Chemicals and reagents
Sodium chloride (S3014), potassium chloride (P9541), sodium phosphate dibasic (S3264), potassium phosphate monobasic (P9791), potassium bicarbonate (12602), ammonium chloride (A9434), ethylene diamine tetra acetic acid di-sodium salt (E6635), bovine serum albumin (BSA) (5482), absolute ethanol (100983), methanol (34860) were purchased from Sigma–Aldrich, St. Louis, MO, United States. CD 34, Sca 1, and CBA Flex Set which contains IL-3 (558346), IL-6 (558301), TNFα (558299), IFN γ (562233), G-CSF (560152), GM-CSF (558347), IL-1α (560157), IL-1β (560232), Mouse/Rat Soluble Protein Master Buffer Kit (558266) were procured from BD Biosciences, United States. May-Grunwalds Stain (S039) and Giemsa Stain Solution (TCL083) were procured from Hi-Media India.
1 L 1X PBS (8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium phosphate dibasic, 0.25 g of potassium phosphate monobasic to 1 L at pH 7.4), 1 L 1X RBC lysis buffer (1 g of potassium bicarbonate, 8 g of ammonium chloride and 0.03 g of di-sodium EDTA), 1% BSA in PBS, 70% ethanol in PBS, and may-grunwald-giemsa stain mixed in 3:1 ratio were prepared in the laboratory.
Animals and groupings
Pathogen free Strain ‘A’ male mice weighing 25.0–30.0 gm were received from Institute of Nuclear Medicine and Allied Science (INMAS) experimental animal facility at the age of 9 weeks, which is equivalent to young adult humans. Six mice per cage were housed under 25 ± 3 °C temperature and relative humidity of 30–70% in 12 h light/dark cycle with standard food and water. The study design strictly adhered to the guidelines approved by Institutional Animal Care and Use Committee (IACUC) of our institute, Institute of Nuclear Medicine and Allied Sciences (INMAS), Defence Research and Development Organization (DRDO), Delhi, India (Institute Animals Ethics Committee number: INM/IAEC/2019/01).
After 1 week of acclimatization under ambient conditions, mice were grouped for the survival assay according to localized gamma radiation dose i.e. 7, 9, 10, 12, 15, 17 and 19 Gy. Eighteen animals were assigned in each group for this assay.
For all other parameters mice were divided into two groups with six animals in each. Group 1, consisted of untreated or sham irradiated animals. Group 2 was exposed to 15 Gy localized irradiation. Mice were sacrificed at different time points (1, 2, 4, 7, 10, 15, 20, 25, and 30 days) post treatment. Hematopoietic stem cell marker CD 34 and Sca1 were measured on 1, 4, 7, and 10th day post-irradiation.
Irradiation
A Cobalt-60 teletherapy (Model: Bhabhatron-II, Panacea Medical Technologies Pvt Ltd, India) machine was used to irradiate the hinds of the three mice at a time (Fig. 1). The cobalt-60 beam was calibrated following IAEA TRS-398 protocol, with measurement done in water phantom (30 cm × 30 cm × 30 cm), for a field size 10 cm × 10 cm at source to surface distance (SSD) of 80 cm. A Farmer type, 0.6 cc volume, ionization chamber was utilised and the same was recently calibrated for absorbed dose to water (NDW) at Secondary Standard Dosimetry Laboratory (SSDL) of India. The measured absorbed dose rate (water, 10 cm × 10 cm, 80 SSD, Dmax) was 1.39 Gy/min.
Representative image of animal exposure with γ-rays in a field size of 2 cm × 35 cm.
The localized bone marrow irradiation was done using single Posteroanterior (PA) beam with 2 cm × 35 cm field size at 80 cm SSD opened through secondary collimators of the machine. The 5 mm thick acrylic re-strainers were used to immobilize each mouse which also provides the build-up thickness for the irradiation. The dose prescription point was the surface of the hinds i.e. 5 mm below the surface of the re-strainer. The whole irradiation platform was placed on 5 cm thick acrylic slab to provide sufficient back scatter and all the air gaps between the three mice were filled with tissue equivalent gel bolus or wet cotton to compensate for missing side scatter. The prescription doses were absorbed dose to water and not corrected for any tissue heterogeneity.
The absolute and relative dosimetry of collimated field (2 cm × 35 cm) were performed using pin point (0.04 cc volume) ionization chamber and EBT3 (ISP, Wayne, USA) Gafchromic dosimetry films in solid water phantom. The response of the pin point chamber and EBT3 films was calibrated against the SSDL calibrated ionization chamber in solid water phantom at the depth of 5 cm and field size 10 cm × 10 cm in the same beam quality (i.e. cobalt-60). The film scanning was performed using Epson 10000XL flatbed scanner as per the manufacturers scanning protocol. The beam profiles were obtained from the scanned films and the full width at half maximum (FWHM) in transverse direction was 1.78 cm for the opening of 2.0 cm. The dose uniformity was better than 4% in central 80% of the field. The absolute dose rate (dose to water, dmax, 2 cm × 35 cm, SSD 80 cm) measured at the centre of the field was 1 Gy/min. The overall uncertainty in dose calculations is 6.5% with enhancement or coverage factor (k = 2) at 95% confidence. The main body of the mice was covered with 15 mm lead to prevent any scatter radiation reaching at other body parts of the animal. The surface dose reaching at the shielded body parts of the mice was measured using 0.4 cc ionisation chamber. The measured mean surface dose was 0.024 ± 0.014% of the open field dose. This dose is negligible and considered to be acceptable.
