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Authors: Kathi Burke, Peter Reifsnyder
Group: Diabetic Complications Consortium
Summary: Albuwell M is an indirect competitive ELISA designed to monitor kidney function in the mouse by measurement of urinary albumin. To complete the assay, sample and rabbit anti-murine albumin antibody are added to albumin coated wells. The antibody interacts and binds with the albumin immobilized to the stationary phase or with albumin in the fluid phase, hence the notion of competitive binding. A subsequent reaction with anti-rabbit -HRP conjugate labels the probe with enzyme. After washing, only the antibody-conjugate bound to the stationary phase remains in the well, and this is detected using a chromogenic reaction. Color intensity is inversely proportional to the logarithm of albumin in the fluid phase. The assay may be completed in less than 2.5 hoursDiabetic Complication:

Proper citation: Kathi Burke, Peter Reifsnyder 2019. Protocol for Albuwell M kit: Murine Microalbuminuria ELISA By Exocell Inc. protocols.io https://dx.doi.org/10.17504/protocols.io.34mgqu6 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 - GSSG (Non-Enzymatic). protocols.io https://dx.doi.org/10.17504/protocols.io.3qpgmvn 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 Analgesics for Rats and Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.3jrgkm6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8e8hthw 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: AFIP Laboratory Methods in Histotechnology, 1994, p. 16, 53-55.

Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Hematoxylin and Eosin Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.3jsgkne Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: The Tail Flick assay is a pain receptive assay in which a mouse is placed within a restraining tube with its tail protruding. The tail is placed on a level surface, a radiant heat is applied to the tail and the latency of the mouse to remove its tail from the heat is recorded. This latency is used as a measure to indicate neurological pathology.Diabetic Complication:

Proper citation: Eva Feldman 2019. Tail Flick. protocols.io https://dx.doi.org/10.17504/protocols.io.7thhnj6 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 - Immunohistochemistry for Tissue Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.7rfhm3n Copy   

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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 https://dx.doi.org/10.17504/protocols.io.8e5htg6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.3bagiie 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 - Western Blot Stripping. protocols.io https://dx.doi.org/10.17504/protocols.io.3s3gngn Copy   

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Authors: Charles Alpers
Group: Diabetic Complications Consortium
Summary: The following table describes the staining color for various structures.Results: Nuclei and Elastic Fibers Black Ground Substance and Mucin Blue Muscle Red Collagen and Reticular Fibers Yellow Fibrinioid and Fibrin Intense RedDiabetic Complications:

Proper citation: Charles Alpers 2019. MOVAT Pentachrome Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.346gqze 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 Reductase. protocols.io https://dx.doi.org/10.17504/protocols.io.3qsgmwe Copy   

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

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Authors: Peter Mundel
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedure for perfusion of mice prior to organ or tissue harvesting .Diabetic Complications:

Proper citation: Peter Mundel 2019. Vascular perfusion of mice. protocols.io https://dx.doi.org/10.17504/protocols.io.8ezhtf6 Copy   

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Authors: Nobuyo Maeda, Oliver Smithies, Nobuyuki Takahashi
Group: Diabetic Complications Consortium
Summary: SummaryIt is extremely important to measure quantitative parameters (e.g. plasma glucose, urinary albumin, etc.) in a way that minimizes the effects of any adventitious variables, such as seasonal changes, dietary differences, any differences in the degrees of backcrossing, or unplanned differences in genotypes. This means that, whenever possible, mice of the critical genotypes should be obtained from genotypically identical matings carried out at or close to the same time. Archival data are not appropriate. The breeding scheme to generate the experimental animals can vary in complexity, depending on the fertility of the two genders, whether the Testgene is to be studied in homozygotes or in heterozygotes, and whether the diabetes is induced genetically or by STZ. However, it is important to remember that, when looking for the effects of the Testgene genotype on diabetic complications, the aim is to compare the Testgene mutant/mutant (or Testgene mutant/wt) diabetic animals with their diabetic litter mates that are wildtype at the Testgene locus (Testgene wt/wt). ReferenceWang, C-H, Li, F, Hiller, S, Kim, H-S, Maeda, N, Smithies, O, and Takahashi, N. A modest decrease in endothelial NOS in mice comparable to that associated with human NOS3 variants exacerbates diabetic nephropathy. Proc. Natl. Acad. Sci. USA 108(5): 2070-2075 (2011) PMID: 21245338 PMCID: PMC3033253.Diabetic Complications:

Proper citation: Nobuyo Maeda, Oliver Smithies, Nobuyuki Takahashi 2019. Breeding Scheme and Selection of Animals for DiaComp Experiments. protocols.io https://dx.doi.org/10.17504/protocols.io.8fphtmn Copy   

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Authors: Jan Breslow, Daniel Teupser
Group: Diabetic Complications Consortium
Summary: Diabetic complication:

Proper citation: Jan Breslow, Daniel Teupser 2019. Mouse dissection and preparation of heart (aortic sinus) and brachiocephalic artery for lesion quantitation by sectioning. protocols.io https://dx.doi.org/10.17504/protocols.io.3a5gig6 Copy   

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Authors: Jan Breslow, Daniel Teupser
Group: Diabetic Complications Consortium
Summary: This protocol describes how the DiaComp members prepare the En Face staining for atherosclerotic lesions quantification.Diabetic Complication:

Proper citation: Jan Breslow, Daniel Teupser 2019. En face preparation and quantification of aortic surface area covered by atherosclerotic lesions. protocols.io https://dx.doi.org/10.17504/protocols.io.8fehtje Copy   

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Authors: Feng Wang, Takamune Takahashi
Group: Diabetic Complications Consortium
Summary: This protocol describes the procedures and data acquisition of quantitative magnetization MR imaging of diabetic mouse kidney.Reference: 1. Zhao, H.J., et al., Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice. J Am Soc Nephrol, 2006. 17(10): p. 2664-2669. 2. Ramani, A., et al., Precise estimate of fundamental in-vivo MT parameters in human brain in clinically feasible times. Magnetic resonance imaging, 2002. 20(10): p. 721-31. 3. Cercignani, M. and G.J. Barker, A comparison between equations describing in vivo MT: the effects of noise and sequence parameters. Journal of Magnetic Resonance, 2008. 191(2): p. 171-83. 4. Pluim, J.P.W., J.B.A. Maintz, and M.A. Viergever, Mutual-information-based registration of medical images: A survey. Ieee Transactions on Medical Imaging, 2003. 22(8): p. 986- 1004. 5. Wang, F., et al., Repeatability and sensitivity of high resolution blood volume mapping in mouse kidney disease. J Magn Reson Imaging, 2014. 39(4): p. 866-71. 6. Smith, S.A., et al., Measurement of T1 and T2 in the cervical spinal cord at 3 tesla. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine, 2008. 60(1): p. 213-9. 7. Henkelman, R.M., et al., Quantitative Interpretation of Magnetization-Transfer. Magnetic Resonance in Medicine, 1993. 29(6): p. 759-766. Diabetic Complication:

Proper citation: Feng Wang, Takamune Takahashi 2019. Quantitative Magnetization Transfer MR Imaging (qMT-MRI) of Diabetic Mouse Kidney. protocols.io https://dx.doi.org/10.17504/protocols.io.8a7hshn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7rhhm36 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 insulin- producing 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. High Dose STZ Induction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8iyhufw Copy   

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