The Effects of Light and Moderate Intensity Exercise on the Femoral Bone and Cerebellum of d -Galactose-Exposed Rats

Abstract

Aging causes the degeneration of organs of the locomotor system, including the cerebellum and bones. Exercise may reverse this deterioration. d-galactose has been frequently used in rodents to accelerate aging. The present study aimed at investigating the effects of exercise on cerebellar and serum growth factors, motor activity, and the number of bone cells of the femoral head of d-galactose-treated rats. Twenty-four male Wistar rats were divided randomly into four groups, that is, three treated groups injected with 300 mg/(mL·kg) body weight (bw) d-galactose solution daily for 4 weeks, and a control group injected with normal saline. Following the 4-week administration of d-galactose solution, two of the treated groups performed light- (45% VO₂max) and moderate- (55% VO₂max) intensity exercise, by running on a treadmill 4 × a week for 4 weeks. Locomotor activity was examined in rotarod and open field tests. The cerebellar and serum Insulin-like Growth Factor 1 (IGF-1) and Brain-Derived Neurotrophic Factor (BDNF) levels were measured using enzyme-linked immunosorbent assay (ELISA). The number of osteoblasts and osteoclasts of femoral head was estimated using unbiased stereological methods. It was found that the number of osteoclasts was higher in the d-galactose-treated group than the normal control and moderate-intensity exercise groups. No significant difference between groups was found in the rotarod and open field test performance, IGF-1 and BDNF levels, as well as number of osteoblasts. In conclusion, a 4-week administration of high-dosed-galactose caused the increase of the number of osteoclasts. A subsequent 4-week moderate-intensity exercise reversed this increase to the normal level.

Introduction

Aging is an enormously complex and progressive deterioration affecting the structure and function of organ systems and tissues,¹ including the locomotor system. Bones and the cerebellum, which are important organs of that system, are not an exception. In maintaining the strength and homeostasis of minerals, bones undergo remodeling, which involves a balance between bone formation by osteoblasts and bone resorption by osteoclasts.²

During aging, the balance is impaired^2,3 and consequently results in fragile bones. The bones will gradually degenerate and the risk of falling and bone disorders, such as osteoporosis, increase.⁴ Similarly, the cerebellum undergoes degeneration during aging and causes impaired motor coordination in elderly.⁵ This deterioration might be partly caused by the decline of the levels of growth factors, such as Insulin-like Growth Factor 1 (IGF-1) and Brain-Derived Neurotrophic Factor (BDNF),⁶ which play a role in maintaining neurons,⁷ including the cerebellar Purkinje cells' dendritic spine.⁸

Physical exercise has been considered to promote neuroprotective mechanisms,⁹ and to slow down the process of osteoporosis¹⁰ during aging. Physical exercise exerts its beneficial effects by increasing neurogenesis, synaptic plasticity, spine density, angiogenesis, neurotransmitters, and growth factors, including BDNF, IGF-1, and vascular endothelial growth factor (VEGF).⁷ Properly administered physical exercise doses are a key to help maximizing positive effects on bone mass.¹¹ Physical exercises that provide sufficient mechanical loads, such as treadmill exercises, are preferable to inhibit bone resorption, prevent bone loss, and maintain bone strength.¹²

A considerable number of studies suggest that mild- and moderate-intensity exercise helps in promoting physical and cognitive health in the elderly.¹³ On the other hand, high-intensity exercise increases the risk of sudden cardiac death, especially in the elderly.¹⁴ High-intensity exercise also increases bone resorption, which in turn causes bone damage.¹⁵ Therefore, moderate intensity is preferable to high intensity exercise regimens for investigating therapeutic effects on cognitive and bone health in animal models of aging.

In rodents, d-galactose has been commonly used to accelerate aging,¹⁶ since it produces signs and symptoms which resemble natural senescence.¹⁷ These include neurodegeneration such as cognitive decline, locomotor disorders,^18
–20 as well as osteoporosis.²¹ It has been found that intraperitoneal injection of 150 mg/kg bw/day of d-galactose for 8 weeks brought about the decrease of femur volume and the increase of femur porosity of rats.²² This might reflect the imbalance between bone formation and resorption.²² This imbalance was observed in rats fed with a MF diet supplemented with 50% (wt/wt) of d-galactose for 6.5 months, in which bone resorption markers (urinary excretion of pyridinoline and deoxypyridinoline) increased, while bone volume and osteoblasts density decreased.²³ However, the density measure is generally prone to the problem of “reference trap,” which is a well-known problem in stereology.^24,25 Therefore, a stereological study is required to evaluate this finding.

Haider et al.²⁶ found that a 1-week intraperitoneal administration of 300 mg/ml/kg bw/day body weight (bw) of d-galactose caused the alterations of behavioral (depression, anxiety, memory), biochemical (antioxidant enzymes, oxidative damage, acetylcholine esterase), and neurochemical (biogenic amines) parameters of rats. On the other hand, Budni et al.²⁷ demonstrated that a 4-week oral administration of 100 mg/kg bw/day of d-galactose impaired the performance in the open field test and caused oxidative damage in the prefrontal cortex and hippocampus of rats.²⁷ The present study applied intraperitoneal injection of 300 mg/(mL·kg) bw of d-galactose (as applied by Haider et al.²⁶), but in a 4-week period (as used by Budni et al.²⁷). We hypothesized that this combined protocol of d-galactose administration was considered to be sufficient to accelerate aging in both the cerebellum and bones.

Previous studies have focused on the effects of physical exercise on the BDNF level¹⁶ or expressions^28,29 in hippocampus of d-galactose-treated rodents. d-galactose has been reported to degenerate the cerebellar myelin sheath¹⁸ and motor learning capability³⁰ of mice. However, no studies have investigated the effects of exercise on the cerebellar growth factors of rats exposed to d-galactose. There was also a number of reports on the beneficial effects of physical exercise on bone resorption or formation of healthy or unhealthy animals.³¹ Many femoral bone studies focused on the femoral neck rather than the femoral head.^32
–34 Of note, femoral head is not less important than femoral neck, since stress and strain on the femoral head determines the mechanical weight load of the femoral neck.³⁵

To our knowledge, there was a lack of stereological research on the effects of physical exercise on the number of osteoclasts or osteoblasts, particularly of the femoral head, of d-galactose-treated rats. It was the aim of the present study to reveal the effects of light- and moderate-intensity exercise on the cerebellar and serum levels of IGF-1 and BDNF, as well as the number of osteoblasts and osteoclasts of femoral bone tissue in d-galactose-induced aging rats.

Materials and Methods

Animals and treatments

Twenty-four male Wistar rats 12 weeks of age (weighing 200–310 g) were used in the present study. The rats were obtained from the animal houses of Universitas Islam Indonesia, the Faculty of Pharmacy, and the Department of Pharmacology and Therapy, Faculty of Medicine, Universitas Gadjah Mada. They were maintained under natural 12-h light–12-h dark cycle and had ad libitum access to water and food. The experimental procedure and animal handling were approved by The Ethics Committee of the Faculty of Medicine, Universitas Gadjah Mada (approval No. KE/38/01/2017; for experiments on cerebellum and behavior), and Integrated Research and Testing Laboratory (approval No. 00007/04/LPPT/lll/2017; for experiments on femoral bones).

