). The other 40 animals (control) were subjected to surgical access but not to PD electrolytic injury (Sham) and distributed in the same subgroups described above. For the PD induction surgery, electrolytic stimulation was used at the following coordinates: anteroposterior (AP) −4.9, mid-lateral (ML) 1.7,and dorsoventral (DV) 8.1. High-intensity progressive resistance training was performed on a vertical ladder five days/week from 30 to 45 minutes for four weeks. For our functional evaluation, the parallel bars and the misstep tests were used at the beginning (after surgery) and at the end of the experiment. After euthanasia, the forelimb biceps and hindlimb flexor hallucis longus muscles were removed. Processing, coloration, and histomorphometry analysis of muscle tissue were performed for all groups. To analyze the data, GraphPad Prism 9.4 was used with one-way analysis of variance (ANOVA) and a p<0.05. Data on muscle fiber count and area in forelimb biceps showed no significant differences, with a 0.853 and 0.4122 p-value, respectively. Flexor hallucis longus muscle fiber count showed a significant difference (p=0.0356), and PA-Exa and PA-Exd averaged the highest means. Hindlimb flexor hallucis longus muscle fiber area also evinced a significant difference (p=0.0306), in which PA-Exd, PA-Exad, and Sham-Exad showed the largest areas. Analysis of hindlegs in the parallel bars test showed that PA-Exad evinced the best functional performance. In the misstep test, we observed an increase in the number of errors animals made for almost all the groups, evincing a significant difference in the number of errors before and after the test only for PA-Exa, PA-Exd, and PA-Sed. We concluded that the animals that underwent high-intensity progressive training showed better performance in their hindlegs than in their fore ones and that animals that exercised before and after surgery benefited more from training.

To analyze the data, GraphPad Prism 9.4 was used with one-way analysis of variance (ANOVA) and a p<0.05.
Data on muscle fiber count and area in forelimb biceps showed no significant differences, with a 0.853 and 0.4122 p-value, respectively. Flexor hallucis longus muscle fiber count showed a significant difference (p=0.0356), and PA-Exa and PA-Exd averaged the highest means.
Hindlimb flexor hallucis longus muscle fiber area also evinced a significant difference (p=0.0306), in which PA-Exd, PA-Exad, and Sham-Exad showed the largest areas. Analysis of hindlegs in the parallel bars test showed that PA-Exad evinced the best functional performance. In the misstep test, we observed an increase in the number of errors animals made for almost all the groups, evincing a significant difference in the number of errors before and after the test only for PA-Exa, PA-Exd, and PA-Sed. We concluded that the animals that underwent high-intensity progressive training showed better performance in their hindlegs than in their fore ones and that animals that exercised before and after surgery benefited more from training.

INTRODUCTION
Parkinson's disease (PD) shows a higher incidence than other neurological disorders, second only to Alzheimer's disease. Discovered by James Parkinson in 1817, it consists of a progressive neurodegenerative comorbidity defined by the presence of primary debilitating motor symptoms, such as bradykinesia-hypokinesia, resting tremors, muscle rigidity, loss of postural reflexes, freezing of gait, facial muscle hypertonia (expressionless face -"parkinsonian mask") and trunk and elbow flexion ("skier's posture"), and secondary motor symptoms, e.g., hypomimia, dysphagia, and micrographia. The disease also shows non-motor symptoms, including bowel dysfunction, fatigue, depression, cognitive decline, sleep disorders, and loss of sense of smell [1][2][3][4] .
Primary parkinsonism shows, as its fundamental biochemistry, decreased nigro-striatal dopaminergic neurotransmission in basal nuclei due to the degeneration of brainstem dopaminergic neurons of the compact part of the substantia nigra, the caudate nucleus, the putamen, and the norepinephrine-containing neurons of the locus ceruleus. Some of the main cells of the nervous system that resist degeneration display eosinophilic cytoplasmic inclusions (Lewis bodies), the characteristic pathological finding of PD. This decrease in dopamine in the nigrostriatal pathway unbalances dopaminergic (decreased) and intrastriatal cholinergic activities (increased), functionally disorganizing the extrapyramidal system (extrapyramidal lesion), resulting in constant muscle contraction due to an excess of acetylcholine in the synaptic cleft and causing the main symptoms of PD: interferences in muscle tone and reduction of postural and involuntary movements 5-8 . According to data from the World Health Organization, PD affects more than 1% of the population aged over 65 years. In Brazil, estimates suggest that about 200,000 people carry the disease and that this number is expected to increase to more than 600,000 individuals by 2030 [9][10][11] .
Considering the aging population and the impact of the disease on the economy and society -since PD can affect the economically active population, social security, and public and private health systems, the development of more accessible and effective treatments, such as physical exercise, is extremely relevant. This preventive and therapeutic alternative for various diseases has been associated with neuroprotective and activating effects of the nigrostriatal dopaminergic system [12][13][14][15] .
Progressive resistance training is a non-pharmacological intervention that has been tested in the treatment of PD. Studies with mild-and medium-intensity physical training have already proven its efficacy in improving the musculoskeletal conditions of PD patients and its neuroprotective effect 14,16,17 . However, high-intensity progressive resistance training are yet to be tested in animals with PD.
Thus, this study aimed to investigate the benefits of high-intensity progressive training on muscle tissue and motor performance before and after PD induction in rats.

