Streptozotocin (STZ) Induced Diabetes Experiment

Streptozotocin (STZ) Induced Diabetes Experiment MATERIALS AND METHODS 3. MATERIALS AND METHODS 3.1 Animal Selection and Care STZ induced diabetes are commonly performed in Male Wistar Kyoto rats. In these models, male Wistar rats at 8 weeks of age (200–300 g) are selected from animal supply facility of SGPGIMS Lucknow, and house in a 12-h light, 12-h dark cycle environment, temperature 25 °C, with standard rat diet and water in metabolic cages for one week prior to STZ treatments. The study had clearance from Institute animal ethics committee. 3.2 Study Design Wistar Rat (Preparation and Validation of animal model of DN) 50 mg/kg STZ induced Diabetic Rat (n=3) Normal Rat (n=3) After 8th week rat are sacrificed for validation of Diabetic nephropathy 3.3 Induction and Validation of Diabetes by Streptozotocin There is a lack of appropriate animal model that could spontaneously develop DN. It has been a significant limitation to find out specific factors that is underlying this disease and also the development of new therapeutic methods or strategies to prevent progressive renal disease in diabetes (Morcelo A. Nobrega et. al. 2004). Most Sprague-Dawley (SD), Wistar-Kyoto (WKY) rats are used as model for performing STZ-induced diabetic nephropathy. Here, in these models, 8 weeks old male rats (200-250gm) are starved or kept on fasting for 18hrs and after that STZ was injected intraperitoneal (WKY-50mg/kg, SD-55mg/kg and SHR-45mg/kg) with sodium citrate buffer (1ml/kg) (Ma G et. al. 2004 and Cooper ME et. al. 1988). STZ given here intraperitoneally to the rats, however, it is a very less common procedure as intravenous injections are comparatively easy to perform in rats and give more reliable results. In addition to the STZ dosage required for inducing diabetes via an intraperitoneal route is relatively higher as compared to other route. After giving STZ, the rats should be given ample amount of drinking water with sucrose for 48 hrs (15g/L), to avoid early mortality of rats as stored insulin is released from damaged pancreatic Islets of Langerhans. After completion of 1 week of STZ induction, the rats must be assessed for hyperglycaemia and also those with fasting blood glucose of over 280 mg/dl (15 mmol/L), which is usually around 90%, and it should be included in the studies of diabetic nephropathy. In order to prevent subsequent development of ketone uria, subsequent injections of long-acting insulin (approx. 2-4 U/rat) should be given daily to maintain the blood glucose levels in a desirable range(300-600 mg/dl, 16-33 mmol/l) (Davis BJ et. al. 2003). Studies exploring the effects of treatment on the development of DN should not be underway until at least 3 weeks after STZ when the kidneys have improved from the acute mild nephrotoxic effects of STZ (Kraynak AR et . al. 1995) This drug i.e., STZ has been shown to induce a diabetic state in 72 hrs (3 days) as documented by examining tail blood samples using a Glucometer (Optimum Exceed) Diabetic animals and non-diabetic control group were kept in metabolic cages individually and separately and under feeding and metabolism control. Glucose in the blood of diabetic rats exceeded that of the non-diabetic control ones. Food consumption was measured in terms of (gm), water consumption was measured in terms of (ml) and urine volume was measured in terms of (ml) on a daily basis and glucose in blood serum were also measured, so that chemical diabetes was verified in rats injected with Streptozotocin. 3.4 Estimation of Creatinine Modified Jaffe’s method was used for colorimetric estimation of creatinine concentration in urine samples. Principle: Creatinine + picric acid Creatinine picrate (Yellow) (Orange) The resultant orange color is measured colorimetrically. However, the intensity of the resultant orange color is directly proportional to the concentration of creatinine in the sample. Method: Draw the Blood from a vein and then transferred into the vial. Centrifuge the blood for 10 minutes and Serum is obtained. Separate out the serum in different eppendrofs. The concentration of creatinine is calculated in the serum sample using the Jaffe’s method, as follows: In a clean dry test tube add 0.5 ml distilled water (blank) or serum (test), add 0.5 ml NaOH and then 0.5 ml picric acid. Mix all the contents of each tube. Left to stand for 15 minutes. The absorbance is measured at ÃŽ »max 500 nm. If a standard creatinine solution (0.55 mg/dl) has an absorbance value of 0.30, then the concentration of creatinine in the provided serum sample is calculated by using the following equation: Ctest = Cstd x 3.5 Estimation of Urinary Albumin Enzyme linked immunosorbent assay (ELISA) for detection of rat albumin in serum, plasma or urine. Other biological fluids that contain Rat Albumin, such as faeces or saliva, may be suitable samples. 