At the outset of the experiment, the rats were randomly divided into four groups, each of which consisted of six rats. The normal control (C) group was injected with normal saline (0.9% NaCl) intraperitoneally. Other rats were treated with intraperitoneal injections of 300 mg/(mL·kg) bw of d-galactose diluted in 0.9% NaCl (Tokyo Chemical Industries) daily for 4 weeks. The GAL group only received injections of d-galactose, whereas GAL-LE and GAL-ME received light- and moderate-intensity physical exercise, respectively, besides d-galactose injections. Of note, the rats that refused to run during the adaptation period were included in the d- galactose-only-treated group.

Exercise training protocol

Following 4 weeks of d-galactose injection, the rats were adapted to physical exercise and treadmill apparatus (Gama Tread version 2010, Faculty of Medicine, Universitas Gadjah Mada) using a modified Brown et al.³⁶ protocol, which was administered for 3–7 days. The rats had to run at the lowest speed (11 m/min) on the treadmill without an inclination (a slope of 0°). Afterward the acclimatized rats were given a physical exercise test protocol to estimate the level of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${{ \dot V}}{{ \rm{O}}_2}{ \rm{max}}$$ \end{document} ³⁷ for a maximum of 3 days. The formula for calculating the \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${ { \dot V}}{{ \rm{O}}_2}{ \rm{max}}$$ \end{document} is as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{{ \dot V}}{{ \rm{O}}_2}{ \rm{max}} = \big ( {{ \rm{speed }} \;[ {{ \rm{m}} / { \rm{min}}} ] } \big ) \times ( { \rm{inclination}} \;[ \% ] \times 100 ) \\\times \left( {{ \rm{BW}} \; \left[ {{ \rm{kg}}} \right] } \right) , \end{align*} \end{document}

where the inclination was a slope of 0° and it is equal to “1” in the equation. The \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${ { \dot V}}{{ \rm{O}}_2}{ \rm{max}}$$ \end{document} of any given rat was determined according to the maximum speed that could be attained by the rat.

Subsequently, for the next 4 weeks, the GAL-LE group had to run at an intensity of exercise of 45% of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${ { \dot V}}{{ \rm{O}}_2}{ \rm{max}}$$ \end{document} and the GAL-ME group had to run at an intensity of exercise of 55% of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${ { \dot V}}{{ \rm{O}}_2}{ \rm{max}}$$ \end{document} on the treadmill. The rats had to run four times a week. The exercise protocol consisted of 5 minutes of warming up (running at the lowest speed), 30 minutes of running at 45% or 55% of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${ { \dot V}}{{ \rm{O}}_2}{ \rm{max}}$$ \end{document} , and 5 minutes of cooling down (running at the lowest speed again).³⁸

Open field test

Twenty-four hours after the last day of exercise, the rats were examined in the open field test. This test assesses the locomotor activity and cerebellar function of rats. The protocol of this test was based on that of other studies^19,27 with slight modifications. The open field apparatus consisted of a 100 × 100 cm plywood arena, which was surrounded by 50-cm-high wooden and glass walls. The floor of the open field was divided into 16 identical squares. A video camera connected to a laptop computer was mounted 200 cm above the open field to record the movements of the animals. One hour before the test, the rats were brought into the test room. Any randomly selected rat was gently placed on the northeast corner of the field and the movements of the rat were recorded for 5 minutes. The number of horizontal (crossings) and vertical (rearings) activities were counted to measure the general locomotor and exploratory behavior of the rat. After each trial, the open field was cleaned with 70% ethanol to remove any odor clues of the test rats.

Rotarod test

The rotarod test was performed to assess the balance, motor coordination, and motor control of rats. The test was conducted once the rats finished the open field test. The protocol and apparatus (model 7700; Ugo Basile) of this test were referred to that described in previous studies.³⁹

Any given rat was placed on the stationary running surface of the rotarod for 1 minute for habituation. The rat was then removed from the rotarod. The rotarod was set to rotate at a speed of 16 rpm and the rat was replaced back on the running surface with its head facing the direction opposite to the direction of rotation of the surface. Therefore, the rat had to walk forward to maintain its position. A stopwatch was turned on at the start of the placement of the rat on the running surface. Each trial lasted for 180 seconds. When the rat fell, it was immediately placed back onto the running surface. The number of falls during these 3 consecutive minutes was recorded. The stopwatch was paused when the rat fell and was started again when it was returned to the running surface.

Tissue preparation

Approximately 24 hours after the rotarod test, the rats were euthanized under deep anesthesia with 40 mg/kg bw of ketamine HCl (PT Guardian Pharmatama, Jakarta, Indonesia). One and a half milliliters of blood was obtained from the orbital sinus of the rats. The blood was incubated for 1 hour at room temperature before centrifugation at 2000 g for 15 minutes at 4°C. The blood serum was collected and stored at −80°C in a freezer. The cerebellum of the rats were removed from their skulls and stored at −80°C in a freezer before homogenization.

Right femoral bones of the rats were also taken and cleaned from the surrounding muscles. The bones were washed using Aqua Dest and 0.9% NaCl solution, weighed, and immersed in 10% formaldehyde in phosphate-buffered solution for 24 hours. The bone specimens were coded and thus the observer was not aware of which group any given specimens originated from.

Cerebellar and serum tissue examination

Twenty milligrams of each cerebellar tissue sample was homogenized in a glass tube containing 600 μL of PRO PREP^™ (Intron Biotechnology) surrounded by ice. The homogenates were incubated for 30 minutes and centrifuged at 12,000 rpm for 15 minutes. The supernatant of these homogenates was used for the determination of the cerebellar IGF-1 and BNDF concentration. The levels of IGF-1 and BDNF of both serum and cerebellum were determined using rat IGF-1 and BDNF Enzyme-Linked Immunosorbent Assay (ELISA) Kits (Elabscience) according to the manufacturer's instruction in a Bio-Rad microplate reader (Benchmark) operated at a wavelength of 450 nm.

Histological procedure

The femoral bones were decalcified using EDTA solution (pH 7) and stored in a 37°C storage for approximately 3–4 weeks. Once the bones became soft, the femoral heads of the bones were isolated from the rest of the femur. The head tissues were subsequently dehydrated in ethanol of graded concentrations, cleared in toluene solution, infiltrated, and eventually embedded in paraffin blocks.

Paraffin blocks containing the tissues were cut using a rotary microtome (Leica RM 2235; Biosystems Nussloch, GmbH) at a nominal thickness of 4 μm. A number between 1 and 50 was randomly selected to determine the first number of a pair of sections being sampled, and thereafter a section together with its adjacent section was taken for every 50 sections. The pairs of sections used for counting the number of osteoblasts were different from those used for osteoclasts, and therefore there were two sets of pairs of sections from each animal. This strategy yielded approximately between 6 and 10 pairs of sections per set, per animal. The sections were mounted on glass slides, dried on a hot plate for the next 24 hours, and deparaffinized using xylol and ethanol. The sections prepared for the counting of osteoblasts were stained using Hematoxylin and Eosin, whereas those for osteoclasts were stained using TRAP (Tartrate-Resistant Acid Phosphatase).

