In vivo retinal and choroidal hypoxia imaging using a novel activatable hypoxia-selective near-infrared fluorescent probe

  • Shinichi Fukuda
  • Kensuke Okuda
  • Genichiro Kishino
  • Sujin Hoshi
  • Itsuki Kawano
  • Masahiro Fukuda
  • Toshiharu Yamashita
  • Simone Beheregaray
  • Masumi Nagano
  • Osamu Ohneda
  • Hideko Nagasawa
  • Tetsuro Oshika
Basic Science



Retinal hypoxia plays a crucial role in ocular neovascular diseases, such as diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusion. Fluorescein angiography is useful for identifying the hypoxia extent by detecting non-perfusion areas or neovascularization, but its ability to detect early stages of hypoxia is limited. Recently, in vivo fluorescent probes for detecting hypoxia have been developed; however, these have not been extensively applied in ophthalmology. We evaluated whether a novel donor-excited photo-induced electron transfer (d-PeT) system based on an activatable hypoxia-selective near-infrared fluorescent (NIRF) probe (GPU-327) responds to both mild and severe hypoxia in various ocular ischemic diseases animal models.


The ocular fundus examination offers unique opportunities for direct observation of the retina through the transparent cornea and lens. After injection of GPU-327 in various ocular hypoxic diseases of mouse and rabbit models, NIRF imaging in the ocular fundus can be performed noninvasively and easily by using commercially available fundus cameras. To investigate the safety of GPU-327, electroretinograms were also recorded after GPU-327 and PBS injection.


Fluorescence of GPU-327 increased under mild hypoxic conditions in vitro. GPU-327 also yielded excellent signal-to-noise ratio without washing out in vivo experiments. By using near-infrared region, GPU-327 enables imaging of deeper ischemia, such as choroidal circulation. Additionally, from an electroretinogram, GPU-327 did not cause neurotoxicity.


GPU-327 identified hypoxic area both in vivo and in vitro.


Hypoxia imaging Ocular ischemia Near-infrared fluorescent probe In vivo imaging 


Compliance with ethical standards


This work was supported in part by research grants KAKENHI 25861614 (to S.F.) and 22790042 (to K.O.) from the Japan Society for the Promotion of Science, Tokyo, Japan, the Japan Science and the Technology Agency, under a program of development of systems and technology for advanced measurement and analysis, and a Research Grant from the Study Group on Chorioretinal Degeneration and Optic Atrophy, the Ministry of Health, Labor and Welfare, Japan.

Financial disclosure

The authors have no financial or proprietary interest in any product mentioned in the article.

Conflict of interest

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or nonfinancial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Animal experiments