Survival assay in mice
For survival assay, 18 mice from each experimental group i.e. 7, 9, 10, 12, 15, 17 and 19 Gy, were irradiated at a fixed dose rate of 1 Gy/min. Groups were inspected daily for their morbidity and mortality status for a total duration of 30 days. All the surviving mice were euthanized humanely using intra-peritoneal injection (i.p.) with dexmedetomidine (0.3 mg/kg) in combination with ketamine (190 mg/kg) at the completion of the observation period.
Validation of hematopoietic syndrome model
The model of hematopoietic syndrome was validated with localized radiation of 15 Gy as this was the maximum dose at which all the animals survived as observed in the survival results. Mice were euthanized using intra-peritoneal injection (i.p.) with dexmedetomidine (0.3 mg/kg) in combination with ketamine (190 mg/kg) at 1, 2, 4, 7, 10, 15, 20, 25, and 30th day time points. Further analysis such as organ weight, cell counting, and serum cytokines measurement assays were conducted on various organs such as femur bone, spleen, thymus, lungs, kidneys (L), kidneys (R), liver and heart and blood at above mentioned time points. Hematopoietic stem cell markers (CD 34 and Sca 1) were measured in bone marrow cells of mice at 1, 4, 7, and 10th day time points.
Body and organ weights
The control and 15 Gy locally irradiated group animals were sacrificed at 1, 4, 7, 10, 15, 20, 25, and 30th day time points. The organs (spleen, thymus, lungs, kidneys (L), kidneys (R), liver and heart) were dissected out and weighed individually. Relative organ weight was calculated as the ratio between organ weight and body weight.
Haematology
The blood samples were collected from untreated and 15 Gy localized radiation group at 1, 4, 7, 10, 15, 20, 25, and 30th day time points in EDTA vial by cardiac puncture. 20 µl of collected blood from each experimental animal was taken for haematology and analysed using Nihon Kohden Celltac α, Tokyo, Japan, a fully automatic 3 part haematology analyzer.
Bone marrow cell counting
Total bone marrow cells were flushed out at 1, 4, 7, 10, 15, 20, 25 and 30th day points in 1 ml PBS from both femurs of the untreated mice and 15 Gy localized radiation group. The PBS containing cells was centrifuged at 2000 rpm for 8 min and the supernatant was discarded. The cells were washed with 1 ml PBS and the cell pellet was re-suspended in 1 ml of PBS. The re-suspended sample was diluted as required. The cells were then counted using improved Neubauer chamber under Olympus BX-63 microscope.
Bone marrow smears study
Bone marrow cells collected at 1, 4, 7, 10, 15, 20, 25, and 30th day post-irradiation from different treatment groups were centrifuged at 2000 rpm for 8 min and re-suspended in 50 μl of Fetal Bovine Serum (FBS). A small drop of cells suspension was dropped on a clean microscopic slide and then smeared thinly over an area of 2–3 cm by pulling another glass slide held at a 45° angle. Cells were fixed with methanol and stained with may-grunwald-giemsa. Slides were then analysed using Olympus BX-63 microscope.
Splenocytes and thymocytes counts
The organs excised at 1, 4, 7, 10, 15, 20, 25, and 30th day time-points were cleaned in chilled PBS. Using sterile-chilled frosted slides both the thymus and spleen were minced into a cell suspension and which was further centrifuged at 2000 rpm for 8 min. After centrifugation the supernatant was discarded. The RBCs were lysed using RBC lysis buffer and the cells were washed with 1 ml PBS. The cells were then counted using improved Neubauer chamber under Olympus BX-63 microscope.
Measurement of hematopoietic stem cell marker CD 34 and Sca1
Bone marrow cells were isolated from both the femurs from various treatment groups at different time points. Cell were washed with chilled PBS and fixed with 70% chilled ethanol and stored overnight at – 20 °C. Following day, five million cells were blocked with 1% BSA-PBS and stained with CD 34 and Sca 1 cell-surface markers for 20 min at room temperature. 10,000 cells from each sample were analyzed using FACS LSR II (BD Biosciences, San Jose, CA) and results were visualized by the BD FACSDiva V7 software (BD Biosciences, San Jose, CA).
Cytokines expression changes
Mice blood samples were collected in BD serum separation tube at different time-points post treatment (1, 4, 7, 10, 15, 25, and 30 days) via. cardiac puncture from different treatment groups. Samples were incubated at room temperature for 2 h and after incubation centrifuged at 6000g for 15 min. Serum was separated and stored at − 80 °C until analysis. The Levels of IL-3, IL-6, TNFα, IFN γ, G-CSF, GM-CSF, IL-1α,and IL-1β cytokines in mice serum were determined by using respective BD Cytometric Bead Array (CBA) Flex Set (BD Biosciences, USA) on dual laser flow analyzer (LSR-II, Becton Dickinson Biosciences, USA) according to manufacturer’s instructions. Results were analysed using FCAP Array V3 software (BD Biosciences, San Jose, CA).
Statistical analysis
All values obtained in our results are represented as mean ± SD of three independent replicates. The 30 day survival data was plotted using Kaplan–Meier analysis. The difference between the experimental groups was evaluated by one-way analysis of variance, with Newman–Keuls multiple comparison test (V, 8.01; GraphPad Prism, San Diego, CA, USA). The comparisons were made among the untreated and 15 Gy locally irradiated groups for all experimental parameters except the survival study. A value of p < 0.05 is considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001, ns = not significant (p > 0.05).
Source: Ecology - nature.com
