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Authors: Frank Brosius
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedures for quantifying the podocyte count and density in the glomerulus.Diabetic Complication:

Proper citation: Frank Brosius 2019. Podocyte Counting and Density Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.8a6hshe 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 - Apoptag (Peroxidase) Sections on Slides. protocols.io https://dx.doi.org/10.17504/protocols.io.3jkgkkw 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 - Hydrogen Peroxide Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.7rghm3w Copy   

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Authors: Daniel Teupser, Jan Breslow
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedures for fixing, sectioning and staining the hearts of mice for signs of atherosclerosis. The following protocol covers each aspect of the procedure. Diabetic Complication:

Proper citation: Daniel Teupser, Jan Breslow 2019. Quantification of atherosclerosis at the aortic sinus. protocols.io https://dx.doi.org/10.17504/protocols.io.3wsgpee Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Describes assay to quantitate NADH Oxidase activity from tissues. Diabetic Complications:

Proper citation: Eva Feldman 2019. NADH Oxidase Activity. protocols.io https://dx.doi.org/10.17504/protocols.io.3jcgkiw Copy   

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Authors: Firouz Daneshgari
Group: Diabetic Complications Consortium
Summary: Cystometry is a test of bladder function in which pressure and volume of fluid in the bladder is measured during filling, storage, and voiding. A cystometry study is performed to diagnose problems with urination, including incontinence and urinary retention.This test should be done on the mice two days after SPT Implantation.Diabetic Complication:Reference 1. Cannon TW, Damaser MS: Effects of anesthesia on cystometry and leak point pressure of the female rat. Life Sci 69:1193-1202, 2001

Proper citation: Firouz Daneshgari 2019. Conscious Cystometry Bladder Function Testing. protocols.io https://dx.doi.org/10.17504/protocols.io.3fegjje 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.This protocol was responsible by Dr. T. L. WoodDiabetic Complication:

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - In Situ Hybridization. protocols.io https://dx.doi.org/10.17504/protocols.io.3m6gk9e 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 - Streptozotocin Treatment For Rats. protocols.io https://dx.doi.org/10.17504/protocols.io.3njgmcn 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 transthoracic echocardiography in awake mice. Diabetic Complication:

Proper citation: E. Dale Abel 2019. Echocardiography: Mouse. protocols.io https://dx.doi.org/10.17504/protocols.io.7s7hnhn Copy   

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Authors: Kathi Burke (Frank Brosius lab), Peter Reifsnyder (Ed Leiter Lab)
Group: Diabetic Complications Consortium
Summary: This assay is designed for companion use with Exocell’s immunospecific ELISAs for urine albumin excretion, allowing expression of results as µg albumin per mg creatinine in the urine. Adaptation of creatinine measurement to microtiter wells enables convenient, simultaneous determination of albumin and creatinine concentrations in the same specimen, using analogous microtiter plate formats. Normalization of albumin excretion in relation to creatinine facilitates classification according to defined ranges of microalbuminuria, the established marker of the early diabetic renal dysfunction. The urine albumin-to-creatinine ration in a random urine specimen is an accepted alternative to cumbersome 24 urine collections in the detection and monitoring of microalbuminuria.The procedure is an adaptation of the alkaline picrate method and entails determination of the differential absorbance in a sample before and after the additions of acid to correct for color generation due to interfering substances.Diabetic Complication:

Proper citation: Kathi Burke (Frank Brosius lab), Peter Reifsnyder (Ed Leiter Lab) 2019. Creatinine Companion Protocol Assay (Exocell). protocols.io https://dx.doi.org/10.17504/protocols.io.3fmgjk6 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 - Intra-Epidermal Fiber Density Determination of Rodent Foot Pad Biopsies. protocols.io https://dx.doi.org/10.17504/protocols.io.7rehm3e Copy   

