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Original Articles

Intravitreal Triamcinolone Acetonide Inhibits Breakdown of the Blood-Retinal Barrier Through Differential Regulation of VEGF-A and Its Receptors in Early Diabetic Rat Retinas

  1. Xinyuan Zhang1,2⇑,
  2. Shisan Bao3,
  3. Donna Lai4,
  4. Robert W. Rapkins1 and
  5. Mark C. Gillies1
  1. 1Retinal Therapeutic Research Group, Save Sight Institute, Department of Clinical Ophthalmology, University of Sydney, Sydney, Australia;
  2. 2Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China;
  3. 3Department of Pathology, University of Sydney, Sydney, Australia;
  4. 4Molecular Biology Facility, Bosch Institute, University of Sydney, Sydney, Australia.
  1. Address correspondence and reprint requests to Dr. Xinyuan Zhang, Save Sight Institute, Department of Clinical Ophthalmology, Level 2, South Block, Sydney Eye Hospital, Macquarie St, Sydney NSW 2000, Australia. E-mail: xzhang{at}eye.usyd.edu.au.
Diabetes 2008 Apr; 57(4): 1026-1033. https://doi.org/10.2337/db07-0982
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  • FIG. 1.
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    FIG. 1.

    mRNA expression ratios for Vegf-a, Flk-1 and Flt-1. A: Sham-treated diabetic retina/sham-treated nondiabetic retina. B: IVTA-treated diabetic retina/sham-treated diabetic retina. C: IVTA-treated nondiabetic retina/sham-treated nondiabetic retina.

  • FIG. 2.
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    FIG. 2.

    Quantification analysis of VEGF-A, FLK-1, and FLT-1 distribution in the retina from different treatment groups. Number of positive cells for VEGF-A (A), FLK-1 (B), and FLT-1 (C) per millimeter (means ± SEM, n = 8). ***P < 0.001.

  • FIG. 3.
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    FIG. 3.

    Retinal thickness was measured on H&E-stained sections at the similar location. The micrograph shows representative samples from IVTA-treated nondiabetic retina (A), sham-treated nondiabetic retina (B), IVTA-treated diabetic retina (C), and sham-treated diabetic retina (D). The yellow bar in A demonstrates one measurement of retinal thickness. E: Table showing mean (± SEM) retinal thickness (*** P < 0.001). Original magnifications ×200. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium layer; OS, inner and outer photoreceptor segment; CH, choroidal layer. To view a high-quality digital representation of this image, go to http://dx.doi.org/db07-0982.

  • FIG. 4.
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    FIG. 4.

    Immunohistochemical analysis of retinal albumin distribution. Albumin was detected (in red) within the vessels in sham-treated nondiabetic retinas (A). A similar pattern of albumin distribution was observed in IVTA-treated diabetic retina, but with slightly higher expression in extra vascular space (B). Diffuse extravascular albumin was observed in the inner nuclear, outer plexiform, and outer nuclear layers in sham-treated diabetic retina (C). Extravasated albumin (arrows in C) was marked green in panel A1, B1, and C1 (corresponding to panels A, B, and C) by IPP4.5. Intravascular albumin was excluded by the analysis (arrow heads in C1). D: Quantification of albumin distribution/leakage in the four treatment groups. Values were presented as IAU. Data are means ± SEM, n = 8; *** P < 0.001. Original magnifications ×400. To view a high-quality digital representation of this image, go to http://dx.doi.org/db07-0982.

Tables

  • Figures
  • TABLE 1

    Primers and product size for real-time RT-PCR

    Target genesGenBank accession no.Forward primerReverse primerProduct size (bp)
    GapdhNM_017008.35′- GAGCTGAATGGGAAGCTCAC-3′5′-AAAGGTGGAGGAGTGGGAGT-3′216
    β-actinNM_031144.25′-AGCCATGTACGTAGCCATCC-3′5′- CTCTCAGCTGTGGTGGTGAA−3′228
    Vegf-aNM_031836.15′-AGAAACCCAATGAAGTGGTG-3′5′- ACTCCAGGGCTTCATCATTG-3′177
    Flt-1NM_019306.15′- TCCCTCAGCCTACCATCAAG-3′5′- GAGAGTCAGCCACCACCAAT-3′207
    Flk-1NM_013062.15′-ACAGCATCACCAGCAGTCAG-3′5′- CCAAGAACTCCATGCCCTTA-3′168
    • The pairs of primers were designed according to rat gene sequences published in GenBank.

  • TABLE 2

    Results from BestKeeper descriptive statistical analysis showing variation in the capability potential (CP) values

    FactornGM (CP)AM (CP)Min (CP)Max (CP)SD (±CP)CV (%CP)
    Gapdh3219.7519.8816.0616.501.668.35
    β-actin3214.0114.0313.0015.800.594.17
    • Housekeeping gene expressions were assessed on total RNA templates isolated from retinas. GM (CP), the geometric mean of CP; AM (CP), the arithmetic mean of CP; Min (CP) and Max (CP), the extreme values of CP; SD (±CP), the SD of the CP; CV (%CP), the coefficient of variance expressed as a percentage of the CP level.

  • TABLE 3

    Results from BestKeeper correlation analysis

    BestKeeper vs.GAPDHβ-actin
    r0.9090.815
    P0.0010.003
    • Measures of correlation between the two candidate gene expressions and the Bestkeeper in an index computed from the best candidate genes.

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Intravitreal Triamcinolone Acetonide Inhibits Breakdown of the Blood-Retinal Barrier Through Differential Regulation of VEGF-A and Its Receptors in Early Diabetic Rat Retinas
Xinyuan Zhang, Shisan Bao, Donna Lai, Robert W. Rapkins, Mark C. Gillies
Diabetes Apr 2008, 57 (4) 1026-1033; DOI: 10.2337/db07-0982

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Intravitreal Triamcinolone Acetonide Inhibits Breakdown of the Blood-Retinal Barrier Through Differential Regulation of VEGF-A and Its Receptors in Early Diabetic Rat Retinas
Xinyuan Zhang, Shisan Bao, Donna Lai, Robert W. Rapkins, Mark C. Gillies
Diabetes Apr 2008, 57 (4) 1026-1033; DOI: 10.2337/db07-0982
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