Animals
A total of 80 male Wistar rats (Rattus norvegicus var. albinus) aged 40 days and weighing between 250 and 450g were used. The animals were kept in polypropylene cages with free access to water and feed under a 12-hour light/dark photoperiod, room temperature between 21 and 22°C, and a 60% to 70% relative humidity.
They were divided into eight groups with 10 rats each and weighed at the beginning of the experiment after PD induction or false surgery, and at the end of the experiment, when they were aged 80 days.
Procedures followed Brazilian ethical standards, the recommendations of international animal protection standards, and the animal experimentation code.
A total of 40 animals were subjected to PDinducing surgery via electrolyte injury and randomly assigned to the following subgroups: trained animals before PD induction (PA-Exa); trained animals after PD induction (PA-Exd); trained animals before and after PD induction (PA-Exad); and sedentary animals with PD induction (PA-Sed).
Moreover, 40 animals (control) were subjected to surgical access but not to PD-inducing electrolyte lesion (sham) and assigned to the following subgroups: animals trained before false surgery (Sham-Exa); animals trained after false surgery (Sham-Exd); animals trained before and after false surgery (Sham-Exad); and sedentary animals that underwent false surgery (Sham-Sed).

Physical training
The animals were adapted to the ladder for three days before training began, performing three attempts per day without any overload. They were positioned in the housing chamber for 60 seconds so they could familiarize themselves with the environment. On their first attempt, they remained 35cm from the chamber; on the second one, 55cm; and on the third, 110cm.
The vertical ladder proposed in Peixinho-Pena et al. 18 is 110-cm long, 18-cm wide, and with a 80° tilt. The housing box at the upper end of the ladder is 20-cm high and wide and has sections.
After adaptation, the rats in the group to be trained on our high-intensity progressive training protocol were subjected to exercise on the ladder five days a week for 30 to 45 minutes in each of the eight sessions with eight climbs for four weeks. The first and second climbs were performed with animals carrying a weight equal to 50% of their body weight; the third and fourth, equal to 75%, the fifth and sixth, to 90%; and the seventh and eighth, to 100% 18,19 . Maximum heart rate (HR max. ) and oxygen saturation (SatO 2 ) were monitored to ensure that exercise reached 80% to 95% of the HR max. of the animal. SatO 2 and HR max. were measured daily via a Contec ® cardiac monitor. Its electrodes were positioned on the tail of the animal to record these measurements.
Sessions were separated by 60-s intervals so animals could rest in the housing chamber. The weight used was fixed to the proximal portion of the tail of the animal 3cm from its caudal root. It had a cylindrical shape and 16cm in length and was fastened with a wool line wrapped by an adhesive rubber tape and was adjusted to protect the skin of the animal 18-20 .

Parkinson's disease induction surgery
Rats were subjected to intraperitoneal anesthesia via ketamine (75mg/kg) and xylazine (10mg/kg). Then, after positioning the head of the animal on a stereotaxis table, trichotomy and cleaning with iodized alcohol in the region of the surgical procedure was performed and their periosteum was removed to enable access the region between the lambda and the bregma. Electrolyte-stimulating electrodes were placed at the −4.9 anteroposterior (AP), 1.7 mid-lateral (ML), and 8.1 dorsoventral (DV ) coordinates 21 , injuring the substantia nigra from a 1-mA current load for 10 seconds. Electrodes were held at the lesion site for about three minutes. Finally, sutures were performed with a surgical wire.

Nervous tissue histochemistry
After the training program, animals were euthanized and their brain and fore and hindlimb skeletal muscles (biceps brachii and flexor hallucis longus), removed. The collection of biological material after euthanasia enabled us to analyze the effect of training on their musculoskeletal tissue.
To confirm the presence of a neurological lesion in the substantia nigra of the mesencephalon, nerve tissue was fixed in a 10% buffered formalin solution for 24 hours and then in 70% alcohol. Finally, it was set in paraffin blocks with 1-mm coronal sections.
Histochemical analysis of the nervous tissue was performed and midbrain, striatum, and motor cortex substantiae nigrae were evaluated. Neuronal cell were counted by Nissl methods, immersing slides in a cresyl violet solution (a neuron-specific marker) to highlight the cytoplasm of neurons and Nissl bodies.
Slides were analyzed by capturing images with a camera attached to a light microscope and the morphometry of the stained material, studied by ImageJ.

Musculoskeletal tissue analysis
Biceps brachii and flexor hallucis longus fragments were dehydrated in an alcoholic gradient for processing and staining with hematoxylin and eosin (HE). Microtomy obtained 4µm thick, half-closed cross sections, with a minimum interval of 40µm between sections. After mounting the slides, five non-matching images were captured per muscle/animal. Intact muscle fibers and muscle fiber area were analyzed and quantified 22 .