3.5.1 Principle: The antigen present in urine sample are allowed to stick to a poly vinyl and then plate is washed to separate antigen and antibodies from remaining sample components. To this plate a corresponding second antibody is added to get fixed to the already adhered first antigen in the plate. To this added second antibody, an enzyme is also tagged is that, when a suitable substrate is added, the enzyme reacts with it to produce a colour. This colour produced is measurable as a function or quantity of antigen present in the urine sample and there by identified. 3.5.2 Chemical and material required: 96-well plate ELISA Coating Buffer ELISA Wash Solution ELISA Blocking Buffer Sample/Conjugate Diluent (ELISA Blocking Buffer + Tween 20) 10% Tween 20 Enzyme Substrate, TMB ELISA Stop Solution Additional Materials Required: Ultrapure water Precision pipettors, with disposable plastic tips Polypropylene, polyethylene or glass tubes to prepare standard and samples Containers to prepare buffers An aspiration device or an automated 96-well plate washer Disposable reagent reservoirs A standard microtiter plate reader for measuring absorbance at 450 nm 3.5.3 Procedure: Added 100  µl of diluted coating antibody to each well. Samples were run in duplicate. Incubated at room temperature (20-25 C) for 1 hour. Washed plate FIVE times. Added 200  µl of Blocking Solution to each well. Incubated at room temperature for 30 minutes. Washed plate FIVE times. Added 100  µl of standard or sample to well. Incubated at room temperature for 1 hour. Washed plate FIVE times. Added 100  µl of diluted HRP detection antibody to each well. Incubated at room temperature for 1 hour. Washed plate FIVE times. Added 100  µl of TMB Substrate Solution to each well. Developed the plate in the dark at room temperature for 15 minutes. Reaction was stopped by adding 100  µl of Stop Solution to each well. Absorbance was measured on a plate reader at 450 nm. 3.5.4 Precautions: Store all reagents at 2-8 ºC. Do not freeze reagents. All reagents must be at room temperature (20-25  ºC) before use. Vigorous plate washing is essential. Use new disposable pipette tips for each transfer to avoid cross-contamination. Minimize lag time between wash steps to ensure the plate does not become Completely dry during the assay. Avoid microbial contamination of reagents and equipment. Automated plate Washers can easily become contaminated thereby causing assay variability. Take care not to contaminate the TMB Solution. Do not expose TMB Substrate solution to glass, foils, or metal. If the solution is blue before use, do not use it 3.6 Oral Glucose Tolerance Test Rats are fasted overnight (12-16 hours) before the test, sedated rats, will be given 50 % dextrose (3ml/kg body weight) intraperitoneally. Whole blood will be collected from the tail vein at 0, 15, 30, 60, 120, and 180 min after the administered of glucose for the measurement of glucose with a glucometer. 3.6.1 Material Required: Glucometer and glucose strips Dextrose Gauge needles Timer and Pen Table of mice for record keeping of glucose values 3.6.2 Preparations before the test: Weigh the animals before the test. The animals are fasted overnight (approximately 16 hours: 5 pm to 9 am). Place each of the animals in a separate fresh cage with no food, but make sure they have water bottles. Make sure that there is no activity in the animal room before and while you are performing the test. Prepare the glucose solution the night before the test. 3.6.3 Procedure: After the overnight fast, blood glucose was determined (time 0) in a drop of blood as following: Rats were placed on top of the cage (let it hold onto the grid). The tail tip was pricked with a needle, wiped off the tail tip with gauze, and the next drop of blood was used for the determination of glucose with a glucometer. Glucose solution was given orally. Blood glucose was determined at 10, 20, 30, 60, 90 and 120 min after the administration of glucose. For the determination of glucose at each of these time points collected a drop of blood as following: wiped the cut end of the tail to break any blood clot that had formed and collected the next drop of blood. If the blood does not appear spontaneously â€Å"milk† the tail by holding the tail between your thumb and index finger and move along the tail from the base of the tail to the tip of the tail while applying gentle pressure. 