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Research Letter |

Subconjunctival Indocyanine Green Identifies Lymphatic Vessels FREE

Clovis Arcoverde Freitas-Neto, MD1,2,3; Rogério A. Costa, MD, PhD1; Ninani Kombo, MD2; Theophilo Freitas, MD3; Juliana Lambert Oréfice, MD, PhD1; Fernando Oréfice, MD1; C. Stephen Foster, MD2
[+] Author Affiliations
1Centro Brasileiro de Ciências Visuais, Belo Horizonte, Brazil
2Massachusetts Eye Research and Surgery Institution, Cambridge
3Hospital de Olhos Santa Luzia, Recife, Brazil
JAMA Ophthalmol. 2015;133(1):102-104. doi:10.1001/jamaophthalmol.2014.3944.
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Published online

The lymphatic system maintains tissue fluid balance and immunity. It also plays an important role in pathologic conditions such as tumor metastasis and inflammation. The anatomists Arnold (in 1847) and Teichmann (in 1861) were the first to visualize and describe the conjunctival lymphatics.1 In 1948, Busacca2 identified a number of anastomosing lymphatics, which penetrated the orbital cavity near the extraocular muscles, using trypan blue dye. A better understanding of the lymphatic system in the eye could provide the basis for developing alternative therapeutic strategies for ocular diseases.3

Newer techniques of fluorescence lymphography using indocyanine green (ICG) are increasingly being used in medical specialties such as vascular surgery and oncology. Lymphography using ICG is useful because there is no endogenous fluorescence in the near-infrared band (780-1500 nm) used for ICG detection and the examination does not cause tissue damage. Indocyanine green is approved for hepatic and ophthalmologic applications.4 We identified conjunctival lymphatic capillaries using lymphography with ICG.

This study was approved by the independent and external Hospital Agamenon Magalhaes Institutional Review Board. One of us (C.A.F.-N.) volunteered as the study participant and provided written informed consent. One vial containing 5 mg of ICG used for internal limiting membrane staining (Ophthalmos) was used to prepare a sterile aqueous ICG solution at 5%. Following topical anesthesia, 0.1 mL of this ICG solution was injected into the subconjunctival area using a 30-gauge needle through the conjunctiva approximately 3 mm away and inferotemporally from the limbus of the right eye.

Ocular imaging was performed using a confocal scanning laser ophthalmoscope (Spectralis HRA + OCT; Heidelberg Engineering Inc). For color photography, a retinal camera (TRC-50IX; Topcon Medical Systems) was used. Ophthalmic evaluation and ocular imaging were performed on a weekly basis until complete absorption of the ICG was verified.

No systemic or local adverse effects were observed following the subconjunctival injection of ICG. The ICG completely cleared from the eye within 4 weeks. Minute lymph vessels (lymphatic capillaries) were identified and differentiated from blood vessels by multimodality diagnostic imaging. Meticulous analysis of the ICG lymphography revealed intermittently dilated lymphatic drainage channels under the conjunctival blood vessels (Figure).

Place holder to copy figure label and caption
Figure.
Indocyanine Green Lymphography Findings

A, Site of contrast injection (asterisk). B, Infrared reflectance showing the conjunctival blood vessels as dark lines. C, Subconjunctival indocyanine green identifies lymphatic vessels below the conjunctival blood vessels (arrowheads). D and E, Fluorescence 24 hours after injection. F, Dilatations of the lymphatic vessels (arrowheads).

Graphic Jump Location

Three decades ago, Rayes et al5 studied the lymphatic distribution in the bulbar conjunctiva of 60 patients by injecting 1% trypan blue dye in different quadrants of the bulbar conjunctiva. The scheme of the lymphatic distribution in normal bulbar conjunctiva was presented and histopathologic studies confirmed the presence of lymphatic tissue. During phacotrabeculectomy surgery, Singh6 observed several vessels through which the dye passed after subconjunctival injection of trypan blue dye. The conclusion was that there were likely lymphatic vessels. These were observed in more than 120 cases of all types of glaucoma. Singh suggested that the excess interstitial subconjunctival fluid is removed by the conjunctival lymphatic system. We present initial data about the use of ICG to identify the microanatomy of the ocular lymphatic system in humans. After a single subconjunctival injection of 0.5 mg of ICG, lymphatic vessels were clearly identified on near-infrared fluorescence.

The lymphatic drainage of the eye remains undefined. Unexpectedly, we could also document that ICG hyperfluorescence drained toward the perilimbal region. The ICG remained in the corneal avascular tissue for several days before complete disappearance. Further topographic mapping studies with near-infrared fluorescence lymphography are needed to better understand normal human conjunctival lymphatic microanatomy and drainage.

