Animals
60 adult female Swiss Albino mice (age: 6-8 weeks, body weight: 25-30 g) bred in Central Animal House, Manipal, Manipal Academy of Higher Education (MAHE) were housed in separate polypropylene cages. Animals were kept at temperatures (22-24° C), 12-hour light/12-hour dark cycle and 40%-60% relative air humidity under standard conditions. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC /KMC/01/2019), and experiments were conducted in accordance with the ethical standards approved by the Ministry of Social Justice and Empowerment (Government of India) and the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).
Experimental deign
Six to eight weeks old Swiss Albino female mice (n=60) were used as experimental model in this study. The animals were divided into short- and long-term groups. In short term animal groups, after finishing drug treatment two weeks recovery gaps were given and in long term animal groups, two months recovery gaps were given after the completion of treatment. During the recovery gap period the animals were kept under observations for any others major anomalies and were provided with regularly normal standard food pellet diet with water.
Study deigns for short term animal groups
In this experiment, 30 adult Swiss albino female mice (age: 6-8 weeks, body weight: 25-30 g) were divided into five groups (n=6/group) and treated as follows.
Normal control: All animals were treated with 0.9 % NaCl, 1 mL/kg.b.w.
Drug susceptible (DS) treatment group: In intensive phase animals were treated with INH (39 mg/kg), RIF (78 mg/kg), EMB (156 mg/kg) and PZA (195 mg/kg) once daily for one week and followed by continuation phase with INH (39 mg/kg), RIF (78 mg/kg) and EMB (156 mg/kg) once daily for three weeks.
INH resistance (INHR) treatment group: In intensive phase animals were treated with Kanamycin (Km) (97.5 mg/kg), Levofloxacin (Lfx) (130 mg/kg), RIF (78 mg/kg), PZA (195 mg/kg), EMB (156 mg/kg) once daily for one week and followed by continuation phase Lfx (130 mg/kg), RIF (78 mg/kg), PZA (195 mg/kg) and EMB (156 mg/kg) once daily for three weeks.
RIF resistance (RIFR) treatment group: In intensive phase animals were treated with Km (97.5 mg/kg), Lfx (130 mg/kg), Ethionamide (Eto) (97.5 mg/kg), Cycloserine (Cs) (97.5 mg/kg) , INH (39 mg/kg) , PZA (195 mg/kg), EMB (156 mg/kg) once daily for one week and followed by continuation phase Lfx (130 mg/kg), Eto (97.5 mg/kg), Cs (97.5 mg/kg) , EMB (156 mg/kg) and INH (39 mg/kg) once daily for three weeks.
Multidrug resistance (MDR) treatment group: In intensive phase animals were treated with Km (97.5 mg/kg), Lfx (130 mg/kg), Eto (97.5 mg/kg), Cs (97.5 mg/kg), PZA (195 mg/kg), EMB (156 mg/kg) once daily up to one week and followed by continuation phase Lfx (130 mg/kg), Eto (97.5 mg/kg), Cs (97.5 mg/kg), EMB (156 mg/kg) once daily for three weeks.
Animals were sacrificed after giving two weeks recovery gap from the last drug treatment. To understand the late effects, mice were sacrificed after giving two months recovery gap from the last drug treatment.
Sample collection
At the end of experiment for both short and long term, animals were sacrificed and dissected to observe internal organs like liver, kidney and spleen were collected for histopathology study. Bone marrow cells were collected from the animals to assess the DNA damage.
Histopathological evaluation of liver, kidney and spleen tissue
Liver, kidney and spleen tissue were washed in normal saline 0.9% NaCl and were fixed in 10 % formalin. Then the tissues were dehydrated by putting in different graded of alcohol and then the tissues were impregnated with molten paraffin wax and sections were taken at 5 µm for staining with Hematoxylin and Eosin (H&E) and Periodic acid-Schiff stain (PAS). Extent of damage to the organ was assessed using modified histological assessment index (HAI) given by [9]. The level of lesion depends on its pathological importance. Every alteration is assessed using a score value ranging from (0 to 6) depending upon the degree and extent of the alteration. Three views per section were observed from six animals of a group. For calculating organ alteration index, formula given by [9] was used. This index represents degree of damage to an organ. It is sum of multiplied importance factors and score values of all changes found within the examined organ. A high index indicates high degree of damage.