3.7 Assessment of Renal Histopathological Injury 3.7.1 Tissue preparation for histology: After 8 weeks the rats were weighed, sacrificed out in accordance to the Institutional animal ethics committee by using suitable anaesthetic agent (Ketamine) and their kidneys were taken out. Left kidneys were perfusion fixed for Histopathological and IHC studies and right kidneys were freezed immediately for western blotting and RT-PCR. Preparation of paraffin blocks of kidney: Kidneys were preserved in Para- formaldehyde is taken out in a glass slab for sectioning. A two cross section of upper half of kidney was done with sterile blade for paraffin embedding. The whole process for preparation of paraffin blocks took two days. Day first: The Formalin fixed kidneys were picked up and place in plastic cassettes was sequentially dipped in alcohol for dehydration. The schedule is as followed: 50% Alcohol →1.5hrs 70%Alcohol →1 hrs. 80% Alcohol →1 hrs. 90% Alcohol →1hrs 100% Alcohol →1hrs (twice) The cassette containing the tissue was left overnight in 100% absolute alcohol. Day Second: On 2nd day we perform the following treatment to tissue containing cassette Cassette was removed from 100% alcohol and dipped for CHCl3 treatments Chloroform (A) 1.5 hours. Chloroform (B) 1.5 hours. Chloroform (C) 1.5 hours. The cassette was then kept in melted paraffin wax (at 58 °C 65 °C in water bath) following two changes of paraffin wax for proper blocking. The steel chocks are kept at the 65 °C electronic heater and the paraffin treated kidney in plastic cassettes are opened and place in pre-heated steel chocks together with melted paraffin wax and closed with cassette. Block was kept at room temperature to solidify the melted wax. The paraffin block containing tissue was sectioned with microtome. The block was fit properly in the Microtome machine 5 µ sections were cut. 3.8 Periodic Schiff’s Acid (PAS) Staining 3.8.1 Principle: PAS (Periodic Acid Schiff) is a method of staining used for the detection of polysaccharides i.e., glycogen and mucosubstances that is glycoprotein’s and glycolipids. PAS stain is a histochemical reaction. In the reaction, periodic acid oxidises vicinal diols in these sugar. It oxidized the aldehyde formed by carbon-carbon bonding that react with fuchsin-sulphurous acid and forms the magenta colour. This periodic acid exposes the glycogen to give a colouring product. The Schiff’s reagent is a very specific agent that only reacts with the carboxylic group compounds. Material required: Glass slides and cover slips Poly Lysine (Sigma Aldrich, USA) Xylene Graduated Alcohol (30%, 50%, 70%, 90% and 100%) Periodic acid Schiff Reagent Haematoxylin Acid alcohol (1% HCl in 70% alcohol) DPX mountant 3.8.3 Protocol for PAS Staining: Keep the Poly-Lysine coated slides on hot plate for 15-20 min. Dip in warm Xylene for 10 min twice. Pass the slide through graded alcohol 100% alcohol- (10 minutes) twice 90% alcohol (5 minutes) 70% alcohol (5 minutes) 50% alcohol (5 minutes) 30% alcohol (5 minutes) Dip in distilled water for 5 min. Place the slide containing section into 0.5 % Periodic acid for 5 minutes. Rinse with distilled water. Schiff Reagent was added for 10 min until deep magenta colour appear. Wash in running tap water for 5 min. Counter stain in haematoxylin Rinse with distilled water. If high stain come, then wash with acid alcohol (1% HCl+70% alcohol) Wash the slide in tap water. Dehydrate in alcohol, and dry the slides. After drying mount the slides by DPX mountant. Masson’s Trichrome Staining 3.9.1 Principle: MT Staining method is used to demonstrate the increase of collagen in diseases. This method of staining uses three dyes of contrasting colours for the selective staining of basic tissue components i.e., muscle, collagen fibers, fibrin, and erythrocytes. The general phenomenon of trichome staining is that smallest dye molecule colours or stains the less porous tissues. However, the penetration of dye of larger molecular size is depends on the expense of small molecules. Some others suggests that the acid dye firstly stained the tissue then the Biebrich Scarlet will binds with the acidophilic components of the tissue, after which when treated with the phosphor acids, the components that are less permeable will retain the red colour, because of the collagen this red colour is pulled out and at the same point of time causing a link with the collagen to bind with the aniline blue. . At the outset it must be made clear that the methods control how ionised acid dyes react with ionised basic tissues. 