Motor performance
To evaluate motor performance, functional tests (parallel bar, open field, and misstep) were applied at the beginning of the experiment, before and after surgery, and at the end of the experiment.
The misstep test was performed for three minutes. A 100×50-cm grilled plate was used with a 3×3cm (9cm 2 ) grid interval. Errors were considered whenever the paws of the animal passed through the bars [23][24][25][26] .
To perform the parallel bars test, two wooden platforms joined by parallel metal bars (115cm) were used. Evaluation lasted five minutes, errors were considered if animals placed both legs on the same bar, between them, or outside them 23,24 .

Statistical analysis
Data were analyzed via the statistical software GramPrism 5.0. The following statistical tests were used: One-way ANOVA (to compare means between groups) and the Tukey's test (to evaluate more than one variable). Results are shown as mean±standard deviation and a 5% level of significance was adopted.

RESULTS
Results of the analysis of substantia nigra of the mesencephalon showed that all groups of animals induced to develop PD showed electrolyte lesions (Figure 1).
Data on forelimb bicep muscle fiber count showed no significant differences (p=0.853). Figure 2B depicts means and standard deviation. Forelimb biceps muscle fiber areas showed no significant differences (p=0.4122) ( Figure 2C).
Flexor hallucis longus muscle fiber count showed significant differences (p=0.0356), in which PA-Exa and PA-Exd averaged the highest means: 37.625 and 37.666, respectively ( Figure 3B). Analysis of hindlimb flexor hallucis longus muscle fiber areas significantly differed between groups (p=0.0306) and PA-Exd, PA-Exad, and Sham-Exad showed the largest areas with 3236.65733µm 2 , 3527.702559µm 2 , and 3950.007703µm 2 means, respectively ( Figure 3C).    We evaluated the effects of our high-intensity progressive resistance training on the functional performance of our sample by the misstep and parallel bar tests, which enabled us to compare the average number of errors animals made at two moments, at the beginning of the experiment and after our physical training protocol. Analysis of hindlegs in the parallel bars test (Table 1) showed that only the PA-Exad group improved their functional performance in this test. On the other hand, all animals in the sham group that underwent progressive resistance training obtained the same result. PA-Exa showed no significant decrease in the number of errors after training. In the misstep test (Table 2), we observed an increase in the number of errors animals made in almost all groups, but we only found a significant difference in the number of errors before and after in PA-EXa, PA-EXd, and PA-Sed in our right forelimb analysis and in the PA-EXa and PA-EXd groups in our left forelimb analysis. These data showed worsened motor coordination in the right forelimb in PA-EXa, PA-EXd, and PA-Sed and of the left forelimb in PA-EXa and PA-EXd. We found no significant differences for other groups, as Table 2 shows.

DISCUSSION
Our data showed that hindleg muscles and motor coordination benefited from progressive physical training but forelimbs showed no modifications. The animals that underwent physical training before and after surgery also had better histomorphometry and motor performance results than the other groups.
Rats and humans with PD undergo changes in their motor coordination, such as impaired control of their extremities, worsened manual dexterity, difficulty transferring weight from one side to the other, and poorer manual reach 21,22 . Such information indicates that rats with PD require longer training to improve their forelimb coordination.
Our evaluation of forelimb muscle fiber count and biceps muscle area showed no significant differences between the evaluated groups, corroborating our assessment of forelimb motor performance in the parallel bar test since we found no differences in motor coordination among groups. Individuals with PD showed changes such as joint range limitations, muscle stiffness, bradykinesia, postural changes, imbalance, pain, plastic spasticity, and altered movement 27,28 . Motor changes to upper limbs debilitate individuals due to asymmetrical rest tremors 29 .
Our analysis of average hindlimb flexor hallucis longus muscle fibers and area evinced a significant difference between rats with higher cell numbers (hyperplasia) and fiber area (hypertrophy) in the PA-Exa, PA-Exd, and PA-Exad, and Sham-Exad, showing that progressive training benefited muscle morphometry. These findings corroborated the data on hindlimb motor performance in the parallel bar test, which evinced an improvement in motor coordination in groups that exercised before and after surgery. Lezcano et al. 21 found significant changes in the hind legs of animals with PD. Thus, intervention strategies that benefit motor performance and muscle hypertrophy are important to recover animals with PD. Some researchers have already shown that mild-and medium-intensity physical training improve the musculoskeletal conditions of individuals with PD 16 . A progressive training program performed in individuals with PD which includes postural and balance training improved posture and freezing of gait 30 .
Thus, progressive resistance training benefits hindleg hypertrophy and motor performance in rats with PD that trained before and after the induction of neurological injury. Thus, this study suggests that, in neurodegenerative diseases such as PD, progressive resistance training is fundamental for hypertrophy and improvement of motor performance.

CONCLUSION
Animals that performed progressive resistance training showed hind legs which better performed in the chosen tests and showed flexor hallux longus muscle hypertrophy. However, we found no improvement in forelimb coordination and hypertrophy of the biceps muscle.
Thus, research requires further studies with longer physical training and further analysis of the factors interfering in the improvement of hindleg results (but not of front limb ones) in these animals.
This study suggests that progressive resistance training on a ladder effectively improves muscle hypertrophy and motor performance in animals with neurodegenerative disease such as PD.