Stereological analyses

Unbiased stereological methods were applied in the estimation of the volume of the femoral head and the total number of osteoblasts and osteoclasts. The volume of the femoral head was calculated using Cavalieri method.⁴⁰ The sections were viewed under an Olympus CX21FSI light microscope (Olympus Singapore PTE, Ltd.) at 40 × magnification. Pictures of parts of the femoral head sections (one section from each pair of sections of an animal) were taken and collated into montages of complete images of femoral head using Adobe Photoshop CS6 software. A virtual grid consisting of points spaced regularly at a distance of 63,000 μm (120 × magnification) between points was made using ImageJ software and superimposed on the montages (Fig. 1). The number of points (P) falling on each montage was counted, and the total number of points (ΣP) for each rat was calculated by summing up all points of all montages of each rat. The range of the total number of points of all subjects was between 100 and 189 points. The volume of the femoral head of each rat was estimated using the following formula⁴⁰: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{{ \rm{V}}_{{ \rm{ref}}}} = { \rm{T}} \cdot \;{ \rm{a}} / { \rm{p}} \cdot \Sigma { \rm{P}} , \end{align*} \end{document}

FIG. 1.

An example of point counting method on a section of femoral head bone. Any points falling on the bone were counted (“P”) and incorporated into the calculation of the volume of the femoral head using Cavalieri's principle. The complete explanation on the procedure is described in the Stereological analyses. Color images are available online.

where V_ref is the volume estimate of femoral head (μm³), T is the distance between disector pairs (μm), and a/p is the area width represented by each point (μm²).

Upon staining with Hematoxylin and Eosin, the cytoplasm and nucleus of osteoblasts showed red and blue color, respectively. TRAP staining showed red color of osteoclasts within a green bone matrix. The counting of osteoblasts and osteoclasts was conducted using the microscope at 400 ×magnification. A systematic random sampling of fields of view was performed in a raster fashion. One out of every 10 fields of view was sampled for osteoblast counting, whereas 1 out of every 5 fields was sampled for osteoclast counting. This sampling method yielded ranges of 74–130 fields of view for osteoclast counting and 102–218 fields of view for osteoclast counting. These fields of view together with their corresponding area in the serial neighboring sections were photographed and printed on normal paper. A rectangular counting frame of 9900 × 143,000 μm (640 × magnification) was superimposed on the printed micrograph. The same frame was also superimposed on the corresponding area of the adjacent section micrograph. The frame consists of two solid lines serving as exclusion lines (left and bottom sides) and two dashed lines serving as inclusion lines (right and top sides) (Fig. 2). The counting of the cells followed the “forbidden line rule.”⁴¹ The counting unit of osteoblasts was the nucleus, whereas the counting unit of osteoclasts was the cell body, since osteoclasts have multiple nuclei. Any osteoblast nuclear or osteoclast cell body profile that appeared in one section (“sampling section”), but not in the neighboring serial section (“look-up section”), was counted as long as it did not touch the exclusion lines or their extensions. To increase the efficiency of the counting, the pairs of section were used in turn. The previous sampling sections were now used as look-up sections, and vice versa. There were between 102–200 osteoblast and 101–131 osteoclast profiles, which were counted from each rat. The numerical densities (Nv) of the cells were estimated using the formula⁴²: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm { Nv } } = { \frac { \Sigma { { \rm Q } ^- } } { { \rm { a } } { \rm { .h } } } } \end{align*} \end{document}

FIG. 2.

An example of the counting of osteoblasts (A) and osteoclasts (B) profile using dissectors and counting frames. Any osteoblast nuclear or osteoclast cell body profile touching the dashed lines (“inclusion lines”) of the counting frame was included in the counting as long as it did not touch the solid lines (“exclusion lines”) or their extension. In addition, the osteoblast nuclear or osteoclast cell body profile must appear in one section (“sampling section”) but not in the neighboring serial section (“look-up section”) to be counted. The examples of cells being counted were marked with ticks (√). Color images are available online.

where ΣQ⁻ is the total of cell profiles of each animal; “h” is the disector height, which was equal to section thickness (4 μm); and “a” is the total area of the counting frames of any given rat (μm²).

The total number of osteoblasts and osteoclasts was finally estimated using the formula Nv × V_ref.⁴²

Statistical analyses

Due to the multiple endpoints measured in this study, a resource equation was used for determining the sample size. A number of n = 5–10 animals per group was estimated to be adequate. Data of body weights and body weight gains during treatment of the rats were analyzed using one-way analysis of variance (ANOVA) procedure. The raw data of the number of falls in the rotarod test and the number of crossings in the open field test were not normally distributed and had some zero values. To fulfill the requirement for a parametric test and to avoid mathematical complications due to some values being zero, the data were transformed using the formula³⁹: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} X \left( t \right) \; = { \rm{ \;square}} \;{ \rm{root\; }} \left( {x \; + \;0.5} \right) \end{align*} \end{document}

where “x” is the number of falls in each trial of the rotarod test or number of crossings in the open field test; and X(t) is the transformed value of “x.” One-way ANOVA procedure was used to analyze the transformed data of the number of falls and the number of crossings, as well as cerebellar and serum IGF-1 and BDNF levels.

Data of the number of rearings in the open field test were also not normally distributed despite their transformation into square root data. Hence data were analyzed using the nonparametric Kruskal–Wallis procedure.

Data of volume, Nv of osteoblasts, as well as the estimated total number of osteoblasts and osteoclasts, were analyzed using one-way ANOVA procedure. Data of Nv of osteoclasts did not pass the normality test despite being transformed, and therefore the data were analyzed using Kruskal–Wallis procedure. The coefficient of error (CE) and coefficient of variance (CV) of the data of the volume of femoral head as well as the number of osteoblasts and osteoclasts were calculated. The stereological procedure was considered efficient if the value of CE_total ²/CV_total ² is between 0.2 and 0.5⁴²

Pearson correlation test was used to examine the correlation between the bone and cerebellar parameters, except the correlation between the number of osteoblasts or osteoclasts and number of rearings. The correlations between these latter parameters were assessed using Spearman correlation test due to the failure of the number of rearing data in the normality test. All statistical analyses were performed using SPSS version 23.0 software. The significance levels were set at p < 0.05. Post hoc least significant difference (LSD) or Mann–Whitney tests were performed wherever necessary.

Results

Table 1 presents the data of the body weights of the rats of all groups. One-way ANOVA of the body weight data before treatment revealed no significant main effect of groups. On the other hand, one-way ANOVA of the body weight data after treatment revealed a significant main effect of group. Post hoc LSD analysis of the body weight data after treatment showed that the body weight of the d-galactose-treated group was significantly lower than that of the light-intensity exercise (p = 0.003) and normal (p = 0.001) control groups. On the other hand, there was no significant difference in the body weight gains between groups.

Table 1.

