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Male mice of age 6-8 weeks were received from in house animal facility of Orchid Research Laboratories and acclimatized for 7 days in 12 hrs light/dark cycle with standard laboratory chow diet and water ad libitum. All animals were handled according to guidelines of Experimental Animal Care issued by Committee for purpose of control and supervision of Experiments on Animals (CPCSEA) (Protocol no for Chronic ASH 17/IAEC-01/PPK/2010, Acute ASH 02/IAEC-01/PCP/2011).
4.2. Chemicals and Reagents
Normal chow diet procured from NutriLab Â® Rodent, Tetragon Chemical Pvt. Ltd., Bangalore. 60 Kcal % high fat diet (cat# D12492) procured from Research diet, New Brunswik, NJ, USA. Ethyl Alcohol AR 99.9% was procured from Changshu Yangyuan Chemicals, China. Vildagliptin, synthesized at Orchid Research Laboratories Ltd (ORLL) and Rosiglitazone was received as a gift sample from Dr Reddy's laboratory.
4.3. Acute ASH Model development
Three Male C57BL/6 mice, weighing18-22g were selected and were dosed with three different concentrations of alcohol i.e., 20%, 40% and 60% respectively. After 18 hours of alcohol administration blood was collected by retro orbital puncture and animals were sacrificed and liver was collected and sent for histopathological examination.
Reports from the histopathology showed dose dependent increase in steatosis, in 20% there was mild steatosis, 40% Marked steatosis and in 60% steatosis grade was Severe. Hence, 40% alcohol have used for screening this molecule.
4.3.1. Experimental design
A rodent ASH model was developed that simulated several features of human alcoholic steatosis. C57BL/6 Female mice aged 5-6 weeks having body weight 18-25g were used for the study. Animals were divided into following groups so that each group contains 6 animals. The animals were fed with normal chow and water.
Group I: Normal control
Group II: Disease control
Group III: Vildagliptin (10mg/kg)
Group IV: Rosiglitazone (10mg/kg)
Group V: Vildagliptin (10mg/kg) + Rosiglitazone (10mg/kg)
The animals were pretreated with vildagliptin and rosiglitazone (10 mg/kg oral) for 4 days prior to ethanol administration. One hour after the last drug administration, mice received ethanol (40%) for all groups except normal control. The animals were found to be sluggish with this dose of alcohol, but they were conscious and regained normal behavior within ~6 hours of alcohol feeding. This dose caused no mortality.
4.3.2. Animal Tissue and Blood Collection
After 18 hours, blood was collected by retro orbital puncture under isoflurane anesthesia. Blood was centrifuged at 6000 rpm for 10min and plasma was separated & sent for biochemical estimation. Then animals were sacrificed by CO2 asphyxiation and liver was weighed and collected. A portion of liver sample was kept in 10% formalin for histopathology study.
4.4. Model development Chronic ASH
4.4.1. Study design I
In model development Male C57BL/6 mice were divided into following four groups according to their body weight (n=10).
Group I: Received normal chow diet and water
Group II: Received 5% alcohol and normal chow diet
Group III: Received normal water and High fat
Group IV: Received 5% alcohol and High fat
In order to induce alcoholic steatosis, pure alcohol was diluted in drinking water. The concentration of alcohol was started from 1% (v/v) alcohol in the first week then gradually increased 2% in the second, 4% in the third, and 5% in the fourth and subsequent weeks for 8 weeks. During the study period alcohol and water were changed thrice a week and intake was recorded. After the completion of induction period animals were sacriï¬ced by CO2 asphyxiation; liver was excised and kept in 10% formalin for histopathological examination.
In the histopathological examination of the liver there was no significant changes observed in alcohol feeding animals (Group II) and mild to moderate grade of steatosis was observed in the high fat fed animals (Group III) , steatosis was significantly less in High fat + alcohol fed (Group IV) animals hence in further study is planned using alcohol alone.
As we observed only mild changes in the liver, in groups fed with alcohol. For that next study plan was to increase the concentration of the alcohol.
4.3.2. Study design II
Male C57BL/6 mice were divided into following four groups according to their body weight (n=10).
Group I: Received normal chow diet and water
Group II: Received 20% alcohol and normal chow diet
Group III: Received 30% alcohol and normal chow diet
Group IV: Received 40% alcohol and normal chow diet
In alcohol fed groups initial concentration of alcohol was 5% in 1st week and in 2nd week the concentration was increased to 10% then on 3rd week the concentration of alcohol was increased to 20% and by the end of that month the 40% concentration was achieved. The alcohol was changed daily in every group. By the end of 2months all the animals were sacrificed and liver and blood samples were collected. The liver was sent for histopathological examination. Reports from the histopathology showed significant induction of steatosis in 40% alcohol fed group and hence 40% alcohol was selected for further study.
4.3.3. Experimental Design
A rodent ASH model was developed that simulated several features of human alcoholic steatohepatitis. C57BL/6 male mice aged 5-6 weeks having body weight 24.29Â±.0.29g were used at the start of this study. Animals were divided into two groups. First group (n=10) was treated as a normal control fed with chow feed and second group (n=55) was treated with alcohol. The concentration of alcohol was 5% on 1st day then gradually increased to 10% from 3rd day, to 20 % from 5th day and by the end of that week 40% concentration was achieved. Daily fresh alcohol were prepared and kept. On 45th day one animal was sacrificed for histopathological examination to confirm ASH. After completion of 8 weeks, animals were grouped according to body weight. Animals fed with normal chow diet (n=10) served as Normal control Group I and animals fed with 40% alcohol were further divided into following groups, n= 8-9.
