Animals and VDD model

This study was conducted on adult male Sprague Dawley rats (age 10–12 weeks; weight 250–300 g). The animals were procured commercially from Vivo Bio-Tech, Hyderabad, India, and obtained informed consent from the company to use the animals in this study. The study protocol was in line with animal ethics guidelines for experiment. The experiments were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The test facility (Dabur Research Foundation, India) was registered with CPCSEA (Registration No. 64/PO/br/s/99/CPCSEA) for an experiment involving animals, Ministry of Environment and Forest, Govt. of India and the study was approved by the Institutional Animal Ethical Committee (IAEC), Dabur Research Foundation (DRF), India (IAEC/39/468) on dated 3rd July 2017.

Rats were housed under controlled environmental conditions at 25 °C in a 12 h light/dark cycle and water ad libitum. Efforts were made to reduce animal suffering and minimize the number of animals used. Normal control animals were fed with standard normal chow diet (type − 1324, batch/lot no. 110,118//0640) and other treatment group animals were fed with a vitamin D₃ deficient diet (VDD; type – C 1017, batch/lot no. 481/2017), obtained from Altromin Spezialfutter GmbH & Co., KG, Germany, for three weeks. To check the rat’s model three weeks after VDD administration, 25(OH) vitamin D₃ was measured in the circulation. Reduced 25(OH) vitamin D₃ levels by about 50–60% in disease control animals (VDD) compared to normal control animals were considered as VDD-induced animals and were selected for further treatment.

Groups and drug administration

The 48 adult rats were each randomly assigned to the following six groups (n = 8): normal control with 0.5% CMC, VDD model with VDD and 0.5% CMC, VDD + calcitriol (0.5 µg/kg body weight per oral) treatment, VDD + vit.D₃ (0.25 mg/kg body weight per oral) treatment, VDD + vit.D₃ (0.5 mg/kg body weight per oral) treatment, and VDD + vit.D₃ (1 mg/kg body weight per oral) in the treatment groups. Dosage of vitamin D was chosen as per Bakhtiari-Dovvombaygi et al. 2021 with slight modification [10]. Each treatment groups’ animals were treated orally for eight weeks following the induction of the VDD model. In the 8th week, all the animals were tested for behavioral parameters such as Y-maze task, muscle grip strength, and knee joint pain assessment. After fasting overnight, rats were anesthetized using isoflurane (Aerrane, Baxter Healthcare Corporation, USA) by inhalation (3% induction, then 2% maintenance, with oxygen) at the rate of 1.5 L/min. Approximately 250 µL of blood from each rat was collected from retro-orbital plexus with the help of micro capillary tube and serum samples were used for biomarkers analysis. After bleeding, cerebrospinal fluid (CSF) was collected with a stereotaxic instrument for the estimation of Klotho and β-endorphin by ELISA. All animals were sacrificed by a CO₂ asphyxiation. Necropsy was performed in a manner that avoided the occurrence of post mortem change in the collected tissues. The organs and tissues were examined in situ before dissecting or collecting tissues. On completion of the gross pathology examination, the livers, kidneys, and adipose tissues were collected and stored in -80 °C for ELISA measurement of 25(OH) D₃, a metabolite of vitamin D₃. To estimate VDR expression, half of the liver and adipose tissues were used.

Y-Maze test

Y-maze testing was used to assess impairment of short-term spatial memory. In the present study, a Y-maze consisting of three arms (35 cm long, 25 cm high, and 10 cm wide) was used with a 120° angle. The arm closest to the experimenter was designated as the start arm, in which rats were placed in each trial. During the first trial (5 min), the novel arm entry was closed and the animals were allowed to explore both open and start arms. Within 30 min of opening the novel arm, animals were allowed to explore all three arms. Exploratory behavior was evaluated for five minutes. In week 8, all the animals were subjected to the Y-Maze test for 5 min. The novelty test includes the estimation of time spent in each arm and number of entries made into each arm. By contrast, spontaneous alternation (i.e., sequential entry into three arms in overlapping triplet sets) includes percentage alternation behavior, was calculated using the following equation: (successive triplet sets/total number of arms entries − 2) X 100 (successive triplet sets : entries into three successive arms).

Muscle grip strength and knee joint pain score

The muscle grip strength was assessed by lifting the tails of rat, so that they could grasp a rigid bar attached to a digital force gauge. In addition, each animal was gently pulled backward by its tail, and the tension reading of the digital force gauge was measured. Before the animals released the bar, that reading was reported in grams as grip strength. The same experimental procedure was repeated five times successively, and the highest muscle grip strength value from each trial was recorded as the grip strength. A pressure application meter (PAM, Ugo Basile, Cat. No. 38,500) was used to measure knee joint pain. By using the PAM probe, pressure was applied to both knee joints of the animal. As a result of pain, the PAM instrument recorded the animal’s limb withdrawal threshold. The data were expressed as a gram force (i.e., pain threshold).

Serum biomarker

The serum biomarkers were determined in all the animals in different treatment groups. Blood was collected and used to isolate serum for the estimation of parathyroid (PTH) [CEA866Ra, Clond-Clone], calcitonin [CEA472Ra, Clond-Clone], thyroxine [CSB-E05082r, CUSABIO], and CRP [CSB-E07922r, CUSABIO] using ELISA.

VDR in liver and adipose tissue

According to the manufacturer’s recommended standard procedure (CUSABIO, USA) [CSB-EL025583RA, CUSABIO], we measured VDR concentrations in liver and adipose tissue homogenates. In brief, about 100 µL of each test sample, including the control and reference standard, were poured into the labeled wells and incubated at 37 °C for an hour. After removing the cover and discarding the liquid, and 100 µL of detection reagent-A working solution was poured into all wells and again incubated at 37 °C for an hour. The plate was then washed three times with 1X washing buffer. Each well was then filled with 100 mL of detection reagent B working solution, sealed, and incubated at 37 °C for 30 min. Following the discarding of the solution, the plate was washed and 90 µL of 3,3’5,5’-Tetramethylbenzidine (TMB) substrate added to each well and incubated at 37 °C for 10 min. After that, 50 µL of stop solution was added to each well, and reading was taken at 450 nm.

Metabolite (25-OH Vit D₃) analysis in multiple matrix

A serum, liver, and kidney homogenate were used from all the animals for estimating the 25-OH Vit D₃ metabolite (CSB-E08098r, CUSABIO) using ELISA, as recommended by the manufacturer.

Klotho and β-endorphin in CSF

CSF was collected by the standard in-house method using a stereotaxic instrument to measure Klotho [CSB-E14958r, CUSABIO] and β-endorphin [CSB-E08206r, CUSABIO] using ELISA.

Statistics

Statistical analysis was performed using Sigma-Plot (V11.0). Data are shown as mean ± standard error of the mean. Multiple comparisons were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc analysis by Tukey’s/Dunnett’s test and between two groups by Student’s t-test. F values p < 0.05 were regarded as statistically significant.