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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:The use of total radical-trapping antioxidant parameter (TRAP) has recently been proposed to explore the antioxidant property of a plasma sample. This assay is a measure of oxidative stress in the animals. This protocol describes the procedure used by the DiaComp to measure TRAP.Diabetic Complications:
Proper citation: Eva Feldman 2019. TRAP Assay. protocols.io dx.doi.org/10.17504/protocols.io.3sqgndw Copy
Authors: Frank Brosius
Group: Diabetic Complications Consortium
Summary: This protocol describes a validated protocol used by some DiaComp members to determine podocyte numbers in rodent glomeruli.Edited by: Brosius LaboratoryDiabetic Complication:Reference: Sanden SK, Wiggins JE, Goyal M, Riggs LK, and Wiggins RC. Evaluation of a thick and thin section method for estimation of podocyte number, glomerular volume, and glomerular volume per podocyte in rat kidney with Wilms' tumor-1 protein used as a podocyte nuclear marker. J Am Soc Nephrol 14: 2484-2493, 2003.
Proper citation: Frank Brosius 2019. Determination of Podocyte Number and Density in Rodent Glomeruli. protocols.io dx.doi.org/10.17504/protocols.io.8eyhtfw Copy
Authors: Zhonghua Qi, Matthew D. Breyer
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedures for estimating glomerular filtration rate in conscious mice based on Fluorescein Isothiocyanate-inulin (FITC-inulin) clearance following a single bolus intravenous injection. Diabetic Complication:Reference:1. Hem, A., Smith, A.J., and Solberg, P. 1998. Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink. Lab Anim 32:364-368. 2. Sturgeon, C., Sam, A.D., 2nd, and Law, W.R. 1998. Rapid determination of glomerular filtration rate by single-bolus inulin: a comparison of estimation analyses. J Appl Physiol 84:2154-2162. 3. Qi, Z., Whitt, I., Mehta, A., Jin, J., Zhao, M., Harris, R.C., Fogo, A.B., and Breyer, M.D. 2003. Serial Determination of Glomerular Filtration Rate in Conscious Mice Using FITC-Inulin Clearance. Am J Physiol Renal Physiol.
Proper citation: Zhonghua Qi, Matthew D. Breyer 2019. Determination of Glomerular Filtration Rate in Conscious Mice using FITC-inulin. protocols.io dx.doi.org/10.17504/protocols.io.3fngjme Copy
Authors: E. Dale Abel
Group: Diabetic Complications Consortium
Summary: Summary:This protocol describes the procedure used by the DiaComp for measuring left ventricular performance in isolated retrogradely perfused mouse hearts.Diabetic Complication:
Proper citation: E. Dale Abel 2019. Measurement of Left Ventricular Performance in Langendorff Perfused Mouse Hearts. protocols.io dx.doi.org/10.17504/protocols.io.7tfhnjn Copy
Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. Diabetic Complication:
Proper citation: Eva Feldman 2019. Biochemical Measures of Neuropathy - Western Blot Stripping. protocols.io dx.doi.org/10.17504/protocols.io.3s3gngn Copy
Authors: Daisuke Katagiri, Takamune Takahashi
Group: Diabetic Complications Consortium
Summary: Summary This protocol describes a protocol to evaluate histological fibrosis in mouse kidney. Diabetic Complication:References 1. Katagiri D, Hamasaki Y, Doi K, et al. Interstitial renal fibrosis due to multiple cisplatin treatments is ameliorated by semicarbazide-sensitive amine oxidase inhibition. Kidney Int 2015; 89: 374 - 385. 2. Hara S, Umeyama K, Yokoo T, et al. Diffuse glomerular nodular lesions in diabetic pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha, an inheritant diabetic gene in humans. PLoS One 2014; 9: e92219.