Mean ± Standard Error of Mean of Body Weight of All Rats Before and After Treatment

Groups	n	Body weight before treatment (g)	Body weight after treatment (g)	Body weight gains (g)
C	6	266.67 ± 7.49	318.33 ± 7.03^a	51.67 ± 4.21
GAL	6	239.17 ± 13.68	258.33 ± 10.54^a,b	19.17 ± 10.68
GAL-LE	6	268.33 ± 11.95	313.33 ± 17.11^b	45.00 ± 9.83
GAL-ME	6	246.67 ± 11.95	285.00 ± 8.37	38.33 ± 9.19
		Results of one-way ANOVA: df = 3, 20, F = 1.598, p = 0.22	Results of one-way ANOVA: df = 3, 20, F = 5.908, p = 0.005	Results of one-way ANOVA: df = 3, 20, F = 2.511, p = 0.088

p = 0.001; ^b p = 0.003.

ANOVA, analysis of variance; bw, body weight; C, 0.9% NaCl intraperitoneal (ip); df, degree of freedom; GAL, 300 mg/(mL·kg) bw d-galactose (ip); GAL-LE, 300 mg/(mL·kg) bw d-galactose (ip) + light -intensity exercise; GAL-ME, 300 mg/(mL·kg) bw d-galactose (ip) + moderate-intensity exercise; n, number of animals.

Data of the results of behavioral tests are presented in Table 2. At a glance, the number of crossings and rearings appeared lower in the galactose-treated group than the other groups. However, one-way ANOVA of the number of falls in the rotarod and number of crossings in the open field revealed no significant main effect of groups. The Kruskal–Wallis test of the number of rearings in the open field also showed no significant difference between groups (Table 2).

Table 2.

Mean ± Standard Error of Mean of Number of Falls in the Rotarod; As Well As Number of Crossings and Number of Rearing in the Open Field of Rats

Groups	n	No. of falls (x)	No. of crossings (x)	No. of rearings (x)
C	6	2.28 ± 0.30	5.75 ± 0.98	23.33 ± 5.97
GAL	6	1.97 ± 0.16	2.47 ± 0.78	11.17 ± 1.83
GAL-LE	6	1.67 ± 0.19	4.70 ± 1.08	20.00 ± 4.92
GAL-ME	6	1.85 ± 0.33	3.22 ± 1.30	11.67 ± 4.07
		Results of one-way ANOVA: df = 3, 20, F = 0.943, p = 0.438	Results of one-way ANOVA: df = 3, 20, F = 1.960, p = 0.153	Results of Kruskal–Wallis test: df = 3, 20, p = 0.237

n, number of animals.

See footnote of Table 1 for detailed description of the groups.

Table 3 shows the data of cerebellar and serum BDNF and IGF-1 levels. One-way ANOVA of these data revealed no significant main effect of groups in the cerebellar and serum BDNF and IGF-1 levels.

Table 3.

Mean ± Standard Error of Mean of the Levels of Insulin-like Growth Factor 1 and Brain-Derived Neurotrophic Factor in the Cerebellum and Serum of Rats (pg/mL)

Groups	n	Cerebellum		Serum
Groups	n	IGF-1	BDNF	IGF-1	BDNF
C	6	271.41 ± 8.99	318.68 ± 65.93	1859.71 ± 125.56	525.45 ± 38.88
GAL	6	272.52 ± 17.33	276.17 ± 7.98	1653.98 ± 139.32	548.77 ± 56.70
GAL-LE	6	252.29 ± 15.26	260.13 ± 13.2	1860.43 ± 146.33	506.72 ± 68.89
GAL-ME	6	250.57 ± 12.00	251.96 ± 6.69	1904.65 ± 62.32	624.53 ± 38.82
One-way ANOVA		df = 3, 20, F = 0.745, p = 0.538	df = 3, 20, F = 0.763, p = 0.528	df = 3, 20, F = 0.837, p = 0.490	df = 3, 20, F = 0.974, p = 0.425

BDNF, brain-derived neurotrophic factor; IGF-1, insulin-like growth factor 1; n, number of animals.

See footnote of Table 1 for detailed description of the groups.

Data of the volume of femoral head as well as the Nvs of osteoblasts and osteoclasts are presented in Table 4. The Kruskal–Wallis test of the volume data demonstrated no significant difference between groups. The CE_total ²/CV_total ² of the data was <0.2, which indicates that the sampling scheme of the stereological procedure is sufficient, but requires a more efficient way in future studies. One-way ANOVA of the data of Nv of osteoblasts also showed no significant main effect of groups. On the other hand, the Kruskal–Wallis test of the Nv of osteoclasts revealed a significant main effect of groups. Post hoc Mann–Whitney of these data showed that the numerical density of osteoclasts of GAL-ME group was significantly lower than that of the GAL and GAL-LE groups (p = 0.006).

Table 4.

Mean ± Standard Deviation of the Volume of Femoral Head and Numerical Density of Osteoblasts and Osteoclasts ( × 10⁻⁵)

Groups	n	Volume (mm³)	CV	CE	N_V OB (/μm³)	N_V OC (/μm³)
C	6	7.28 ± 1.645	0.226	0.03	1.1 ± 0.27	0.49 ± 0.11
GAL	6	7.43 ± 1.637	0.220	0.03	1.1 ± 0.19	0.66 ± 0.13^a
GAL-LE	6	7.74 ± 1.444	0.187	0.03	1.2 ± 0.37	0.58 ± 0.07^b
GAL-ME	6	7.89 ± 1.343	0.170	0.03	1.2 ± 0.21	0.42 ± 0.05^a,b
Kruskal–Wallis: df = 3, 2, p = 0.833		CV_total = 0.20, CV_biol = 0.20	CE_total = 0.03, CE_total ²/CV_total ² = 0.02	One-way ANOVA: df = 3, 20, F = 0.249, p = 0.861	Kruskal–Mann–Whitney–Wallis: df = 3, 20, p = 0.016

p = 0.006; ^b p = 0.006.

CE, coefficient of error; CV, coefficient of variance; CV_biol, biological coefficient of variation; CV_total, total coefficient of variation; n, number of animals;.

See footnote of Table 1 for detailed description of the groups.

Figures 3 and 4 present the dot plot diagram of the number of osteoblasts and osteoclasts, respectively. One-way ANOVA of the data of the number of osteoblasts showed no significant main effect of groups. On the other hand, one-way ANOVA of the data of the number of osteoclasts showed a significant main effect of groups. Post hoc LSD test of these data revealed that the estimated total number of osteoclasts was significantly higher in the GAL group than that of the C (p = 0.037) and GAL-ME (p = 0.017) groups. The sampling procedure of both sets of data was sufficient (CE_total ²/CV_total ² <0.2) (Table 5).^43,44 The correlation analyses showed no significant correlation between any bone and cerebellar or behavioral parameters (Table 6).

FIG. 3.

The mean ± standard deviation of the number of osteoblasts of all groups of rats. The data were analyzed using one-way ANOVA procedure (df = 3, 20; F = 0.249; p = 0.861). See footnote of Table 1 for detailed description of the groups. ANOVA, analysis of variance.

FIG. 4.

The mean ± standard deviation of the number of osteoclasts of all groups of rats. The data were analyzed using one-way ANOVA procedure (df = 3, 20: F = 3.146: p = 0.048). See footnote of Table 1 for detailed description of the groups.

