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Authors: Frank Brosius
Group: Diabetic Complications Consortium
Summary: This protocol describes a protocol used by some DiaComp members to detect podocyte nuclei in rodent glomeruli.Edited by: Brosius LaboratoryDiabetic Complications:

Proper citation: Frank Brosius 2019. WT-1 Staining Protocol for Podocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.8e2htge Copy   

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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: Kumagai et al., Brain Research 1996, 706:313-317

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Immuno-Electron Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.3jtgknn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.3g7gjzn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8i2huge Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7s9hnh6 Copy   

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Authors: Michael Hill
Group: Diabetic Complications Consortium
Summary: Summary:This protocol presented an induction of Type 1 Diabetes, and myocardial Infarction, performed an evaluation of in vivo residual LV function by echocardiography, and measurement of blood glucose and tissue weights of rats.Diabetic Complication:

Proper citation: Michael Hill 2019. Induction of Type 1 Diabetes, Myocardial Infarction, Echocardiography, Measurement of Blood Glucose and Tissue Weights. protocols.io https://dx.doi.org/10.17504/protocols.io.8fmhtk6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8fahtie Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: This protocol measures and quantifies the animals sensitivity to pain in the hind paw. These measurements can be used to evaluate the pathology present in the nervous system.Diabetic Complication:

Proper citation: Eva Feldman 2019. Plantar Analgesia Test for Hind Paw and Tail. protocols.io https://dx.doi.org/10.17504/protocols.io.8a3hsgn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7tfhnjn Copy   

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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:

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Transmission Electron Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.3nzgmf6 Copy   

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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:

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Dosages of Tranquilizers and Anesthetics for Mice, Rats, Hamsters/Gerbils. protocols.io https://dx.doi.org/10.17504/protocols.io.3jqgkmw Copy   

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Authors: Tim Nichols, Dave Clemmons
Group: Diabetic Complications Consortium
Summary: These assays are performed on all the pig models studied by the DiaComp to assess their serum chemistry and blood cell counts.Diabetic Complications:

Proper citation: Tim Nichols, Dave Clemmons 2019. Serum, Blood and Body Weight Measurements in Pigs. protocols.io https://dx.doi.org/10.17504/protocols.io.32agqae Copy   

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Authors: E. Dale Abel
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedure used by the DiaComp for measuring left ventricular hemodynamic parameters in intact mice.Diabetic Complication:

Proper citation: E. Dale Abel 2019. Measurement of Left Ventricular Hemodynamic Parameters in Intact Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.7tdhni6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8a2hsge Copy   

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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 https://dx.doi.org/10.17504/protocols.io.3gygjxw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8izhuf6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8eyhtfw Copy   

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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 - Lowry Protein Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.3sggnbw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.3fngjme Copy   

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Authors: Willa Hsueh, Alan Collins
Group: Diabetic Complications Consortium
Summary: This Rat / Mouse Insulin ELISA kit is used for the non radioactive quantification of insulin in mouse and rat sera. Plasma samples may also be used but application to samples of other biological fluids may need validation by the user.Diabetic Complications:

Proper citation: Willa Hsueh, Alan Collins 2019. Plasma Insulin (LINCO ELISA). protocols.io https://dx.doi.org/10.17504/protocols.io.8a4hsgw Copy   

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