Diabetic Retinopathy: Retina‐Specific Methods for Maintenance of Diabetic Rodents and Evaluation of Vascular Histopathology and Molecular Abnormalities

Alexander Veenstra1, Haitao Liu1, Chieh Allen Lee2, Yunpeng Du2, Jie Tang3, Timothy S. Kern3

1 These authors contributed equally to this work, 2 Case Western Reserve University and Case Medical Center, Cleveland, Ohio, 3 Veterans Administration Medical Center Research Service 151, Cleveland, Ohio
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140190
Online Posting Date:  September, 2015
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Diabetic retinopathy is a major cause of visual impairment, which continues to increase in prevalence as more and more people develop diabetes. Despite the importance of vision, the retina is one of the smallest tissues in the body, and specialized techniques have been developed to study retinopathy. This article summarizes several methods used to (i) induce diabetes in mice, (ii) maintain the diabetic animals throughout the months required for development of typical vascular histopathology, (iii) evaluate vascular histopathology of diabetic retinopathy, and (iv) quantitate abnormalities implicated in the development of the retinopathy. © 2015 by John Wiley & Sons, Inc.

Keywords: diabetes; streptozotocin; retinopathy; retina; mouse

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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Diabetes Using Streptozotocin
  • Basic Protocol 2: Clinical Measures of Diabetes Severity
  • Basic Protocol 3: Isolation of Fresh Retina
  • Basic Protocol 4: Isolation of Retinal Vessels for Histology
  • Basic Protocol 5: Measurement of Retinal Superoxide
  • Basic Protocol 6: Quantification of Diabetes‐Induced Adherent Leukocytes in Retinal Vasculature (Leukostasis)
  • Basic Protocol 7: Immunohistochemistry of Vasculature and Leukocytes in Retinal Whole Mounts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Induction of Diabetes Using Streptozotocin

  • Mice or rats (see discussion above)
  • Streptozotocin (STZ; MP Biomedicals) solution: prepare immediately before use in citrate buffer (see below) at a concentration that will deliver 60 mg/kg body weight for mice or 55 mg/kg body weight for rats, in a volume of 200 μl citrate buffer)
  • Citrate buffer: 47 mM sodium citrate/53 mM citric acid in distilled water, pH 4.5 (may be stored frozen for 6 months)Insulin
  • For mice: Dilute NPH insulin 1:10 with diluent (provided free by Eli Lilly and Co upon request) and refrigerate
  • For rats: Use NPH insulin (Humulin; Eli Lilly and Co) undiluted
  • Animal balance
  • Additional reagents and equipment for injection of mice (Donovan and Brown, ) and measuring fasting blood glucose ( protocol 2)

Basic Protocol 2: Clinical Measures of Diabetes Severity

  • Blood glucose test strips and glucose meter (most glucose meters will be appropriate)
  • Method to measure HbA1c (or glycated hemoglobin); we currently use the VARIANT Classic system (BioRad Clinical Diagnostics)
  • Animal balance to measure body weight.
  • Additional reagents and equipment for blood collection from rodents (Donovan and Brown, )

Basic Protocol 3: Isolation of Fresh Retina

  • Mouse or rat (see discussion in protocol 1 introduction)
  • Small dissecting scissors (curved, blunt‐end, 22‐mm; Roboz, cat. no. RS‐5983)
  • Pink dental wax (Electron Microscopy Sciences, cat. no. 72670)
  • Dissecting microscope
  • Micro‐forceps (Dumont #5, biological‐grade tip; Electron Microscopy Sciences, cat. no. 72700‐D or Dumont, electronic‐grade tip, Electron Microscopy Sciences, cat. no. 0103‐2‐PO)
  • Razor blades (GEM single edge stainless steel, Teflon coated; Electron Microscopy Sciences, cat. no. 71970)
  • Micro‐spatula (Fine Science Tools, FST 10091‐12)
  • Micro‐dissecting spring scissors (Vannas, 3‐mm straight; Roboz, cat. no. RS‐5620)
  • Additional reagents and equipment for rodent anesthesia (Donovan and Brown, ) or euthanasia (Donovan and Brown, )

