Study design: preliminary and main experiments
All animal procedures for experiments were approved by the Animal Care and Use Committee of Nagoya University (license number; 28106) and carried out according to the National Institutes of Health's Guide to the Management and Use of Laboratory Animals. All experiments were performed painlessly under pentobarbital sodium or isoflurane anesthesia and every effort was made to minimize mouse distress. This study consisted of two stages of experiments. First, we investigated the vitamin D and bone metabolism in ovariectomized mice, a model of postmenopausal osteoporosis, without irradiation as a preliminary experiment. Based on the results of this preliminary treatment, we determined the timing of UV-LED irradiation. Under the determined conditions, UV-LED was irradiated to ovariectomized mice to investigate the effects on vitamin D and bone metabolism, bone mineral density and strength, in addition to the muscle volume and strength as the main experiment.
Preliminary Experiments
Mice and Diet
Inbred C57BL/6 female mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan). The mice were shielded from normal fluorescent UVB in a 12 hour light-dark cycle, and kept at a temperature of 25 ° C. Mice were fed a standard wheat-based diet until the age of 12 weeks. To make an experimental 25-hydroxyvitamin D [25 (OH) D] starved mouse group, Mice were fed AIN93G as a vitamin D-containing diet and AIN93GA-2 (Oriental Yeast Co., Ltd., Tokyo, Japan) as a vitamin D-deficient diet until 24 weeks of age when the study protocol was completed.25. AIN93G contains 1000 IU / kg of vitamin D, 0.50% calcium and 7.00% total fat, whereas AIN93GA-2 contains 0 IU / kg, 0.50% and 7.00%, respectively. To create the postmenopausal osteoporosis model, we ovariectomized mice at 16 weeks of age as described in the previous reports2426. Finally, the mice were randomly divided into four groups (n = 6): (1) Vit.D + OVX (ovariectomy)- as normal control; (2) Vit.D + OVX+; (3) Vit.D- OVX-; (4) Vit.D- OVX+.
Ovariectomy or sham surgery
16-week-old mice were randomly assigned to OVX or sham surgery group, anesthetized with intraperitoneal pentobarbital, and operated on. In short, OVX was performed by a bilateral dorsoventral approach as previously reported27. Each ovary was cauterized and resected at the tip of the uterine horn. The sham surgery also made an incision to expose the ovaries, but did not remove the tissues. After ovarian and sham surgery, mice were randomly divided into groups based on experimental design.
Serum Metabolites
Levels of serum 25(OH)D and 1,25(OH)2D were determined at 12 weeks of age (pre-vitamin D diet), 15 weeks (pre-OVX/sham), and 20, 24 weeks (4, 8 weeks after OVX/sham, respectively). The levels were determined with radioimmunoassay kits (SRL, Tokyo, Japan) according to the manufacturer's instruction. Blood samples were taken from the orbital plexus and used for measurement for six mice in each group, and stored at − 20 ℃ until quantification. Levels of the vitamin D were classified as follows: deficient, 25(OH)D < 25 nmol/L or sufficient, 25(OH)D > 90 nmol/L, as described previously28.
Analyses with Micro-Computed Tomography (CT)
The distal femoral metaphysis was used to assess the effect of ovariectomy on mouse trabecular and cortical microarchitecture. Analysis by micro-CT scan of the metaphysis of the right distal femur was performed every 4 weeks from 12 to 24 weeks (pre- to 8 weeks after OVX) of age for six mice alive in each group, by high resolution micro-CT scanner with a specific software (SkyScan 1176; Bruker, Kontich, Belgium), according to the previous reports 1229. Mice were anesthetized with Isoflurane (2.5% flow) and maintained below 2.5% using a nose-cone setup for imaging. Each scan was performed with a power supply voltage of 50 kV, a current of 500 µA, a rotation step of 0.5 °, a full rotation of over 180 °, and a 0.5 mm aluminum filter for reduced beam hardening. The exposure time was 0.89 s, and the pixel size was 9 µm. The scan also included phantom bones to analyze bone mineral density (250 mg / cm3 and 750 mg / cm3) to standardize grayscale values and maintain consistency between assessments. Three-dimensional (3D) microstructural images were reconstructed using NRecon software (Bruker, Kontich, Belgium), and morphometric parameters were analyzed using the SkyScan CT Analyzer (CTAn) software for trabecular and cortical bone of the femur. To determine cancellous bone morphometry parameters, the volume of interest (VOI) from 0.17 mm from the growth plate of the femur towards the diaphysis (2 mm high), including the trabecular and medullary cavity. To determine cortical bone morphometry parameters, the VOI started at the proximal end of the trabecular bone, and set up to 2 mm towards the central shaft (height 2 mm), targeting only the cortical shell. Bone parameters (bone volume fraction [BV (bone volume) /TV (trabecular volume), %], trabecular thickness [Tb.Th, µm], number [Tb.N, 1/mm], spacing [Tb.Sp, mm], bone mineral density [BMD, mg/cm3], and cortical thickness [Ct.Th, mm]) were calculated based on guidelines for analyzing bone microstructure in rodents with micro-CT30.