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Supplementary material

417_2016_3476_MOESM1_ESM.pdf (129 kb)
Supplemental Figure 1 1H NMR (400 MHz, DMSO-d6) and 13C NMR (125 MHz, CD3OD) for 5-methoxy-1,2,3,3-tetramethyl-3H-indolium iodide (2). (PDF 128 kb)
417_2016_3476_MOESM2_ESM.pdf (194 kb)
Supplemental Figure 2 1H NMR (400 MHz, CD3CN) and 13C NMR (125 MHz, CD3CN:CD3OD:CDCl3) for 1-(3,5-dinitrobenzyl)-5-methoxy-2,3,3-trimethyl-3H-indolium iodide (3). (PDF 193 kb)
417_2016_3476_MOESM3_ESM.pdf (217 kb)
Supplemental Figure 3 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) for 5-methoxy-1,3,3-trimethyl-2-{6-(N-phenylacetamido)hexa-1,3,5-trienyl}-3H-indolium iodide (4). (PDF 216 kb)
417_2016_3476_MOESM4_ESM.pdf (247 kb)
Supplemental Figure 4 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 125 MHz) for 2-[7-{1-(3,5-dinitrobenzyl)-5-methoxy-3,3-dimethylindolin-2-ylidene}hepta-1,3,5-trienyl]-5-methoxy-1,3,3-trimethyl-3H-indolium iodide (GPU-327). (PDF 247 kb)
417_2016_3476_MOESM5_ESM.pdf (155 kb)
Supplemental Figure 5 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) for 2-{7-[1-(3,5-diaminobenzyl)-5-methoxy-3,3-dimethylindolin-2-ylidene]hepta-1,3,5-trienyl}-5-methoxy-1,3,3-trimethyl-3H-indolium iodide (GPU-328). (PDF 155 kb)
417_2016_3476_MOESM6_ESM.pdf (49 kb)
Supplemental Figure 6 HPLC chromatogram for (A) 1-(3,5-dinitrobenzyl)-5-methoxy-2,3,3-trimethyl-3H-indolium iodide (3), (B) 5-methoxy-1,3,3-trimethyl-2-{6-(N-phenylacetamido)hexa-1,3,5-trienyl}-3H-indolium iodide (4), (C) 2-[7-{1-(3,5-dinitrobenzyl)-5-methoxy-3,3-dimethylindolin-2-ylidene}hepta-1,3,5-trienyl]-5-methoxy-1,3,3-trimethyl-3H-indolium iodide (GPU-327), and (D) 2-{7-[1-(3,5-diaminobenzyl)-5-methoxy-3,3-dimethylindolin-2-ylidene]hepta-1,3,5-trienyl}-5-methoxy-1,3,3-trimethyl-3H-indolium iodide (GPU-328). All analyses were performed on Waters symmetry C18 (3.5 μm, 4.6 × 75 mm) column, and flow rate was 1.0 ml/min at r.t. Respective analysis conditions are described as follows. (A) eluent: H2O/CH3CN containing 0.1% trifluoroacetic acid 95/5 to 5/95 over 0 to 15 min, then 5/95 from 15 to 20 min, retention time: 6.93 min. The absorbance at 254 nm was observed. (B): eluent: H2O/CH3CN containing 0.1% trifluoroacetic acid 90/10 to 5/95 over 0 to 15 min, then 5/95 from 15 to 20 min, retention time: 8.87 min. The absorbance at 520 nm was observed. (C): eluent: H2O/CH3CN containing 0.1% trifluoroacetic acid 65/35 to 25/75 over 0 to 15 min, then 10/90 from 15 to 20 min, retention time: 11.53 min. The absorbance at 760 nm was observed. (D): eluent: H2O/CH3CN containing 0.1% trifluoroacetic acid 65/35 to 25/75 over 0 to 15 min, then 25/75 to 10/90 from 15 to 20 min, retention time: 6.77 min. The absorbance at 760 nm was observed. (PDF 48 kb)
417_2016_3476_MOESM7_ESM.pdf (114 kb)
Supplemental Table 1 (PDF 113 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Shinichi Fukuda
    • 1
  • Kensuke Okuda
    • 2
  • Genichiro Kishino
    • 1
  • Sujin Hoshi
    • 1
  • Itsuki Kawano
    • 3
  • Masahiro Fukuda
    • 4
  • Toshiharu Yamashita
    • 5
  • Simone Beheregaray
    • 1
  • Masumi Nagano
    • 5
  • Osamu Ohneda
    • 5
  • Hideko Nagasawa
    • 3
  • Tetsuro Oshika
    • 1
  1. 1.Department of Ophthalmology, Faculty of MedicineUniversity of TsukubaIbarakiJapan
  2. 2.Laboratory of Bioorganic & Natural Products ChemistryKobe Pharmaceutical UniversityKobeJapan
  3. 3.Laboratory of Pharmaceutical and Medicinal ChemistryGifu Pharmaceutical UniversityGifuJapan
  4. 4.Department of Molecular Therapy, National Institute of NeuroscienceNational Center of Neurology and PsychiatryKodairaJapan
  5. 5.Laboratory of Regenerative Medicine and Stem Cell Biology, Graduate School of Comprehensive Human ScienceUniversity of TsukubaIbarakiJapan

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