Basic Protocol 4: Isolation of Retinal Vessels for Histology

  • Mouse or rat (see discussion in protocol 1 introduction)
  • 10% buffered formalin (Sigma, cat. no. HT501128, or equivalent)
  • Elastase solution (see recipe)
  • Activating solution (100 mM Tris·Cl, pH 8.5)
  • Periodic acid solution: 35 mM periodic acid/12 mM sodium acetate in H 2O
  • Schiff reagent base (Sigma, cat. no. 3952016)
  • Harris modified hematoxylin (Sigma‐Aldrich, cat. no. HHS128); filter before use to remove precipitates
  • 7.4 mM ammonium hydroxide
  • 70%, 80%, 95%, and 100% ethanol
  • Xylene
  • Permount mounting medium
  • Microcentrifuge tubes or 48‐well plates
  • Lid from petri dish
  • Dissecting microscope
  • Two “brushes” made from single sable hairs or cat whiskers glued to the end of glass a rod with fingernail polish (an additional brush glued at both ends of the hair to the rod so that it forms a loop is helpful (Fig. )
  • Micro‐forceps (Dumont #5, biological‐grade tip; Electron Microscopy Sciences, cat. no. 72700‐D or Dumont, electronic‐grade tip; Electron Microscopy Sciences, cat. no. 0103‐2‐PO)
  • Superfrost Plus treated microscope slides (Fisher, cat. no. 12‐550‐15; use slides from unopened box or clean as described in under step 20, below)
  • Glass transfer pipet: snap off the thin end of a Pasteur pipet (borosilicate glass, Fisher, cat. no. 1367820B), fire‐polish to remove jagged edges, and fit the “broken” end with a rubber bulb (Fisher, cat. no. 0344821)
  • Staining jars
  • 22 × 40–mm glass coverslips
  • Mounting dish (glass, ∼4‐in. radius × 1.25‐in. high, having fluid exit for water to drain out from underneath the glass microscope slide that is placed in the mounting dish while attaching the isolated vasculature to the microscope slide)
  • Additional reagents and equipment for isolating retina ( protocol 3)

Basic Protocol 5: Measurement of Retinal Superoxide

  • Freshly isolated retina ( protocol 3)
  • Krebs‐HEPES buffer (see recipe)
  • 5 mM lucigenin (see recipe)
  • 10× cell lysis buffer (Cell Signaling, cat. no. 9803)
  • Bradford protein assay kit
  • Probe type sonicator/cell disrupter
  • Luminometer (e.g., GlowMax 20/20 from Promega)

Basic Protocol 6: Quantification of Diabetes‐Induced Adherent Leukocytes in Retinal Vasculature (Leukostasis)

  • Saline: 0.9%, veterinary grade (Baxter, cat. no. 04925‐04‐10)
  • 1 mg/ml concanavalin A–FITC (Vector FL‐1001) in PBS (see next line)
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. P3813)
  • Infusurge 4010 pressure infuser (Ethox, Inc.)
  • IV catheter set with regulating clamp (70 in., Baxter, cat. no. 2C5417s)
  • Two 4‐way stopcock luer‐lock IV line valves (Baxter, 2C6204)
  • Two IV extension sets (3.9 ml, 36 in., luer‐lock; Baxter, cat. no. 2C2667)
  • One IV extension set (2.3 ml, 21 in., Luer‐lock; Baxter, cat. no. 2C2662)
  • 0.22‐μm syringe filter
  • 20‐G gavage needle (1.25‐mm O.D. barrel tip, 30 mm length; Fine Science Tools, cat. no. 18060‐20)
  • 10‐ml syringe
  • Hemostats
  • Fluorescence microscope with FITC filter
  • Additional reagents and equipment for rodent anesthesia (Donovan and Brown, ) and isolating the retina ( protocol 3)

Basic Protocol 7: Immunohistochemistry of Vasculature and Leukocytes in Retinal Whole Mounts

  • Concanavalin A working solution (see recipe)
  • Fixation buffer: 2% paraformaldehyde in PBS pH 7.2
  • Triton X‐100
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. P3813)
  • Demasking buffer (see recipe)
  • Blocking buffer (see recipe)
  • Primary antibodies:
  • Rabbit anti‐collagen IV (1:1000 dilution; Abcam, cat. no. Ab6586)
  • Donkey anti‐Rabbit AF488 conjugated (1:1000 dilution; Jackson ImmunoResearch, cat. no. 711‐545‐152)
  • Rat IgG2b anti‐CD45 clone 30 F‐11 PE conjugated (1:500 dilution; BD, cat. no. 553081)
  • Goat Anti‐PE (1:1000 dilution; Rockland, cat. no. 600‐101‐387), pre‐labeled with Dylight 594 (Thermo Fisher, cat. no. 46412)
  • Goat anti‐Concanavalin A (1:500 dilution; Vector Labs, cat. no. AS2004), pre‐labeled with Dylight 650 (Thermo Fisher, cat. no. 62274)
  • TBST (see recipe)
  • Hoechst 3342 (1:1000 dilution Thermo Fisher Scientific, cat. no. 62249)
  • Tris‐buffered saline (TBS; optional, see recipe)
  • Antifade mounting medium
  • Microscope slide with well (see annotation to step 17)
  • Clear nail polish
  • Glass coverslips, thickness #1, 22 × 40 mm
  • Fluorescence microscope
  • Additional reagents and equipment for perfusion of mouse ( protocol 6) and isolation of the retina ( protocol 3)
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