Table 5.

Coefficient of Variation and Coefficient of Error of the Total Number of Osteoclasts and Osteoblasts of Rats

Groups	n	Osteoblasts		Osteoclasts
Groups	n	CV	CE	CV	CE
C	6	0.261	0.09	0.269	0.10
GAL	6	0.339	0.09	0.279	0.10
GAL-LE	6	0.484	0.10	0.280	0.10
GAL-ME	6	0.187	0.08	0.167	0.10
		CV_total = 0.34	CE_total = 0.09	CV_total = 0.25	CE_total = 0.10
		CV_biol = 0.32	CE_total ²/CV_total ² = 0.07	CV_biol = 0.23	CE_total ²/CV_total ² = 0.15

n, number of animals.

See footnote of Table 1 for detailed description of the groups.

Table 6.

The Correlations Between the Number of Osteoclasts or Osteoblasts and the Brain-Derived Neurotrophic Factor, Insulin-like Growth Factor 1, or Behavioral Parameters of Rats

	(r)	p
No. of osteoblasts
Cerebellar IGF-1 level	(+) 0.190	0.374^a
Serum IGF-1 level	(−) 0.063	0.771^a
Cerebellar BNDF 1 level	(−) 0.108	0.614^a
Serum BDNF level	(+) 0.040	0.852^a
No. of rearings	(−) 0.044	0.838^b
No. of crossings	(+) 0.109	0.613^a
No. of falls	(−) 0.072	0.738^a
No. of osteoclasts
Cerebellar IGF-1 level	(+) 0.161	0.452^a
Serum IGF-1 level	(−) 0.98	0.647^a
Cerebellar BNDF 1 level	(−) 0.015	0.946^a
Serum BDNF level	(−) 0.63	0.770^a
No. of rearings	(+) 0.031	0.884^b
No. of crossings	(+) 0.003	0.990^a
No. of falls	(+) 0.184	0.184^a

Pearson correlation coefficient.

Spearman's rank correlation coefficient.

(+), positive correlation; (−), negative correlation.

Discussion

The main finding of the present study was that d-galactose caused the increase of the estimated total number of osteoclasts. In d-galactose-exposed rats, moderate-intensity exercise suppressed the number of osteoclasts back to normal level. d-galactose also brought about the decrease of body weights. On the other hand, d-galactose and exercise did not exert any effects on the total number of osteoblasts, number of falls, crossings, and rearings, as well as cerebellar and serum BDNF and IGF-1 levels. In addition, there was no correlation whatsoever between any of the bone and cerebellar parameters. This result suggests that independent pathways of d-galactose effects are at play on the bones and the cerebellum, although IGF-1 is thought to affect both osteoblasts^45,46 and osteoclasts,^45,47 besides the cerebellum.⁸

The finding in the present study that the body weight of d-galactose-treated rats was lower than that of normal control group corroborates that of another study.⁴⁸ This decrease in body weight may represent the basic characteristics of aging, such as decreased food intake as well as body and organ weight loss.⁴⁹ Similar to other studies,^26,28,50,51 there was no significant difference between groups in the body weight gain observed in the present study. In contrast, another study on mice by Tang and He⁵² found a significant reduction of body weight gain following d-galactose treatment. However, caution should be taken in interpreting all of these studies, since they used different species for testing and applied different dosage and duration of d-galactose, as well as type, dosage, and duration of exercise, all of which may affect the body weights. For example, mice are known to have a two- or three-fold higher metabolic rate than rats,⁵³ and therefore, they are more susceptible than rats to the decrease of calorie intake.

To the best of our knowledge, there is a lack of stereological studies on the effects of exercise on the estimated total number of osteoclasts and osteoblasts in d-galactose-treated rats. Our study found that moderate-intensity physical exercise returned the number of osteoclasts of d-galactose-treated rats to normal. During aging, the activity of osteoclasts increases, and hence bone resorption is more dominant than bone formation.^54,55 A previous investigation by Iura et al.⁵⁶ reported that physical exercise reduced the number of osteoclasts per total surface bone area. A number of studies have also reported that a long-term moderate exercise maintains bone mass by decreasing bone resorption.³¹ Our stereological estimation on the total number of osteoclasts confirmed these findings. This decrease in the number of osteoclasts may suggest a decrease in bone resorption activity, which in turn may help in reducing bone loss. Physical exercise has been found to prevent bone loss⁵⁷ and improve bone profiles, including bone volume, trabecular number and thickness, bone mass density, mechanical strength, geometry of ovariectomized rats,⁵⁸ or osteoporosis condition.⁵⁹

The present study demonstrates that there was no change of the total number of osteoblasts following d-galactose exposure and exercise. Studies revealed that the administration of d-galactose as low as 20 mg/kg bw for 5 months²¹ and as high as 1 g/kg bw for 42 days⁶⁰ caused bone aging as represented by various parameters (bone weights, mineral content, and biomechanics). Our present study which administered d-galactose at a dose of 300 mg/kg bw for a shorter duration than those studies (4 weeks) yielded an increase in the number of osteoclasts but no change in the number of osteoblasts. These results may suggest that there are differential effects of d-galactose administered at various doses and durations on different bone parameters.

Two studies on mice models of osteoporosis reported that an intermittent 4 week of treadmill exercise combined with ethanolic extract of Spilanthes acmella ⁶¹ or an intermittent 6-week program of swimming⁶² increased the average number of osteoblasts following dexamethasone exposure⁶¹ or during the perimenopause period.⁶² However, caution should be exercised in interpreting the results of these studies, since they estimated the average number of osteoblasts in five fields of view per animal. Such density estimates are not an appropriate surrogate measure of the total number of osteoblasts, since it is subject to changes of the volume of reference space (bone).^24,25

The absence of the increase of the number of osteoblasts following exercise in the present study might be due to the relatively short duration and small direct mechanical load of muscles on the femoral head. Of note, the effects of physical exercise on bone are determined by the intensity, duration, and type of the exercise.⁶³ It has been found that a 12-week routine treadmill exercise enhanced bone mass of ovariectomized rats.⁶⁴ Other studies also reported that long-term moderate exercise preserves bone mass by increasing bone formation.³¹ Such an increase may indicate an enhancement of the number of osteoblasts. However, an unbiased stereological study is required to confirm or refute this finding.

The negative findings in the effects of d-galactose on the cerebellar IGF-1 and BDNF levels as well as the locomotion performance in the rotarod and open field tests might have arisen from several causes, such as insufficient dosage or improper route of administration of d-galactose. The current study, however, applied a subchronically high dose of d-galactose administered intraperitoneally, which has been considered to be a successful model of administration.⁶⁵ Thus far, there seems to be no single standard protocol of d-galactose administration, which is capable of inducing aging-like degeneration in all brain areas.^66,67 Therefore, inconsistencies of results may appear between studies. This disagreement may be brought about by not only the differences in the administration method of d-galactose,^{16,20,26,27,66

–71} but also the types,^66,70 strains,^16,26 sex,⁶⁶ and age⁷⁰ of test animals used, and different responses of different brain areas toward d-galactose exposure.