Histopathology of liver tissue
After preparation of block on molten paraffin wax, 5 µm sections was taken at microtone and was stained with H & E to observe the pathological changes for both short and long-term groups. Prime focus was given on necrosis, nuclear alteration, WBC infiltration, vacuolization and sinusoidal dilation. All the observations were done in blinded manner. Scoring of Kupffer cells were also done under compound microscope.
Morphometric measurement of liver tissue
By using image pro software, measurements were taken on central vein diameter and sinusoidal diameter. All the data was presented in graph.
Histopathology of kidney tissue
After preparation of block on molten paraffin wax, 5 µm sections was taken and staining was done with H & E to observe the pathological changes for both short and long-term groups. The main importance was given on mesangial cells proliferation, vacuolization, WBC infiltration and dilation of Bowman’s space. All the observation was done in blinded manner.
Morphometric measurement of kidney tissue
By using image pro software, measurements were taken for glomerular diameter, Bowman’s space diameter and Bowman’s capsule diameter. All the data were presented in graph.
Histopathology of spleen tissue
After preparation of block on molten paraffin wax, 5 µm sections was taken at microtone and was stained with H&E to observe the pathological changes for both short and long-term groups of spleen tissue.
Collection of bone marrow cells
Both the femur bone was collected from all short-term animals. By using 1 mL syringe, PBS (Phosphate Buffered Saline) was injected from one end of femur bone and from other end bone marrow cells were collected in 2 mL eppendorf tube. The tube was centrifuged three times at 2000 rpm for 10 minutes to get clean sediment. Each time supernatant was discarded and the sediments were mixed with PBS and finally bone marrow slides were made by spreading the sediments on glass slide.
Expression of γ-H2AX in bone marrow cells to assess the DNA damage
The bone marrow cells were washed and fixed in 4 % paraformaldehyde overnight. The supernatant was discarded after centrifuging the cells at 1000 rpm for 10 min. The pellet was washed in PBS three times to remove the traces of fixative. The cells were incubated with permeabilization buffer containing 0.25 % Triton X-100 in PBS for 15 min at room temperature. After centrifugation, the cell pellet was incubated with blocking solution containing 1 % bovine serum albumin in PBS with 0.1 % Tween-20 for 30 min at 37 ºC. The cells were incubated with 1:50 anti-phospho histone H2AX antibody (primary antibody) in blocking solution at 4 ºC. The cells were washed again with PBS and incubated with 1:200 goat anti-rabbit IgG H&L Alexa Fluor 488 (secondary antibody) for 2 h at room temperature. The cells were washed three times with PBS, transferred to clean grease free microscopic slides and were mounted using Fluoroshield™ with DAPI. A clean coverslip was placed on the cells and observed under fluorescence microscope (Zeiss Imager A1-AX10) at 400x magnification for the signals. Total 500 cells were counted from each group to assess the DNA damage.
Micronucleus assay
After preparation of bone marrow slides, the slides were stained with Acridine Orange dye. The scoring was done according to the criteria given by [16]. The nucleus was considered as a micronucleatic cells that were present inside the cytoplasm and counted as micronucleus if the size 1/3rd of the main nucleus. Scoring was done for 2000 lymphocytes.
Apoptotic cells assay
After preparation of bone marrow slides the section was stained with Acridine Orange dye to observe apoptotic cell. Identification of apoptotic cell was done according to the following criteria; cell shrinkage, chromatin condensation, DNA fragmentation, membrane blabbing and formation of apoptotic bodies. All the scoring was done in blinded manner and the data was presented in graph.
Statistical analysis
Using the Statistical Package for the Social Sciences (SPSS version 16.0; SPSS), data was expressed as mean ± standard deviation and analyzed by one way analysis of variance (ANOVA) followed by post hoc Tukey test. A level for p ≤ 0.05 was considered to be statistically significant (p ≤ 0.05).