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Authors: Sandra Rieger
Group: Diabetic Complications Consortium
Summary: Zebrafish embryos develop in their eggshell (the chorion) until hatching (~48-72 hours post fertilization). Because the chorion is impermeable to water, it needs to be removed prior to metronidazole treatment and β-cell ablation in Tg(ins:NTR-mCherry) transgenic zebrafish embryos. This can be achieved either with forceps or by enzymatic removal using Pronase. The earliest time of removal should be after completion of gastrulation to avoid bursting of the yolk and potential infections.Diabetic Complication:

Proper citation: Sandra Rieger 2019. Dechorionation of zebrafish embryos with Pronase for metronidazole-mediated ß-cell ablation. protocols.io https://dx.doi.org/10.17504/protocols.io.8exhtfn Copy   

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Authors: Daniel Teupser, Jan Breslow
Group: Diabetic Complications Consortium
Summary: This protocol is used to isolate the various lipid fractions from blood plasma using ultracentrifugation. The actual measured concentrations are performed separately once the isolations are complete.NOTE: This protocol is not applicable for ApoE knockout mice.Diabetic Complications:

Proper citation: Daniel Teupser, Jan Breslow 2019. Ultracentrifugal separation of VLDL, LDL and HDL. protocols.io https://dx.doi.org/10.17504/protocols.io.33bgqin Copy   

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Authors: Daniel Teupser, Jan Breslow
Group: Diabetic Complications Consortium
Summary: This protocol is used to isolate the various lipid fractions from blood plasma using ultracentrifugation. The actual measured concentrations are performed separately once the isolations are complete.NOTE: This protocol IS applicable for ApoE knockout mice.Diabetic Complication:

Proper citation: Daniel Teupser, Jan Breslow 2019. Ultracentrifugal separation of HDL alone and calculation of non-HDL. protocols.io https://dx.doi.org/10.17504/protocols.io.32mgqc6 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:Yarborough et al, Cancer Res. 56:683-688, 1996

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Immunofluorescence Method for 8DG Localizaiton. protocols.io https://dx.doi.org/10.17504/protocols.io.3jwgkpe Copy   

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Authors: Ed Leiter
Group: Diabetic Complications Consortium
Summary: Used to calculate the insulin content in the pancreas.Diabetic Complications:

Proper citation: Ed Leiter 2019. Pancreatic Insulin Content by Acid-Ethanol Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.3jjgkkn 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 - Genotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.3qdgms6 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 the extent of atherosclerotic lesions in diabetic cardiovascular disease models. Each measurement/assay is listed below with the details of how the data is collected. Details of the reagents and materials are in the assay.Diabetic Complication:

Proper citation: Tim Nichols, Dave Clemmons 2019. Atherosclerotic Morphometry in Pigs. protocols.io https://dx.doi.org/10.17504/protocols.io.7s6hnhe Copy   

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Authors: Ian Simpson
Group: Diabetic Complications Consortium
Summary: ♦ NONcNZO10/LtJ (NON/NZ) mice are the mice used for a Type 2 diabetes model. The mice are given 5K20 diet at weaning and the mice are usually exhibiting hyperglycemia by 10-11 weeks of age (see Figure 1) 90- 95 % of the mice will become diabetic at 20 weeks. ♦ NON/ShiLtJ (NON/Shi) mice are the mice used for a control to the diabetic mice. The mice are given 5K20 diet at weaning and should not become diabetic. But 5-10% of the mice will become diabetic at 20 weeks. ♦ Breeding, housing, and experimentation are performed in a pathogen-free environmentDiabetic Complications:

Proper citation: Ian Simpson 2019. Breeding NONcNZO10/LtJ (NON/NZ) and NON/ShiLtJ (NON/Shi) mice. protocols.io https://dx.doi.org/10.17504/protocols.io.556g89e Copy   

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Authors: Eva Feldman, Omorodola Abatan
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, Omorodola Abatan 2019. Neuropathy Phentoyping Protocols - Insulin Implantation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7rdhm26 Copy   

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