In addition, a possible time-, dose-, or sex-dependent manner of paradoxical behavioral responses of rodents toward d-galactose administration may generate unpredictable results in a study. For instance, oral and subcutaneous administrations of 200 mg/kg bw/day of d-galactose for up to 4 weeks exerted beneficial effects on the learning and memory of rats, whereas that administration for up to or more than 8 weeks nullified these effects or might worsen the learning and memory.⁶⁵ Subcutaneous injections of a relatively high dose (100 mg/kg bw) of d-galactose for 56 days on male mice caused sensorimotor impairment, but the low dose (50 mg/kg bw) gave rise to an improvement in the learning and memory. On the other hand, such administration of d-galactose on female mice caused a dose-dependent deterioration of the motor and spatial learning, but enhanced memory.⁷²

The dose and route of administration of d-galactose regimen used in the present study referred to the protocol by Haider et al.²⁶ but applied at a longer duration. Haider et al.²⁶ showed deficits in various depression- and anxiety-like behavioral parameters following a 1-week exposure of d-galactose. On the other hand, our study did not observe any alteration in the locomotion behavior, although there seemed to be numerical deficits in the crossing and rearing behaviors. The decrease of rearing behavior is considered to indicate the loss of muscle power (sarcopenia).⁷³ It has been thought that different brain regions age at different onset and rate.⁷⁴ A behavioral, stereological, and electrophysiological study reported that the cerebellum underwent aging earlier than hippocampus.⁷⁵ However, another study found that the cerebellum ages at a slower rate than other brain regions.⁷⁶ Such elusiveness may also occur in the artificially induced aging by d-galactose.

To summarize, moderate-intensity, but not low-intensity, exercise may return the d-galactose-induced decrease of the number of osteoclasts back to normal level. The administration of d-galactose in the present study failed to induce aging-like deterioration in the number of osteoblasts, cerebellar growth factors, and locomotion. This failure might have stemmed from factors, such as the species, sex, and age of the experimental animals; dose, duration, or route of administration of d-galactose; as well as differences of response of different types of organ tissues. Future studies may be directed toward formulating standardized protocol of d-galactose administration to induce aging in different types of tissue. Further investigations on the effects of physical exercise on different aspects or parameters of aging of d-galactose-induced aging on the bone and cerebellum are also warranted.

Footnotes

Acknowledgments

This research was funded by a Research Grant of Public Funding of Universitas Gadjah Mada (No. UPPM/221/M/05/04/05.17). The results reported in the present study were parts of Nisa Karima and Kurnia Putri Utami's thesis. The authors would like to thank Suparno and Dwi Kurniawati (Department of Physiology, Faculty of Medicine, Universitas Gadjah Mada), Rina Susilowati and Sumaryati (Department of Histology, Faculty of Medicine, Universitas Gadjah Mada) for the consultation and technical assistances, as well as Erik C. Hookom (English Clinic) for language editing.

Author Disclosure Statement

No competing financial interests exist.

References

Nigam

, Knight

, Bhattacharya

, Bayer

. Physiological changes associated with aging and immobility. J Aging Res, 2012; 2012:468469.

Phan

, Xu

, Zheng

. Interaction between osteoblast and osteoclast: impact in bone disease. Histol Histopathol, 2004; 19:1325–1344.

Cavanaugh

, Blanchard-Fields

. Adult Development and Aging, 7th Ed. Boston, MA: Cengange Learning, 2014.

Zumerchik

. Encyclopedia of Sports Science, Vol. 2. New York: Simon and Schuster MacMillan, 1997.

Seidler

, Bernard

, Burutolu

, Fling

, Gordon

, Gwin

, Kwak

, Lipps

. Motor control and aging: links to age-related brain structural, functional, and biochemical effects. Neurosci Biobehav Rev, 2010; 34:721–733.

Adlard

, Perreau

, Cotman

. The exercise-induced expression of BDNF within the hippocampus varies across life-span. Neurobiol Aging, 2005; 26:511–520.

van Praag

. Exercise and the brain: something to chew on. Trends Neurosci, 2009; 32:283–290.

Ito

. The Cerebellum: Brain for an Implicit Self, 1st Ed. Upper Saddle River, New Jersey (NJ): Pearson Education, Inc, 2012, p. 314.

Perluigi

, Swomley

, Butterfield

. Redox proteomics and the dynamic molecular landscape of the aging brain. Ageing Res Rev, 2014; 13:75–89.

10.

Toumi

, Benaitreau

, Pallub

, Mazor

, Hambli

, Ominsky

, Lespessailles

. Effects of anti-sclerostin antibody and running on bone remodeling and strength. Bone Rep, 2015; 2:52–58.

11.

Farr

, Laddu

, Going

. Exercise, hormones, and skeletal adaptations during childhood and adolescence. Pediatr Exerc Sci, 2014; 26:384–391.

12.

Iwamoto

, Takeda

, Sato

. Effect of treadmill exercise on bone mass in female rats. Exp Anim, 2005; 54:1–6.

13.

Tse

ACY

, Wong

TWL

, Lee

. Effect of low-intensity exercise on physical and cognitive health in older adults: a systematic review. Sports Med Open, 2015; 1:37.

14.

Thompson

. Benefits and risks associated with physical activity. In: Pescatello

, Arena

, Riebe

, Thompson

(eds): ACSM'S Guidelines for Exercise Testing and Prescription. Baltimore, MD: Wolter Kluwer/Lippincot Williams & Wilkins, 2014, p. 456.

15.

de Sousa

, Pereira

, Fukui

, Caparbo

, Da

. Carbohydrate beverages attenuate bone resorption markers in elite runners. Metabolism, 2014; 63:1536–1541.

16.

Austin

, Effects of d-Galactose Treatment and Moderate Exercise on Spatial Memory in Rats. Kalamazoo: Department of Psychology, Western Michigan University, 2012, p. 62.

17.

Song

, Bao

, Li

. Advanced glycation in d-galactose induced mouse aging model. Mech Ageing Dev, 1999; 108:239–251.

18.

Zhu

, Ren

, Zhang

. Effects of d-galactose on the structure of nerve fibers in cerebellar white matter. Pakistan J Zool, 2015; 47:187–192.

19.

, Zhou

, Hu

, Gu

, Wu

, Cao

, Ke

, Liu

. Reduced numbers of cortical GABA-immunoreactive neurons in the chronic d-galactose treatment model of brain aging. Neurosci Lett, 2013; 549:82–86.

20.

Banji

, Banji

OJF

, Dasaroju

, Kumar

. Curcumin and piperine abrogate lipid and protein oxidation induced by d-galactose in rat brain. Brain Res, 2013; 1515:1–11.

21.

Pei

, Hui

, Dong

. Influence of canthaxanthin on d-galactose induced osseous changes of rat [in Chinese]. Zhongguo Gu Shang, 2008; 21:613–616.

22.

Hung

Y-T

, Tikhonova

, Ding

S-J

, Kao

P-F

, Lan

HH-C

, Liao

J-M

, Chen

J-H

, Amstislavskaya

, Ho

Y-J

. Effects of chronic treatment with diosgenin on bone loss in a d-galactose-induced aging rat model. Chin J Physiol, 2014; 57:121–127.

