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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 - Basic Breeding to Harvest. protocols.io https://dx.doi.org/10.17504/protocols.io.3jngkme Copy   

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Authors: Dale Abel
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedure used by the DiaComp for inducing pressure overload hypertrophy in mice.Diabetic Complication:

Proper citation: Dale Abel 2019. Aortic Banding in Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.7rvhm66 Copy   

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Authors: Sandra Rieger
Group: Diabetic Complications Consortium
Summary: To induce hyperglycemia in larval zebrafish, utilize a genetic β-cell ablation system involving the Tg(ins:NTR-mCherry) transgenic zebrafish strain (courtesy of Dr. Michael Parsons, Johns Hopkins University). This strain expresses the bacterial enzyme Nitroreductase (NTR), which is encoded by the nfsB gene, in β-cells of the pancreas starting around 24 hours post fertilization (hpf). Because NTR is genetically fused to the red fluorescent protein mCherry, β-cells expressing NTR can be visualized with fluorescence microscopy. Upon addition of the prodrug metronidazole (MTZ) to the embryo medium, NTR converts MTZ into a toxin, which selectively ablates NTR-expressing β-cells (Curado et al., Nature Protocols 3, 948-954 (2008). This can be monitored by the disappearance of the fluorescence under a confocal microscope. Diabetic Complication:

Proper citation: Sandra Rieger 2019. Ablation of ß-cells in Tg(ins:NTR-mCherry) transgenic zebrafish using metronidazole. protocols.io https://dx.doi.org/10.17504/protocols.io.8fkhtkw 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 -- Glutathione S-Transferase. protocols.io https://dx.doi.org/10.17504/protocols.io.3sbgnan Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Plasma concentrations of thiobarbituric acid reactive substances (TBARS) are an index of lipid peroxidation and oxidative stress. The protocol describes how the DiaComp quantitates TBARS in the animal models.Diabetic Complication:

Proper citation: Eva Feldman 2019. Thiobarbituric acid reactive substances (TBARS) Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.3sngnde Copy   

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Authors: Eva Feldman, Frank Brosius
Group: Diabetic Complications Consortium
Summary: Describes the protocol used by the DiaComp to detect and quantify catalase activity in a tissue. Diabetic Complications:

Proper citation: Eva Feldman, Frank Brosius 2019. Catalase. protocols.io https://dx.doi.org/10.17504/protocols.io.3e6gjhe Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: To confirm the presence of diabetic neuropathy, nerve conduction velocity (NCV) studies are performed. The animals are anesthetized with 30/2.5 mg/kg ketamine/xylazine to prevent discomfort. Body temperature is monitered with a dermal temperature probe and maintained at 32º C with a warming lamp during NCV. Body temperature is maintained at 37ºC after NCV using a warming pad to ease animal stress from anesthetic. The nerve studies last less than 30 min per rat or mouse. The electrodes are cleaned with 70% alcohol between animals to maintain pathogen-free status.Diabetic Complication:

Proper citation: Eva Feldman 2019. Nerve Conduction Velocity Tests. protocols.io https://dx.doi.org/10.17504/protocols.io.7tghnjw 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 - Nerve Conduction Velocity. protocols.io https://dx.doi.org/10.17504/protocols.io.7rbhm2n 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 - Animal Perfusion/Tissue Fixation. protocols.io https://dx.doi.org/10.17504/protocols.io.3jmgkk6 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 - (H2) DCFDA. protocols.io https://dx.doi.org/10.17504/protocols.io.3n7gmhn 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 - Glutathione Assay (GSH). protocols.io https://dx.doi.org/10.17504/protocols.io.3qjgmun Copy   

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Authors: Timothy Kern
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedure used by the DiaComp to isolate rodent eyes to phenotype for Diabetic Retinopathy. Diabetic Complication:

Proper citation: Timothy Kern 2019. Isolation of rodent eyes for analysis of diabetic retinopathy. protocols.io https://dx.doi.org/10.17504/protocols.io.8fjhtkn Copy   

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Authors: Ian Simpson
Group: Diabetic Complications Consortium
Summary: In this series of studies we wished to determine whether Metformin was able to establish and maintain euglycemia in RCS10 mice and would reduce infarct volume following a stroke. Metformin is a widely prescribed agent to treat Type II diabetes and as illustrated in Figure 2, when Metformin (0.8-1.3 g/kg/D) is included in the water, it is able to establish and maintain euglycemia in the RCS10 mice over a 4- week period. However, as illustrated in in Figure 3, restoration of euglycemia with metformin did not improve ischemic outcome.Diabetic Complications:

Proper citation: Ian Simpson 2019. Restoration of euglycemia in the RCS10 mice with Metformin. protocols.io https://dx.doi.org/10.17504/protocols.io.8e4htgw 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 - DHE. protocols.io https://dx.doi.org/10.17504/protocols.io.3n8gmhw Copy   

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Authors: Frank Brosius
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedures for quantifying the percent area of the glomerulus that is PAS stained and is expressed as the mesangial index.Diabetic Complication:

Proper citation: Frank Brosius 2019. Mesangial Index Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.8e7hthn 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 - Cryoembedding. protocols.io https://dx.doi.org/10.17504/protocols.io.3jpgkmn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7s8hnhw 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 - Aconitase. protocols.io https://dx.doi.org/10.17504/protocols.io.3n4gmgw 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 - Glutathione Peroxidase. protocols.io https://dx.doi.org/10.17504/protocols.io.3qrgmv6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8e9hth6 Copy   

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