Streptozotocin (STZ) Induced Diabetes Experiment

Streptozotocin (STZ) Induced Diabetes ExperimentMATERIALS AND METHODS3. MATERIALS AND METHODS3.1 Animal survival and C arSTZ induced diabetes atomic number 18 commonly coiffeed in Male Wistar Kyoto rats. In these models, male Wistar rats at 8 calendar weeks of age (200300 g) are selected from animal supply facility of SGPGIMS Lucknow, and house in a 12-h light, 12-h non-white cycle environment, temperature 25C, 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 keister (Preparation and proof of animal model of DN) 50 mg/kg STZ induced Diabetic blackleg (n=3) Normal Rat (n=3)After 8th week rat are sacrificed for validation of Diabetic nephropathy3.3 Induction and check of Diabetes by StreptozotocinThere is a lack of appropriate animal model that could spontaneously develop DN. It has been a signifi sightt limitation to find aside specific factors that is underlying this disease and also the development of refreshing 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 employ as model for performing STZ-induced diabetic nephropathy. Here, in these models, 8 weeks old male rats (200-250gm) are avid or unbroken 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 intraperitone bothy 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 summation to the STZ dosage call for for inducing diabetes via an intraperitoneal route is relatively higher as compared to other route. After loose STZ, the rats should be given ample amount of drinking water with su crose for 48 hrs (15g/L), to avoid primeval 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 inception 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 later(prenominal) 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 enviable 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 a spread oute mild nephrotoxic effects of STZ (Kraynak AR et. al. 1995)This do drugs i.e., STZ has been shown to induce a diabetic state in 72 hrs (3 days) as docume nted by examining lowlife blood ingests using a Glucometer (Optimum Exceed) Diabetic animals and non-diabetic control group were kept in metabolic cages apiece and crystallisely and under feeding and metabolism control. Glucose in the blood of diabetic rats exceeded that of the non-diabetic control ones. Food inhalation was measured in terms of (gm), water consumption was measured in terms of (ml) and urine heap was measured in terms of (ml) on a daily basis and glucose in blood blood serum were also measured, so that chemical diabetes was verified in rats injected with Streptozotocin.3.4 Estimation of CreatinineModified Jaffes method was employ for excuseimetric estimation of creatinine concentration in urine samplings. dominionCreatinine + picric irate 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. r uleDraw the Blood from a vein and whence transferred into the vial.Centrifuge the blood for 10 minutes and blood serum is obtained.Separate out the serum in different eppendrofs.The concentration of creatinine is metrical in the serum sample using the Jaffes method, as followsIn a clean dry test tube augment 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 separately tube.Left to stand for 15 minutes.The absorbance is measured at max 500 nm.If a standard creatinine solvent (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 equationCtest = Cstd x 3.5 Estimation of Urinary AlbuminEnzyme linked immunosorbent analyse (enzyme-linked-immunosorbent serologic essay) for let onion of rat albumin in serum, plasma or urine. Other biologic fluids that contain Rat Albumin, such as faeces or saliva, may be suitable samples.3 .5.1 PrincipleThe antigen present in urine sample are allowed to stick to a poly vinyl and then collection 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 early antigen in the plate. To this added second antibody, an enzyme is also tagged is that, when a suitable substrate is added, the enzyme fight downs with it to produce a tinct. 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 required96-well plateELISA Coating loverELISA purify SolutionELISA steming BufferSample/Conjugate Diluent (ELISA Blocking Buffer + Tween 20)10% Tween 20Enzyme Substrate, TMBELISA Stop SolutionAdditional Materials RequiredUltrapure waterPrecision pipettors, with usable shaping tipsPolypropylene, polyethylene or glass tubes to prepare standard and samplesContainers to pre pare buffersAn aspiration device or an automated 96-well plate washerDisposable reagent reservoirsA standard microtiter plate reader for measuring absorbance at 450 nm3.5.3 ProcedureAdded 100 l of thin coating antibody to each well. Samples were run in duplicate.Incubated at room temperature (20-25 C) for 1 hour. wash offed plate FIVE times.Added 200 l of Blocking Solution to each well.Incubated at room temperature for 30 minutes.Washed plate FIVE times.Added 100 l of standard or sample to well.Incubated at room temperature for 1 hour.Washed plate FIVE times.Added 100 l of diluted HRP detection antibody to each well.Incubated at room temperature for 1 hour.Washed plate FIVE times.Added 100 l 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 l of Stop Solution to each well.Absorbance was measured on a plate reader at 450 nm.3.5.4 PrecautionsStore all reagents at 2-8C. Do not choke up reagents .All reagents must be at room temperature (20-25 C) before use.Vigorous plate washing is essential. subroutine new disposable pipette tips for each transfer to avoid wipe-contamination.Minimize lag time amongst wash steps to ensure the plate does not becomeCompletely dry during the assay.Avoid microbic contamination of reagents and equipment. Automated plateWashers can easily become contaminated thereby causing assay variability.Take care not to contaminate the TMB Solution. Do not expose TMBSubstrate etymon to glass, foils, or metal. If the outcome is blue before use, do not use it3.6 Oral Glucose Tolerance TestRats are fasted over iniquity (12-16 hours) before the test, sedated rats, will be given 50 % dextrose (3ml/kg body weight) intraperitoneally. unit of measurement blood will be collected from the merchantman 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 g lucose stripsDextroseGauge