23.

Inaba

, Terada

, Nishizawa

, Shioi

, Ishimura

, Otani

, Morii

. Protective effect of an aldose reductase inhibitor against bone loss in galactose-fed rats: possible involvement of the polyol pathway in bone metabolism. Metabolism, 1999; 48:904–909.

24.

Howard

, Reed

. Unbiased Stereology. Three-Dimensional Measurement in Microscopy, Microscopy Handbook, 2nd Ed. Oxford: BIOS Scientific Publishers, 2005, p. 277.

25.

Braendgaard

, Gundersen

. The impact of recent stereological advances on quantitative studies of the nervous system. J Neurosci Methods, 1986; 18:39–78.

26.

Haider

, Liaquat

, Shahzad

, Sadir

, Madiha

, Batool

, Tabassum

, Saleem

, Naqvi

, Perveen

. A high dose of short term exogenous d-galactose administration in young male rats produces symptoms simulating the natural aging process. Life Sci, 2015; 124:110–119.

27.

Budni

, Pacheco

, da Silva

, Garcez

, Mina

, Bellettini-Santos

, de Medeiros

, Voss

, Steckert

, Valvassori

SDS

. Oral administration of d-galactose induces cognitive impairments and oxidative damage in rats. Behav Brain Res, 2016; 302:35–43.

28.

Nam

, Kim

, Yoo

, Yim

, Kim

, Choi

, Kim

, Jung

, Won

M-H

, Hwang

, Seong

, Yoon

. Physical exercise ameliorates the reduction of neural stem cell, cell proliferation and neuroblast differentiation in senescent mice induced by d-galactose. BMC Neurosci, 2014; 15:1–12.

29.

, Xu

, Shen

, Li

, Gao

, Wei

S-G

. Moderate exercise prevents neurodegeneration in d-galactose-induced aging mice. Neural Reg Res, 2016; 11:807–815.

30.

, Wu

, Yang

, Sheng

, Chen

, Li

, Liu

. Progressive impairment of motor skill learning in a d-galactose-induced aging mouse model. Pakistan J Zool, 2014; 46:215–221.

31.

, Liu

, Lu

. The mechanisms underlying the beneficial effects of exercise on bone remodeling: roles of bone-derived cytokines and microRNAs. Prog Biophys Mol Biol, 2016; 122:131–139.

32.

Jarvinen

TLN

, Pajamaki

, Sievanen

, Vuohelainen

, Tuukkanen

, Jarvinen

, Kannus

. Femoral neck response to exercise and subsequent deconditioning in young and adult rats. J Bone Miner Res, 2003; 18:1292–1299.

33.

Allison

, Folland

, Rennie

, Summers

, Brooke-Wavell

. High impact exercise increased femoral neck bone mineral density in older men: a randomised unilateral intervention. Bone, 2013; 53:321–328.

34.

Abe

, Narra

, Nikander

, Hyttinen

, Kouhia

, Sievänen

. Exercise loading history and femoral neck strength in a sideways fall: a three-dimensional finite element modeling study. Bone, 2016; 92:9–17.

35.

Gao

, He

, Jiang

, Chang

, Zhu

, Luo

, Zhou

, Ma

, Yan

. Salidroside ameliorates cognitive impairment in a d-galactose-induced rat model of Alzheimer's disease. Behav Brain Res, 2015; 293:27–33.

36.

Brown

, Johnson

, Armstrong

, Lynch

, Caruso

, Ehlers

, Fleshner

, Spencer

, Moore

. Short-term treadmill running in the rat: what kind of stressor is it?. J Appl Physiol, 2007; 103:1979–1985.

37.

Brooks

, White

. Determination of metabolic and heart rate responses of rats to treadmill exercise. J Appl Physiol, 1978; 45:1009–1115.

38.

Cunha

, Ilha

, Centenaro

, Lovatel

, Balbinot

, Achaval

. The effects of treadmill training on young and mature rats after traumatic peripheral nerve lesion. Neurosci Lett, 2011; 501:15–19.

39.

Suryanti

, Partadiredja

, Atthobari

. Red sorrel (Hibiscus Sabdariffa) prevents the ethanol-induced deficits of Purkinje cells in the cerebellum. Bratisl Lek Listy, 2015; 116:109–114.

40.

Gundersen

, Jensen

. The efficiency of systematic sampling in stereology and its prediction. J Microsc, 1987; 147(Pt 3):229–263.

41.

Gundersen

HJG

. Notes on the estimation of the numerical density of arbitrary particles: the edge effect. J Microsc, 1977; 111:219–223.

42.

Yuliani

, Mustofa, Partadiredja

. Turmeric (Curcuma longa L.) extract may prevent the deterioration of spatial memory and the deficit of estimated total number of hippocampal pyramidal cells of trimethyltin-exposed rats. Drug Chem Toxicol. 2018; 41:62–71.

43.

Utami

. Efek latihan fisik terhadap osteoblastus dan osteoklastus pada tikus Wistar jantan model penuaan yang diinduksi d-galaktosa [in Indonesian]. [The Effect of Physical Exercise on Osteoclasts and Osteoblasts of Wistar Rats Exposed with d-Galactose]. Yogyakarta: Department of Physiology, Universitas Gadjah Mada, 2017, p. 127.

44.

Karima

. Pengaruh d-galaktosa dosis tinggi selama 4 minggu terhadap kadar Insulin-like Growth Factor-1 (IGF-1) dan Brain Derived Neurotrophin Factor (BDNF) serta aktivitas motorik tikus Wistar jantan [in Indonesian]. [The Effect of a 4-Week High Dose of d-Galactose on Insulin-like Growth Factor-1 (IGF-1) and Brain Derived Neurotrophin Factor (BDNF) As Well As Motor Activity of Male Wistar Rats]. Yogyakarta: Department of Physiology, Universitas Gadjah Mada, 2017, p. 96.

45.

Guerra-Menéndez

, Sádaba

, Puche

, Lavandera

, de Castro

, de Gortázar

, Castilla-Cortázar

. IGF-I increases markers of osteoblastic activity and reduces bone resorption via osteoprotegerin and RANK-ligand. J Transl Med, 2013; 11:1–12.

46.

Lau

, Adachi

. Bone aging. In: Nakasato

, Yung

(eds): Geriatric Rheumatology: A Comprehensive Approach. New York: Springer Science & Business Media, 2011, pp. 11–15.

47.

Guntur

, Rosen

. IGF-1 regulation of key signaling pathways in bone. Bonekey Rep, 2013; 2:1–6.

48.

Khan

SHS

. Biomolecular and Physiological Study of Some Antiaging Factors in Animal Cell Senescence Markers in Aged Rats. Mosul: Department of Biology, University of Mosul, 2015, p. 211.

49.

Alliot

, Boghossian

, Jourdan

, Veyrat-Durebex

, Pickering

, Meynial-Denis

, Gaumet

. The LOU/c/jall rat as an animal model of healthy aging?. J Gerontol Biol Sci Am, 2002; 57:B312–B320.

50.