Main Experiments
Ovariectomy-induced osteoporotic mice and treatment groups
Thirty-two C57BL/6 female mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan). They were fed either a vitamin D containing or deficient diet from 12 weeks of age, OVX were performed in all mice at 16 weeks of age, and irradiated with UV from 24 to 48 weeks of age based on the results of the preliminary experiment. At 24 weeks of age before irradiation, 32 mice were divided into 4 groups: oral vitamin D-repletion without UV irradiation (Vit.D + UV-) as a control, oral vitamin D-repletion with UV irradiation (Vit.D + UV+), oral vitamin D-deficiency without UV irradiation (Vit.D- UV-), and oral vitamin D-deficiency with UV irradiation (Vit.D- UV+). Each group comprised 8 mice. At the age of 48 weeks, the mice were victimized, and specimens were collected, and subjected to RT-PCR analysis, mechanical tests, and histological assays.
UV Irradiation
In collaboration with Dr. Hiroshi Amano of our hospital, a UV lamp equipped with a surface mount device of an LED system developed by Nikkiso Co., Ltd. (Tokyo, Japan) was used as a UV source. We adjusted to a wavelength of 316 nm of the LED module, because 316 nm, which is in the UVA wavelength range, was already determined to provide Vitamin D efficiently, and confirmed to be less harmful in our previous work 14. As previously reported, a 2 × 4 cm a dorsal part of the skin was cleanly shaved as the area to be irradiated 31. Mice were irradiated in a transparent acrylic box with a bottom area of 4 × 6 cm. The lamp was placed 10 cm above the back of the mouse. The irradiance of the dorsal area inside the box by the LED module was calculated using a UV radiometer USR-45DA-10 (Ushio Inc., Tokyo, Japan). The reflectance coefficient in the box was calculated to be 1.77. UV irradiation dose was adjusted to 1000 J / m2 twice a week based on the determination in the previous study 14.
Serum Metabolites
Levels of serum 25(OH)D were determined at 15 weeks of age (pre-OVX/sham), 22 weeks (pre-UV irradiation), and 40, 48 weeks (12, 24 weeks’ UV irradiation), and levels of serum 1,25(OH)2D at 48 weeks of age. Serum inorganic phosphorus (IP) and calcium (Ca) concentrations were measured immediately after blood collection with standard colorimetric methods using a DryChem (FujiFilm, Tokyo, Japan). Levels of serum 1–84 parathyroid hormone (PTH) were measured using a sandwich ELISA kit (Immutopics, San Clemente, USA). Sera were stored at − 80℃, and subjected to the measurement for PTH.
Analyses using Micro-Computed Tomography (CT)
In the same way as in the preliminary experiment, we evaluated the effect of UV irradiation on trabecular and cortical microarchitectures in ovariectomized mice. The right distal femur metaphysis was scanned with micro–CT at 15 weeks of age (pre-OVX/sham), 22 weeks (pre-UV irradiation), and 29, 36, 42, 48 weeks (5, 12, 18, 24 weeks’ UV irradiation) for eight mice alive in each group, Bone parameters measured in the preliminary experiment were also measured in the main one.