needlesTimer and PenTable of mice for demonstrate keeping of glucose values 3.6.2 Preparations before the testWeigh 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 dash off 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 closing 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 determin ation 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 milk the tail by holding the tail in the midst of 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 Injury3.7.1 Tissue breeding for histologyAfter 8 weeks the rats were weighed, sacrificed out in accordance to the Institutional animal ethics committee by using suitable anesthetic(a) 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 foils of kidneyKidneys were hold in Para- formaldehyde is taken out in a glass slab for sectioning. A two cross section of upp er half of kidney was done with sterile blade for paraffin embedding.The whole process for preparation of paraffin blocks took two days.Day firstThe Formalin fixed kidneys were picked up and place in plastic cassettes was sequentially dipped in inebriant for dehydration. The schedule is as followed50% alcoholic beverage1.5hrs70%Alcohol1 hrs.80% Alcohol1 hrs.90% Alcohol1hrs100% Alcohol 1hrs ( doubly)The cassette containing the tissue was left-hand(a) overnight in 100% absolute alcohol.Day SecondOn 2nd day we perform the following treatment to tissue containing cassetteCassette was haved from 100% alcohol and dipped for CHCl3 treatments anesthetize (A) 1.5 hours.Chloroform (B) 1.5 hours.Chloroform (C) 1.5 hours.The cassette was then kept in melted paraffin wax (at 58C 65C in water bath) following two changes of paraffin wax for proper blocking.The steel chocks are kept at the 65C 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 sections were cut.3.8 Periodic Schiffs red- importunate (PAS) detection3.8.1 PrinciplePAS (Periodic vitriolic Schiff) is a method of staining used for the detection of polysaccharides i.e., glycogen and mucosubstances that is glycoproteins and glycolipids. PAS stain is a histochemical action. 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 Schiffs reagent is a very specific agent that only reacts with the carboxylic group compounds.Material required Glass soarings and give chase slipsPoly Lysine (Sigm a Aldrich, USA)XyleneGraduated Alcohol (30%, 50%, 70%, 90% and 100%)Periodic acidSchiff ReagentHaematoxylinAcid alcohol (1% HCl in 70% alcohol)DPX mountant3.8.3 Protocol for PAS StainingKeep the Poly-Lysine coated slides on hot plate for 15-20 min.Dip in warm Xylene for 10 min twice.Pass the slide through class-conscious alcohol100% alcohol- (10 minutes) twice90% 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 data track tap water for 5 min.Counter stain in haematoxylinRinse 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.Massons Trichrome Staining3.9.1 PrincipleMT Staining m ethod is used to demonstrate the increase of collagen in diseases. This method of staining uses three dyes of contrasting act upon for the selective staining of basic tissue components i.e., muscle, collagen fibers, fibrin, and erythrocytes. The general phenomenon of trichome staining is that menialest 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 ruby-red will binds with the acid-loving 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 phenylamine 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 requiredBouins solutionXyleneGraduated Alcohol (30%, 50%, 70%, 90% and 100%)Weigerts iron hematoxylin solutionBiebrich scarlet acid fusinphosphomolybedic-phosphotungustic acid solutionAniline blue solution 3.9.3 Protocol for MT StainingKeep 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 alcohol100% alcohol- (10 minutes) twice90% 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 Bouins Solution for 30 minutes to 1 hr. at 56C to improve the staining prime(a) although this step is not necessary.Rinse in racetrack tap water for 5-10 minutes to remove the icteric color.Stain in Weigerts iron hematoxylin working solution for 10 minutes.Rinse in running warm tap water for 10 minutes.Wash in D/WStain with Biebrich Scarlet Acid Fusin for 5 minutes.Differentiate in phosphophospho molybedic-phosphotungustic acid solution for 10-15 minutes.Transfer the section directly (without rinse) to phenylamine 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 nifty slides in xyleneMount with DPX mountant.NoteNuclei-Black, Muscle Fibre-Red, Collagen-Blue, Cytoplasm-RedModified Immunohistochemistry3.10.1 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 i ncluding 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 requiredPoly-Lysine coated slide.Xylene1X- PBS buffer.(Sigma Aldrich Inc., USA)Graduated Alcohol (30%, 50%, 70%, 90% and 100%)DPX mountant for microscopyAcid alcohol (1% HCl in 70% alcohol)citrate bufferSodium BorohydrateHydrogen bleachBlocking solutionPrimary antibodySecondary antibodyStreptovidin HRP3.10.3 Protocol for Modified ImmunohistochemistryCut the section of 3 to 5 m.Warm the slide on hot plate (55c) 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. vaporization using 100% Alcohol twice for 10 minutes each and 90, 80, 40, 50 and 30 part for 5 each in coupling jar.Wash thri ce in TBS or PBS for 5 minutes each.Antigen convalescence -10 minutes for 98c 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 retrievalCool 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 solution0.3% Triton X and 5% sheep serum in 1xPBS or 1xTBSFor 2ml (6l Triton, 100 l sheep serum, 19 of 94 l (1xPBS or 1xTBS)) 14. Blocking for 30minutes at 37c 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 4c(Note 1200 dilution for fibronectin 1500 dilution of Collagen IV).Wash the slides thrice in 1xTBS or 1xPBSSecondary antibody added on sl ide and keep it for 30 to 1hour. But parafilm on it. (Note 1200 dilution)Wash the slide thrice in 1xTBSor 1XPBS for 5 minutes.Streptovidin HRP (1200) in 1xTBS or 1xPBS for 20 minutes at populate temperature.Three wash in 1xTBS 5 minutes each.DAB (Till color in Brown) (15 minutes)23.1xPBS (5minutes) wash24. Counterstain for 30 seconds with Hematoxylene (Directly put on slide)25. 1xTBS or PBS immediate wash26. Dehydrate and dry and mount