Use of in vivo near-infrared laser confocal endomicroscopy with indocyanine green to detect the boundary of infiltrative tumor

Laboratory investigation

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Object

Infiltrative tumor resection is based on regional (macroscopic) imaging identification of tumorous tissue and the attempt to delineate invasive tumor margins in macroscopically normal-appearing tissue, while preserving normal brain tissue. The authors tested miniaturized confocal fiberoptic endomicroscopy by using a near-infrared (NIR) imaging system with indocyanine green (ICG) as an in vivo tool to identify infiltrating glioblastoma cells and tumor margins.

Methods

Thirty mice underwent craniectomy and imaging in vivo 14 days after implantation with GL261-luc cells. A 0.4 mg/kg injection of ICG was administered intravenously. The NIR images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope probe. Histological samples were acquired from matching imaged areas for correlation of tissue images.

Results

In vivo NIR wavelength confocal endomicroscopy with ICG detects fluorescence of tumor cells. The NIR and ICG macroscopic imaging performed using a surgical microscope correlated generally to tumor and peritumor regions, but NIR confocal endomicroscopy performed using ICG revealed individual tumor cells and satellites within peritumoral tissue; a definitive tumor border; and striking fluorescent microvascular, cellular, and subcellular structures (for example, mitoses, nuclei) in various tumor regions correlating with standard clinical histological features and known tissue architecture.

Conclusions

Macroscopic fluorescence was effective for gross tumor detection, but NIR confocal endomicroscopy performed using ICG enhanced sensitivity of tumor detection, providing real-time true microscopic histological information precisely related to the site of imaging. This first-time use of such NIR technology to detect cancer suggests that combined macroscopic and microscopic in vivo ICG imaging could allow interactive identification of microscopic tumor cell infiltration into the brain, substantially improving intraoperative decisions.

Abbreviations used in this paper: 5-ALA = 5-aminolevulinic acid; BBB = blood-brain barrier; BNI = Barrow Neurological Institute; FOV = field of view; ICG = indocyanine green; NIR = near infrared; ROI = region of interest; VWF = visible-wavelength fluorescence.

Article Information

Address correspondence to: Mark C. Preul, M.D., Division of Neurological Surgery, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, 350 West Thomas Road, Phoenix, Arizona 85013. email: neuropub@chw.edu.

Please include this information when citing this paper: published online September 16, 2011; DOI: 10.3171/2011.8.JNS11559.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    A: Photographs showing bioluminescence at various time points. The tumor burden was quantified by the in vivo photon count. The color scheme represents signal intensity, with red representing the highest-intensity photon count and purple representing low density of tumor signal. B: Bar graph of GL261-luc tumor cell growth in an albino C57BL/6 mouse. Bioluminescence was measured every 3 days and plotted as the in vivo photon count versus days postimplantation. p/sec/cm2/sr = photons/sec/cm2/steradian. Used with permission from Barrow Neurological Institute.

  • View in gallery

    Indocyanine green fluorescence yielding visualization of brain tumor macroscopically. A: Intraoperative view of a B6 (Cg)-Tyrc-2 J/J mouse brain tumor 14 days after stereotactic implantation of GL261-luc glioma cells. A large necrotic mass with significant peritumoral edema abuts the surface of the right frontal lobe under regular white light. B: After the ICG was injected, the tumor was macroscopically visible with NIR light, and is delineated by an intense, heterogeneous central fluorescent region and a less bright peripheral halo. Intravascular ICG denotes vessels. Used with permission from Barrow Neurological Institute.

  • View in gallery

    Timing characteristics of brain tumor saturation with ICG. A: Macroscopically visible tumor with NIR light, delineated by an intense, heterogeneous central fluorescent region and a less bright peripheral halo 1 minute after ICG injection. B: Intensity of fluorescence in the central region of tumor increased 15 minutes after injection. Regions of focal brightness, morphologically consistent with normal brain on confocal endomicroscopy, mostly representing leakage of ICG related to surgical trauma (arrow). C: Immediately after ICG injection, fluorescence was visible in only a few clusters of tumor cells. D: Fifteen minutes after ICG injection, the entire tumor mass was saturated with ICG, expressing bright fluorescence of tumor cells. Bars = 100 μm. Used with permission from Barrow Neurological Institute.

  • View in gallery

    Confocal images of the normal brain area obtained with the endomicroscope demonstrating predominantly intravascular ICG distribution, with fluorescent small vessels and capillary networks. Individual erythrocytes are visible within capillaries. Bar = 100 μm. Used with permission from Barrow Neurological Institute.

  • View in gallery

    A: Image obtained with the handheld miniaturized confocal endomicroscope, using ICG to reveal the atypical morphological features of tumor cells, including pleomorphic enlarged nuclei, mitoses, and multinucleated cells. B: Confocal image (upper panel) obtained during surgery highlighting cellular tumor with necrosis (arrow) and mitosis (arrowhead). H & E–stained section (lower panel) of tissue from the same ROI revealing corresponding histological characteristics. Bars = 50 μm. Used with permission from Barrow Neurological Institute.

  • View in gallery

    Confocal images obtained from the tumor border. A: Confocal image obtained during scanning of the operative bed, demonstrating infiltrative tumor (arrows) extending into adjacent edematous brain parenchyma (arrowhead) consistent with residual tumor and positive surgical margins. B: Tissue section from the same ROI showing corresponding histological characteristics. Bars = 100 μm. Used with permission from Barrow Neurological Institute.

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