Mechanical test
The mechanical strength of the right femur was measured by a three-point bending test using a mechanical strength analyzer (MZ500D; Maruto, Tokyo, Japan). Four mice were analyzed in each group. The central diaphysis of the femur was placed on two supports located 6 mm apart on the test device. At the midpoint between the two supports, a three-point bending test load was applied in the anteroposterior direction. Load-displacement curves were recorded at crosshead velocities of 2.0 mm / s. Mechanical parameters [ultimate load (N), stiffness (N/mm), displacement of fracture (mm), and work to failure (N*mm)] were measured with CTR win. Ver. 1.05 software (System Supply, Nagano, Japan).
Real-time RT-PCR Analysis
To evaluate the effects of UV irradiation on the control of the metabolism of vitamin D (25(OH)D and 1,25(OH)2D), mRNA expression levels of involved enzymes, which mediate the vitamin D metabolic pathway, were analyzed. We also evaluated expression levels of various bone turnover markers to analyze the effects of UV irradiation on the control of bone metabolism. Liver samples were obtained at 48 weeks of age and used for real time RT-PCR to calculate mRNA levels of vitamin D 25-hydroxylase (Cyp27a1). Kidney samples obtained at 48 weeks of age were subjected to the analysis of mRNA levels of 25 hydroxyvitamin D-1-alpha hydroxylase (Cyp27b1) and 1,25-dihydroxyvitamin D 24-hydroxylase (Cyp24a1). Tibia samples at 48 weeks of age were obtained to analyze mRNA levels of alkaline phosphatase (ALP), Osteocalcin, Runt-related transcription factor 2 (Runx2), Osterix, receptor activator of NFκB ligand (RANKL), nuclear factor of activated T cells (NFATc1) and nuclear factor kappa B (NFκB). RNA was extracted from liver, kidney, and tibia of each mouse using the RNeasy Mini Kit (Qiagen, Hilden, Germany) under the instruction of the supplier’s description. After reverse transcription, the cDNA was used for real-time RT-PCR using a LightCycler 480 (Roche Diagnostics, Mannheim, Germany), with 480 SYBR Green I Master (Roche Diagnostics, Mannheim, Germany), with 0.5 µM of the specific sense and antisense primers. Amplification protocol was as follows; denaturation of the template cDNA for 10 min at 95 °C, 45 cycles of a denaturation step for 10 s at 95 °C and an annealing step for 10 s at 60 °C and an extension step for 10 s at 72 °C. All PCRs contained a negative control that did not include a cDNA template. To confirm the specificity of the amplified products, the PCR products were subjected to melting curve analysis with LightCycler 480 and 2% agarose / TAE gel electrophoresis to measure Tm and amplicon sizes, respectively. To allow relative quantification after PCR, the LightCycler 480 software (Roche Diagnostics, Mannheim, Germany) was diluted from the specified gradient to calculate real-time efficiency. The levels of mRNA in the sample were calculated as relative values normalized at the level of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The primer pairs for Gapdh, Cyp27a1, Cyp27b1, Cyp24a1, ALP, Osteocalcin, Runx2, Osterix, RANKL, NFATc1, and NFκB were designed according to previous reports 3233.
Bone Histology
To assess bone formation in non-decalcified bone, the resected left bone was analyzed by Villanueva Goldner staining. A 48-week-old sample was fixed with 70% ethanol for 3 days, dehydrated stepwise with ethanol, and embedded in glycol methacrylate without decalcification (Aichi Pathologic Laboratory, Aichi, Japan). A 30 µm coronary section was made from the embedded tissues, stained with Villanueva Goldner, and analyzed under a light microscope.
Grip Strength measurement
Grip strength of the forefoot of mice was tested in each group at 48 weeks of age using a grip strength meter (Columbus Instruments, OH, USA), and recorded in Newtons (N). In brief, the tail of the mouse was held by the examiner's finger, and the forearm of the mouse held the handle. The examiners pulled the mouse body parallel to the floor with their fingers. Three sets of five tests were performed on each mouse, with short breaks between sets. Mean values of grip strength were determined.
Statistics
Results are expressed as mean ± standard deviation (SD). The Kruskal-Wallis test and the Mann-Whitney U test were applied to compare the results. SPSS statistics version 24 (IBM Corp. Armonk, NY) were used for all statistical analyses. P < 0.05 was considered as statistically significant.