Human model
Patients
Three patients with inevitable RT to the adrenal gland after standard optimization for intensity-modulated radiation therapy (IMRT) were prospectively enrolled. The patients were required to have a World Health Organization performance status of 0 to 2. All patients were treated with curative intent.
Blood analysis
Serum levels of cortisol at 08:00 and 16:00, aldosterone, and ACTH were measured on the day before RT, in the middle of the RT course, and 1 week after RT using radioimmunoassay kits. The cortisol levels at 16:00 were considered baseline cortisol levels according to the circadian rhythm.
RT technique
Patients underwent computed tomography (CT) simulation in the supine position and were immobilized with an alpha cradle. The adrenal glands were contoured while planning the CT images. The adrenal glands were located superior and anteromedial to the upper pole of the kidneys and appeared as a triangular or Y-shaped organ. IMRT was used for all patients. The treatment plans were generated using 6-MV - or 10-MV photons. All patients were treated with linear accelerators, and dosimetric parameters, including the mean dose of the left adrenal gland, were collected using RT planning systems (Eclipse Treatment Planning System v.13; Varian Medical Systems Inc., Palo Alto, CA, USA). Dose distributions for the planning and dose-volume histograms (DVHs) were recorded for evaluation (Fig. 1).
Fatigue severity scale
Fatigue was scored according to the validated fatigue severity scale (FSS), a short questionnaire for rating the levels of fatigue on the day before RT and 1 week after RT [21, 22]. The questionnaire contained nine statements that attempted to explore the severity of fatigue symptoms. Patients were required to circle a number from 1 to 7, depending on how appropriate they felt the statement applied to them during the past week. A low value indicated that the statement was not applicable, whereas a high value indicated agreement. The scoring was performed by calculating the average response to the questions. Patients with fatigue reported an averaged high value.
Ethical statement
This study was approved by the institutional review board of our institution with ethics committee approval and informed consent (IRB number: 20MMHIS132e).
Experimental animal model
Experimental animals
Four-week-old male BALB/c mice were purchased from the National Laboratory Animal Center, Taiwan. Mice were maintained at a temperature of 22 ± 1°C and humidity of 55 ± 10% with 12-hours of light (artificial illumination; 07:00-19:00). The mice were fed a commercial rodent diet (LabDiet 5001, PMI Nutrition International LLC, Brentwood, MO, USA) and allowed access to purified water in a water bottle.
After one week of pre-feeding, the mice were randomly grouped into four groups of four mice per group as follows: the sham group, the 0 Gy group, 0.5 Gy group, and 2 Gy group. The mice in the sham group only had fur shaved around the left flank area as controls. The mice in the 0 Gy group were surgically explored for the identification of the left adrenal gland after fur shaving. The mice in the 0.5 Gy group received a radiation dose of 0.5 Gy on the left adrenal gland after surgical exploration and fur shaving, whereas the mice in 2 Gy group received a dose of 2 Gy.
After grouping, the blood of all mice was collected at 15:00 and 20:00 for the measurement of plasma levels of stress hormones. The cortisol levels at 15:00 were considered the baseline cortisol levels according to the circadian rhythm. Surgery and radiation were arranged on the next day (Fig. 2a and 2b). The swimming endurance test was performed for exhaustion assessment to represent fatigue after wound healing around 5 days after surgery and RT. Then another blood sampling was performed at 15:00 and 20:00 on the next day. Histopathology of the adrenal gland was sampled 7 days after surgery and RT.
All procedures were approved by the Experimental Animal Committee at MacKay Memorial Hospital (MMH-A-S-103-06), in compliance with the National Institute of Health Guide for Care and Use of Laboratory Animals (NIH Publication, 8th edition, 2011). All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.
Blood analysis
Blood samples were collected via retro-orbital blood sampling and detected using Drew Hemavet HV950 (Drew Scientific, Inc., Dallas, TX, USA). For serum biochemical parameters, blood samples were centrifuged (1000×g for 15 min at 4°C), and the serum was stored at −80°C until use. Serum cortisol levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits from Arbor Assays (Ann Arbor, MI, USA).
RT technique
After surgical exploration, intraoperative RT was administered to the left adrenal glands of the mice using a linear accelerator (IX, Varian, Palo Alto, CA) with an electron beam (6 MeV, 90%, dose rate: 400 MU/min).
Exhausted swimming test
The swimming ability of all mice was confirmed using the swimming test before the experiment. The swimming test was stratified according to studies performed by Matsumoto et al. An acrylic pool (90×45×45 cm) filled with water to a depth of 38 cm was used. The temperature of the water was maintained at 25 ± 0.5°C with a water heater and thermostat. The mice were allowed to swim until they failed to rise to the surface of the water to breathe within 7 s. The mice were rescued at the time to avoid drowning. For the swimming endurance test, all mice were loaded with lead wire weighted to 7% of body weight. These mice were then placed in a water tank for swimming (Fig. 2c). The duration of starting swimming to fail to rise within 7 s was recorded as the swimming endurance time [20, 23].
Histopathology
Formalin-fixed and paraffin-embedded adrenal glands from each group of mice were sectioned. Sections 4 µm thick were stained with hematoxylin and eosin (H&E).
Statistical Analysis
Statistical analysis was performed using SigmaPlot version 12.0 (Systat Software, Inc., CA, USA). Numerical data were expressed as mean ± standard deviation. A paired t-test was performed for FSS, serum levels of cortisol, aldosterone, and ACTH, which were measured before, during, and after RT for the human data and serum levels of cortisol in mice, which were measured before and after RT. The swimming endurance time of different groups of mice was recorded, and the significance was analyzed using one-way ANOVA. Differences were considered significant if p < 0.05.