Huang

C-C

, Chiang

W-D

, Huang

W-C

, Huang

C-Y

, Hsu

M-C

, Lin

W-T

. Hepatoprotective effects of swimming exercise against d-galactose-induced senescence rat model. Evid Based Complement Alternat Med, 2013; 2013(Article ID 275431):1–9.

51.

Jeong

, Liu

, Kim

H-S

. Dried plum and chokeberry ameliorate d-galactose-induced aging in mice by regulation of Pl3k/Akt-mediated Nrf2 and Nf-kB pathways. Exp Gerontol, 2017; 95:16–25.

52.

Tang

, He

. Treatment of d-galactose induced mouse aging with Lycium barbarum polysaccharides and its mechanism study. Afr J Tradit Complement Altern Med, 2013; 10:12–17.

53.

Radermacher

, Haouzi

. A mouse is not a rat is not a man: species-specific metabolic responses to sepsis—a nail in the coffin of murine models for critical care research?. Intensive Care Med Exp, 2013; 1:1–5.

54.

JafariNasabian

, Inglis

, Reilly

, Kelly

, Ilich

. Aging human body: changes in bone, muscle and body fat with consequent changes in nutrient intake. J Endocrinol, 2017; 234:R37–R51.

55.

Chung

P-L

, Zhou

, Eslami

, Shen

, LeBoff

, Glowacki

. Effect of age on regulation of human osteoclast differentiation. J Cell Biochem, 2014; 115:1412–1419.

56.

Iura

, McNerny

, Zhang

, Kamiya

, Tantillo

, Lynch

, Kohn

, Mishina

. Mechanical loading synergistically increases trabecular bone volume and improves mechanical properties in the mouse when BMP signaling is specifically ablated in osteoblasts. PLoS One, 2015; 10:e0141345.

57.

Renno

ACM

, Gomes

ARS

, Nascimento

, Salvini

, Parizoto

. Effects of a progressive loading exercise program on the bone and skeletal muscle properties of female osteopenic rats. Exp Gerontol, 2007; 42:517–522.

58.

Sun

, Fengbo

, Ma

, Zhao

, Zhang

, Li

, Lv

, Meng

. The effects of combined treatment with Naringin and treadmill exercise on osteoporosis in ovariectomized rats. Sci Rep, 2015; 5:1–9.

59.

Yuan

, Chen

, Zhang

, Wu

, Guo

, Zou

, Chen

, Sun

, Shen

, Zou

. The roles of exercise in bone remodeling and in prevention and treatment of osteoporosis. Prog Biophys Mol Biol, 2016; 122:122–130.

60.

Qin

D-Y

, Wu

, Cui

, Liu

. The effect of d-galactose on bone metabolism in mice and its mechanism. Chin Pharmacol Bull, 2003; 19:1017–1019.

61.

Laswati

, Agil

, Widyowati

. Efek pemberian spilanthes acmella dan latihan fisik terhadap jumlah sel osteoblas femur mencit yang diinduksi deksametason [in Indonesian]. [Effect of Spilanthes acmella and exercise on osteoblast cells femur in mice dexamethasone induced]. Media Litbangkes, 2015; 25:43–50.

62.

Karmaya

MINM

, Tirtayasa

. Pemberian kalsium laktat dan berenang meningkatkan osteoblast pada epiphysis tulang radius mencit perimenopause [in Indonesian]. [Orally lactate calcium and swimming increase osteoblast in epiphysis radial perimenopause mice bone]. Jurnal Veteriner, 2014; 15:39–45.

63.

Gombos

, Bajsz

, Pék

, Schmidt

, Sió

, Molics

, Betlehem

. Direct effects of physical training on markers of bone metabolism and serum sclerostin concentrations in older adults with low bone mass. BMC Musculoskelet Disord, 2016; 17:1–8.

64.

Simões

, Zamarioli

, Blóes

, Mazzocato

, Pereira

LHA

, Volpon

, Shimano

. Effect of treadmill exercise on lumbar vertebrae in ovariectomized rats: anthropometrical and mechanical analyses. Acta Bioeng Biomech, 2008; 10:39–41.

65.

Chogtu

, Arivazhahan

, Kunder

, Tilak

, Sori

, Tripathy

. Evaluation of acute and chronic effects of d-galactose on memory and learning in Wistar rats. Clin Psychopharmacol Neurosci, 2018; 16:153–160.

66.

Hao

, Huang

, Gao

, Marshall

, Chen

, Xiao

. The influence of gender, age and treatment time on brain oxidative stress and memory impairment induced by d-galactose in mice. Neurosci Lett, 2014; 571:45–49.

67.

Cardoso

, Magano

, Marrana

, Andrade

. d-Galactose high-dose administration failed to induce accelerated aging changes in neurogenesis, anxiety, and spatial memory on young male Wistar rats. Rejuvenation Res, 2015; 18:497–507.

68.

S-C

, Liu

J-H

, Wu

R-Y

. Establishment of the mimetic aging effect in mice caused by d-galactose. Biogerontology, 2003; 4:15–18.

69.

Budni

, Garcez

, Mina

, Bellettini-santos

, da Silva

, da Luz

, Schiavo

, Batista-Silva

, Scaini

, Streck

, Quevedo

. The oral administration of d-galactose induces abnormalities within the mitochondrial respiratory chain in the brain of rats. Metab Brain Dis, 2017; 32:811–817.

70.

Hadzi-Petrushev

, Stojkovski

, Mitrov

, Mladenov

. d-Galactose induced changes in enzymatic antioxidant status in rats of different ages. Physiol Res, 2015; 60:61–70.

71.

Rodrigues

, Biasibetti

, Zanotto

, Sanches

, Schmitz

, Nunes

, Pierozan

, Manfredini

, Magro

DDD

, Netto

, Wyse

ATS

. d-Galactose causes motor coordination impairment, and histological and biochemical changes in the cerebellum of rats. Mol Neurobiol, 2017; 54:4127–4137.

72.

Baeta-Corral

, Castro-Fuentes

, Giménez-Llort

. Sexual dimorphism in the behavioral responses and the immunoendocrine status in d-galactose-induced aging. J Gerontol A Biol Sci Med Sci, 2018; 73:1147–1157.

73.

Altun

, Bergman

, Edström

, Johnson

, Ulfhake

. Behavioral impairments of the aging rat. Physiol Behav, 2007; 92:911–923.

74.

Rapp

, Bachevalier

. Cognitive development and aging. In: Squire

, Berg

, Bloom

, Lac

, Ghosh

, Spitzer

(eds): Fundamental Neuroscience. Amsterdam: Elsevier, 2013, pp. 919–945.

75.

Woodruff-Pak

, Foy

, Akopian

, Lee

, Zach

, Nguyen

KPT

, Comalli

, Kennard

, Agelan

, Thompson

. Differential effects and rates of normal aging in cerebellum and hippocampus. Proc Natl Acad Sci U S A, 2010; 107:1624–1629.

76.

Horvath

, Mah

, Lu

, Woo

, Choi

O-W

, Jasinska

, Riancho

, Tung

, Coles

, Braun

, Vinters

, Coles

. The cerebellum ages slowly according to the epigenetic clock. Aging, 2015; 7:294–305.