Proper citation: Daisuke Katagiri, Takamune Takahashi 2019. Histological Evaluation of Renal Fibrosis in Mice. protocols.io dx.doi.org/10.17504/protocols.io.3gygjxw Copy
Authors: E. Dale Abel
Group: Diabetic Complications Consortium
Summary: Summary:This protocol describes the procedure used by the DiaComp for cardiac performance, oxygen consumption and substrate metabolic rates in isolated working mouse hearts.Diabetic Complication:References:1. Belke, D.D., Larsen, T.S., Gibbs, E.M., Severson, D.L. 2001.Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter. Am J Physiol Endocrinol Metab.280:E420-7.2. Belke, D.D., Larsen, T.S., Gibbs, E.M., Severson, D.L. 2000.Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice. Am J Physiol Endocrinol Metab.279:E1104-13.3. Belke, D.D., Larsen, T.S., Lopaschuk, G.D., Severson, D.L. 1999.Glucose and fatty acid metabolism in the isolated working mouse heart. Am J Physiol.277:R1210-7.4. Larsen, T.S., Belke, D.D., Sas, R., Giles, W.R., Severson, D.L., Lopaschuk, G.D., Tyberg, J.V. 1999.The isolated working mouse heart: methodological considerations. Pflugers Arch.437:979-85.5. Belke D.D., Betuing S, Tuttle M.J., Graveleau C, Young M.E., Pham M., Zhang D., Cooksey R.C., McClain D.A., Litwin S.E., Taegtmeyer H., Severson D., Kahn C.R., Abel E.D. 2002. Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression. J Clin Invest 109:629-639.6. Mazumder P.K., O’Neill B.T., Roberts M.W., Buchanan J., Yun U.J., Cooksey R.C., Boudina S., Abel E.D. 2004. Impaired Cardiac Efficiency and Increased Fatty Acid Oxidation in Insulin Resistant ob/ob mouse hearts. Diabetes. 53: 2366-2374.7. Suga H: Ventricular energetics. Physiol Rev 70:247-277, 1990
Proper citation: E. Dale Abel 2019. Phenotypic Characterization of the Working Heart. protocols.io dx.doi.org/10.17504/protocols.io.8a2hsge Copy
Authors: Frank Brosius
Group: Diabetic Complications Consortium
Summary: This protocol is used by DiaComp members to induce diabetes in a number of the animal models developed by the consortium. STZ is toxic to the insulinproducing beta cells of the pancreas and used to induce a diabetes similar to a type I diabetic (Reference). Some reports suggest cellular toxicity outside of the pancreas. STZ also exhibits broad spectrum antibacterial properties and alters the gut microbiota. Please ensure that appropriate controls are included in all studies and complementary models considered (e.g. the Ins2Akita mouse).Diabetic Complications:
Proper citation: Frank Brosius 2019. Low-Dose Streptozotocin Induction Protocol (mouse). protocols.io dx.doi.org/10.17504/protocols.io.8izhuf6 Copy
Authors: Firuoz Danashgeri
Group: Diabetic Complications Consortium
Summary: This protocol describes the proper procedure for harvesting bladders from rats or mice for use by the DiaComp Uropathy group.Diabetic Complication:
Proper citation: Firuoz Danashgeri 2019. Removal and Preservation of the Urinary Bladder. protocols.io dx.doi.org/10.17504/protocols.io.8fahtie Copy
Authors: Donald McClain
Group: Diabetic Complications Consortium
Summary: This is the standard protocol for measuring insulin action in terms of total body glucose disposal. It is the "gold standard" for quantifying insulin resistance/sensitivity.Diabetic Complications:
Proper citation: Donald McClain 2019. Glucose "Clamp" for measurement of whole body insulin-stimulated glucose disposal. protocols.io dx.doi.org/10.17504/protocols.io.7s9hnh6 Copy
Authors: Timothy Nichols, David Clemmons
Group: Diabetic Complications Consortium
Summary: This assay is used by the DiaComp to measure glucose tolerance and insulin sensitivity in pigs.Diabetic Complications:Reference:1. Bergman RN, Finegood DT, Ader M: Assessment of insulin sensitivity in vivo. Endocr Rev 1985, 6:45-86.
Proper citation: Timothy Nichols, David Clemmons 2019. Frequently sampled Insulin glucose tolerance test. protocols.io dx.doi.org/10.17504/protocols.io.8e8hthw Copy
Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Phenotyping of Rodents for the Presence of Diabetic Neuropathy In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.Diabetic Complication:Reference: AFIP Laboratory Methods in Histotechnology, 1994, p. 16, 53-55.
Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Hematoxylin and Eosin Staining. protocols.io dx.doi.org/10.17504/protocols.io.3jsgkne Copy
Authors: Sandra Rieger
Group: Diabetic Complications Consortium
Summary: To assess hyperglycemia in larval zebrafish, we analyzed the expression of genes implicated in glucose metabolism, such as insulin, insulin receptor, glucagon, and phosphoenolpyruvate carboxylase (pepck), using quantitative PCR (qPCR). We analyzed gene expression following treatment of Tg(ins:NTRmCherry) transgenic larvae either with 0.5 % DMSO (controls) or 10 mM metronidazole (see Protocol 2) for β-cell ablation at 3 and 8 days post fertilization (dpf). Diabetic Complication:
Proper citation: Sandra Rieger 2019. Quantitative PCR analysis to assess gene expression changes in hyperglycemic larval zebrafish. protocols.io dx.doi.org/10.17504/protocols.io.3bagiie Copy
Authors: Ian Simpson
Group: Diabetic Complications Consortium
Summary: This document describes the procedure for inducing a stroke in diabetic RCS10 mice, their non-diabetic controls and NON/ShiLtJ control mice. Our initial objective was to use those RCS10 mice that did not become diabetic as the controls for the experiment to compare the effect of diabetes on stroke outcome and that data is illustrated in Figure 2. However, with further breading the numbers of non- diabetic RCS 10 mice at twenty weeks of age were so few that it became necessary to use the NON/ShiLtJ mice which are 85% genetically identical to the RCS10 mice and stroke outcome is illustrated in Figure 3.Diabetic Complications:
Proper citation: Ian Simpson 2019. Induce Stroke via Hypoxia-Ischemia. protocols.io dx.doi.org/10.17504/protocols.io.8e5htg6 Copy
Authors: Ed Leiter
Group: Diabetic Complications Consortium
Summary: This protocol describes a number of the measurements that are made on the mouse anatomy.Edited by: DiaComp MembershipDiabetic Complications:
Proper citation: Ed Leiter 2019. Morphometry: Mouse. protocols.io dx.doi.org/10.17504/protocols.io.7rhhm36 Copy
Authors: E. Dale Abel
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedures used by the DiaComp for evaluating mitochondrial function.Diabetic Complication:References 1. Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA. Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta. 1987;892:191-6. 2. Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem. 1998;184:81-100. 3. Ouhabi R, Boue-Grabot M, Mazat JP. Mitochondrial ATP synthesis in permeabilized cells: assessment of the ATP/O values in situ. Anal Biochem. 1998;263:169-75.
Proper citation: E. Dale Abel 2019. Evaluation of Mitochondrial Function. protocols.io dx.doi.org/10.17504/protocols.io.7s8hnhw Copy
Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. Diabetic Complication:
Proper citation: Eva Feldman 2019. Biochemical Measures of Neuropathy - Glutathione Peroxidase. protocols.io dx.doi.org/10.17504/protocols.io.3qrgmv6 Copy
Authors: Donald McClain
Group: Diabetic Complications Consortium
Summary: This is the standard protocol for most routine glucose tolerance testing. It is performed on awake mice, fasted for 6 hr.Diabetic Complications:
Proper citation: Donald McClain 2019. Intraperitoneal Glucose Tolerance Testing (IPGTT). protocols.io dx.doi.org/10.17504/protocols.io.3g7gjzn Copy
Authors: Kumar Sharma
Group: Diabetic Complications Consortium, Metabolomics Protocols & Workflows
Summary: This protocol describes the procedures for collecting, preparing and performing the DiaComp Creatinine Clearance assay via high performance liquid chromatography (HPLC).Diabetic Complication:
Proper citation: Kumar Sharma 2019. Creatinine Clearance by HPLC. protocols.io dx.doi.org/10.17504/protocols.io.8e9hth6 Copy
Authors: Sandra Rieger
Group: Diabetic Complications Consortium
Summary: This protocol describes a method to mount live 8 day-old zebrafish larvae in agarose for capturing fluorescent transgene-expressing peripheral somatosensory axons with confocal imaging. We have previously described a similar method in the Journal of Visualized Experiments (1).Diabetic Complication:References: 1. G. S. O'Brien, S. Rieger, S. M. Martin, A. M. Cavanaugh, C. Portera-Cailliau, A. Sagasti, Twophoton axotomy and time-lapse confocal imaging in live zebrafish embryos. J Vis Exp, (2009). 2. S. Rieger, A. Sagasti, Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin. PLoS Biol 9, e1000621 (2011); published online EpubMay (10- PLBI-RA-9260R3 [pii] 10.1371/journal.pbio.1000621). 3. A. J. Pittman, M. Y. Law, C. B. Chien, Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points. Development 135, 2865-2871 (2008); published online EpubSep
Proper citation: Sandra Rieger 2019. Confocal imaging of live larval zebrafish for assessing peripheral neuropathy. protocols.io dx.doi.org/10.17504/protocols.io.8i2huge Copy
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