Corresponding Author: Clovis Arcoverde Freitas-Neto, MD, Massachusetts Eye Research and Surgery Institution, 355 Main St, Eighth Floor, Cambridge, MA 02142 (cfreitas@hospitalsantaluzia.com.br).

Published Online: October 2, 2014. doi:10.1001/jamaophthalmol.2014.3944.

Author Contributions: Drs Freitas-Neto and F. Oréfice had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Freitas-Neto, Costa, F. Oréfice, Foster.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Freitas-Neto, Costa, Kombo, Freitas, Foster.

Critical revision of the manuscript for important intellectual content: Freitas-Neto, Costa, Kombo, J. L. Oréfice, F. Oréfice, Foster.

Obtained funding: Foster.

Administrative, technical, or material support: Freitas-Neto, Costa, Kombo, Freitas, Foster.

Study supervision: Costa, J. L. Oréfice, F. Oréfice, Foster.

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Foster reported having been a consultant for Abbvie, Alcon Laboratories, Allergan, Ista Pharmaceuticals, LUX Biosciences, Novartis Pharmaceuticals, and Bausch & Lomb Surgical; having received grants or having grants pending from Abbvie, Alcon Laboratories, Allergan, Eyegate, LUX Biosciences, Novartis Pharmaceuticals, pSivida, sanofi-aventis, XOMA, and Santen; having received honoraria for lectures including service on speakers bureaus for Alcon Laboratories, Allergan, Inspire, Ista Pharmaceuticals, and LUX Biosciences; and having stock or stock options in Eyegate. No other disclosures were reported.

Teichmann  L. Das Saugader System. Leipzig, Germany: Engelmann; 1861:1-121.
Busacca  A.  Les vaisseaux lymphatiques de la conjonctive bulbaire humaine étudiés par la méthode des injections vitales de bleutripan. Arch d’opht. 1948;8:10.
Nakao  S, Hafezi-Moghadam  A, Ishibashi  T.  Lymphatics and lymphangiogenesis in the eye. J Ophthalmol. 2012;2012:783163.
PubMed   |  Link to Article
Mihara  M, Hara  H, Araki  J,  et al.  Indocyanine green (ICG) lymphography is superior to lymphoscintigraphy for diagnostic imaging of early lymphedema of the upper limbs. PLoS One. 2012;7(6):e38182.
PubMed   |  Link to Article
Rayes  A, Oréfice  F, Rocha  H.  Distribuição da rede linfática da conjuntiva bulbar humana normal, estudada através de injeções conjuntivais de azul de tripan a 1%. Arq Bras Oftalmol. 1980;43(5):188-200.
Singh  D.  Conjunctival lymphatic system. J Cataract Refract Surg. 2003;29(4):632-633.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure.
Indocyanine Green Lymphography Findings

A, Site of contrast injection (asterisk). B, Infrared reflectance showing the conjunctival blood vessels as dark lines. C, Subconjunctival indocyanine green identifies lymphatic vessels below the conjunctival blood vessels (arrowheads). D and E, Fluorescence 24 hours after injection. F, Dilatations of the lymphatic vessels (arrowheads).

Graphic Jump Location

Tables

References

Teichmann  L. Das Saugader System. Leipzig, Germany: Engelmann; 1861:1-121.
Busacca  A.  Les vaisseaux lymphatiques de la conjonctive bulbaire humaine étudiés par la méthode des injections vitales de bleutripan. Arch d’opht. 1948;8:10.
Nakao  S, Hafezi-Moghadam  A, Ishibashi  T.  Lymphatics and lymphangiogenesis in the eye. J Ophthalmol. 2012;2012:783163.
PubMed   |  Link to Article
Mihara  M, Hara  H, Araki  J,  et al.  Indocyanine green (ICG) lymphography is superior to lymphoscintigraphy for diagnostic imaging of early lymphedema of the upper limbs. PLoS One. 2012;7(6):e38182.
PubMed   |  Link to Article
Rayes  A, Oréfice  F, Rocha  H.  Distribuição da rede linfática da conjuntiva bulbar humana normal, estudada através de injeções conjuntivais de azul de tripan a 1%. Arq Bras Oftalmol. 1980;43(5):188-200.
Singh  D.  Conjunctival lymphatic system. J Cataract Refract Surg. 2003;29(4):632-633.
PubMed   |  Link to Article

Correspondence

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