3.9.2 Material required: Bouin’s solution Xylene Graduated Alcohol (30%, 50%, 70%, 90% and 100%) Weigert’s iron hematoxylin solution Biebrich scarlet acid fusin phosphomolybedic-phosphotungustic acid solution Aniline blue solution 3.9.3 Protocol for MT Staining: Keep the Poly-Lysine coated slides on hot plate for 15-20 min. Dip in warm Xylene for 10 min twice. Pass the slide through graded alcohol 100% alcohol- (10 minutes) twice 90% alcohol (5 minutes) 70% alcohol (5 minutes) 50% alcohol (5 minutes) 30% alcohol (5 minutes) Dip in distilled water for 5 min. For formalin fixed tissue, re-fix in Bouin’s Solution for 30 minutes to 1 hr. at 56 ºC to improve the staining quality although this step is not necessary. Rinse in running tap water for 5-10 minutes to remove the yellow color. Stain in Weigert’s iron hematoxylin working solution for 10 minutes. Rinse in running warm tap water for 10 minutes. Wash in D/W Stain with Biebrich Scarlet Acid Fusin for 5 minutes. Differentiate in phosphophosphomolybedic-phosphotungustic acid solution for 10-15 minutes. Transfer the section directly (without rinse) to aniline blue solution and stain for 5-10 minutes. Rinse directly in D/W and differentiate in 1% Acetic acid solution for 2 minutes. Rinse slides, dehydrate through Alcohol Clean slides in xylene Mount with DPX mountant. NoteNuclei-Black, Muscle Fibre-Red, Collagen-Blue, Cytoplasm-Red Modified Immunohistochemistry 3.10.1 Principle: Immunohistochemistry (IHC) is the process whereby antibodies are used to detect proteins (antigens) in cells within a tissue section (for instance liver, pancreas or the heart). Immunohistochemistry exploits the principle that in biological tissues antibody binds to the specific antigens. This tool is used to localize specific antigens in tissue sections with labelled antibodies based on antigen-antibody interactions. This antigen-antibody interaction can be visualized in number of ways i.e., the immune reactive products can be visualized by a marker including fluorescent dyes, enzymes in general; radioactive elements or colloidal gold. This IHC technique is widely used by the researcher in basic research for understanding the differentially expressed proteins and for the localization and distribution of biomarkers in different- different parts of biological tissues. 3.10.2 Material required: Poly-Lysine coated slide. Xylene 1X- PBS buffer.(Sigma Aldrich Inc., USA) Graduated Alcohol (30%, 50%, 70%, 90% and 100%) DPX mountant for microscopy Acid alcohol (1% HCl in 70% alcohol) citrate buffer Sodium Borohydrate Hydrogen Peroxide Blocking solution Primary antibody Secondary antibody Streptovidin HRP 3.10.3 Protocol for Modified Immunohistochemistry: Cut the section of 3 to 5  µm. Warm the slide on hot plate (55 °c) for 30’. Dip the slide in warm Xylene twice for 10 minutes. Wash the slides three times in TBS or PBS for 5 minutes each. Dehydration using 100% Alcohol twice for 10 minutes each and 90, 80, 40, 50 and 30 percent for 5’ each in coupling jar. Wash thrice in TBS or PBS for 5 minutes each. Antigen retrieval -10 minutes for 98 °c in citrate buffer pH-6 (Note: 0.294 gm in 100 ml MQ with pH=6, 1 10mm citrate buffer) Here we are using pressure cooker for Antigen retrieval Cool the citrate Buffer slides. Give the Sodium Borohydrate treatment in coupling jar (To remove Background). (Dark Condition) (Note:- 1% NaBH4) Wash slides thrice in TBS or PBS for 5 minute each. Hydrogen Peroxide Blocking (3% H2O2 in Methanol or water –Dip the slides in it and keep it for 30’). Wash thrice in TBS or PBS for 5 minutes each. Blocking solution[0.3% Triton X and 5% sheep serum in 1xPBS or 1xTBS] For 2ml (6 µl Triton, 100  µl sheep serum, 19 of 94  µl (1xPBS or 1xTBS)) 14. Blocking for 30minutes at 37 °c in lab (some time 1hour also) Wash the slides thrice in TBS or PBS for 5’ each. Primary antibody in TBS or PBS solution, left overnight at 4 °c (Note: 1:200 dilution for fibronectin 1:500 dilution of Collagen IV). Wash the slides thrice in 1xTBS or 1xPBS Secondary antibody added on slide and keep it for 30’ to 1hour. But parafilm on it. (Note: 1:200 dilution) Wash the slide thrice in 1xTBSor 1XPBS for 5 minutes. Streptovidin HRP (1:200) in 1xTBS or 1xPBS for 20 minutes at Room temperature. Three wash in 1xTBS 5 minutes each. DAB (Till color in Brown) (15 minutes) 23.1xPBS (5minutes) wash 24. Counterstain for 30 seconds with Hematoxylene (Directly put on slide) 25. 1xTBS or PBS immediate wash 26. Dehydrate and dry and mount