Group II Disease control
Group III Vildagliptin 10 mg/kg
Group IV Rosiglitazone 10 mg/kg
Group V Vildagliptin 10 mg/kg + Rosiglitazone 10 mg/kg
188.8.131.52. Animal Tissue and Blood Collection
On 20th day Animals were kept for overnight fasting (12 h) and on 21st day blood collected by retro orbital puncture under isoflurane anesthesia. Blood was centrifuged at 6000 rpm for 10min and plasma was separated & stored at -800c for biochemical estimation. Then animals were sacrificed by CO2 asphyxiation and liver, inguinal and epididymal fat was removed and weighed. A portion of liver sample was kept in 10% formalin for histopathology study; at the same time liver sample stored for laboratory assay at -800C.
184.108.40.206. Bio chemical assays in plasma
The collected plasma samples were analyzed for different biochemical parameters -. Glucose, ALT, AST, ALP, Bilirubin, Triglyceride and Cholesterol, Protein was measured by random access clinical chemistry analyzer Erba XL 300 using commercially available kit.
220.127.116.11. Measurement of TG content in liver tissue
0.1 M KCL (Mol wt 74.55, for 0.1M 745.5.mg in 100ml water)
Triton X- 100
Liver Tissue Homogenate Procedure
100 mg tissue was taken in 1 ml methanol and homogenized. Homogenate was centrifuged at 4000 rpm at 40 C for 5 min. Supernatant was taken out in separate 15 ml Tarson tube. To the pellet add 2ml CHCl3-Methanol mixture (2:1) and mixed by homogenizing it. It was centrifuged at 4000 rpm at 40 C for 5 min. The supernatant was mixed with the supernatant got in first centrifugation. 1 ml 0.1 M KCL added to the supernatant and mixed well by vortexing and then centrifuged 4000 rpm at 40 C for 5 min. Two layers were formed, 1 ml of organic layer (from bottom) taken out without disturbing upper layer in 1.5 ml eppendroff. Sample was evaporated in Turbovap LV evaporator. Residues were reconstituted with 400ïl of mixture of N-butyl alcohol: Triton: Methanol (3:1:1) mixed properly by vortexing. Sample was used for TG estimation, using commercial kit (ERBA diagnostics Mannheim GmbH). Values were expressed in mg/g of tissue.
Triglyceride Estimation Procedure
Aliquate 10ïl, 5ïl, 2.5ïl, 1.25ïl and 0.62ïl of standard which contain 1000, 500, 250, 125 and 62.5 mg/dl TG concentration respectively.
2 ïl of sample or water as blank is added to well.
Add 200 ïl of reagent to all wells.
Shake plate for 2min and then read the plate using spectrophotometer at 505nm.
Working reagent preparation
4 ml of Reagent 1 mixed with 1 ml of Reagent 2 (4:1)
18.104.22.168. TNF Î± PROCEDURE
Preparation of Reagents
Wash Buffer Concentrate
50 ml of wash buffer in 1500ml of distilled water (DW), (i.e:- 10 ml in 300 ml of DW-up to the mark)
Streptavidin- HRP solution
Add 30 ïl of Streptavidin- HRP concentrate in 12 ml of Streptavidin- HRP dilution buffer. (i.e:- 2.5ïl of Streptavidin- HRP concentrate in 1ml of Streptavidin- HRP dilution for each strip of well)
Prepare, not more than 15 mins prior to use
TNF Î± standard Preparation
Reconstitute the TNF Î± standard with distilled water for serum sample (2450 pg/ml)
Take two tubes add 600 ïl of standard diluent. Pipette 100ïl of reconstituted std to first tube (350 pg/ml), mix well. From this pipette out 100ïl and add to second tube (50pg/ml)
Add 50ïl of standard. diluent, standard and sample to respective well.
Add 50ïl of biotinylated Ab reagent, cover with adhesive plate,
Incubate for 2 h RT
wash with wash buffer (400 ïl) for 5 times
add 100ïl of Streptavidin- HRP conjugate, cover with adhesive plate
Incubate for 30 mins RT.
Wash with wash buffer (400 ïl) for 5 times
Add 100ïl of TMB substrate Don't cover with adhesive plate
Incubate for 30 mins
Add 100ïl of stop solution; determine the OD within 30 mins at 450 nm.
For mice serum dilution must required, 1: 4 and 1:5 dilution preferred.
DF= Ratio of final volume / aliquot volume
For ex: 1:1 means, final volume- 2, and aliquot is 1 then 2/1= 2; DF= 2
22.214.171.124. .Histopathology of Liver
The tissues are most commonly fixed for light microscopy with 10% neutral buffered formalin. The tissues were trimmed in to 2-3mm thickness after 24-48 hrs of fixation. After the tissue was trimmed it was transferred through baths of progressively more concentrated ethanol to remove the water and followed by a hydrophobic clearing agent (such as xylene) to remove the alcohol, and finally molten paraffin wax, the infiltration agent, which replaces the xylene. After the tissues were dehydrated, cleared, and infiltrated with the embedding material, they were ready for external embedding. During this process the tissue samples were placed into molds along with liquid embedding material (such as agar, gelatine or wax) which was then hardened. For light microscopy, a steel blade mounted in a microtome was used to cut 4-5 micron thick tissue sections which were mounted on a glass microscope slide. Hematoxylin and eosin (H&E) is the most commonly used light microscopical stain in histology and histopathology. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus; eosin, an acidic dye, stains the cytoplasm pink (Edna, B. P et al., 1992; Carson FL 1996).
126.96.36.199. Statistical Analysis
Data was presented as mean Â±S.E.M. Significance of difference between groups was analyzed by one way ANOVA followed by Dunnett's multiple comparison test and Bonferroni multiple comparisons. Disease control group was compared with normal control and treatment groups were compared with disease control group. p<0.05 was considered statistically significant. Graph pad prism 4 software was employed for analysis.