Study of the biodistribution of fluorescein in glioma-infiltrated mouse brain and histopathological correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence guidance

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OBJECT

Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood.

METHODS

The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation.

RESULTS

The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell–specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI–guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI.

CONCLUSIONS

Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.

ABBREVIATIONSBBB = blood-brain barrier; GTR = gross-total resection; IQR = interquartile range; NHA = normal human astrocyte; PBS = phosphate-buffered saline; PDGF = platelet-derived growth factor; PDGFB = PDGF beta; PDGFR = PDGF receptor; PDGFRA = PDGFR alpha; RCAS = replication-competent avian sarcoma-leukosis; RFP = red fluorescent protein; tva = tumor virus A.

Article Information

Correspondence Aaron A. Cohen-Gadol, Goodman Campbell Brain and Spine, Department of Neurological Surgery, Indiana University, 355 W. 16th St., Ste. 5100, Indianapolis, IN 46202. email: acohenmd@gmail.com.

ACCOMPANYING EDITORIAL DOI: 10.3171/2014.10.JNS141788.

INCLUDE WHEN CITING Published online April 3, 2015; DOI: 10.3171/2015.2.JNS132507.

DISCLOSURE Aaron A. Cohen-Gadol has a consulting agreement with Carl Zeiss Meditec AG. The compensation from this arrangement is donated to a not-for-profit educational organization, The Neurosurgical Atlas.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Fluorescein uptake by NHAs after 5 and 15 minutes of exposure in vitro. Nuclear DNA stained with DAPI (blue) and fluorescein staining (green) of the cell body are observed. Bar = 100 μm. Figure is available in color online only.

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    Fluorescein distribution in U251 malignant glioma cell culture after 5, 15, 30, and 60 minutes of exposure. Nuclear DNA stained with DAPI (blue). Fluorescein staining (green) is not enhanced in the nuclei or cell bodies of U251 cells compared with background. Some slight granular cytoplasmic staining is observed at 15 minutes but is absent at later time points. Bar = 45 μm. Figure is available in color online only.

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    Upper: In vivo cranial window fluorescence microscopy images at 5 minutes and 25 minutes after tail vein administration of fluorescein and Alexa Fluor 647 10-kD dextran. Red fluorescence represents mCherry (tumor cell) signal, blue fluorescence represents Alexa Fluor 647 dextran signal, and green fluorescence represents fluorescein signal. Note that the dextran extravasates into the brain parenchyma at 25 minutes, whereas fluorescein staining is seen at 5 minutes and becomes more diffuse by 25 minutes. Bar = 200 μm. Lower: Magnified in vivo cranial window images at 5, 15, and 25 minutes after fluorescein administration showing lack of fluorescein (green) uptake by engrafted U87 glioma cells (red). The upper row shows the fluorescein channel and the lower row shows the merge of mCherry and fluorescein channels. Bar = 100 μm. Figure is available in color online only.

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    In vivo fluorescence intensity measurements across tumor-infiltrated brain and adjacent parenchyma in 3 individual mice bearing U87 mCherry–expressing intracranial xenografts. The images were obtained 15 minutes after fluorescein administration at 10× magnification. Fluorescence intensity graphs are on the left and represent the intensity at intervals along the linear region of interest demonstrated on the merged 3-channel image on the right. Red fluorescence represents mCherry (tumor cell) signal, blue fluorescence represents Alexa Fluor 647 dextran signal, and green fluorescence represents fluorescein signal. Figure is available in color online only.

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    Left: Low-magnification coronal sections at the site of cranial window imaging in Fig. 6 reveal a glioma in the virus-injected right hemisphere that immunolabels for GFAP, PDGFRA, and RFP. Bar = 2 mm. Sections are oriented so the top is dorsal and the bottom is ventral. Right: Magnified (20×) image of tumor cells in the right hemisphere stained with H&E or immunolabeled for GFAP, PDGFR, or RFP. Bar = 100 μm. Figure is available in color online only.

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    In vivo cranial window fluorescence microscopy of fluorescein biodistribution into brain after intravenous administration. The right frontal lobe of mice is visualized 4 weeks after injection of PBS or RCAS-PDGFB-RFP viral injection. Transformed cells (glioma) express RFP and are labeled in red; fluorescein is labeled in green; and Alexa Fluor 647 10-kD dextran is labeled in blue. Fluorescence intensity measurements for RFP (red), fluorescein (green), and Alexa Fluor 647 dextran (blue) fluorescence are graphed against the distance along the linear region of interest with origin on the left of the white line shown. Bar = 200 μm. Figure is available in color online only.

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    A: A right occipital glioblastoma in a 67-year-old man presenting with visual decline. B: The fluorescent margin of the tumor against the normal brain is demonstrated; note the correlation between the fluorescent margin and the enhancing margin of the tumor based on intraoperative neuronavigation data (inset). C: Also note minimal fluorescent signal in the resection cavity and CSF, filling the defect. D: Postoperative MRI revealing gross-total removal of the enhancing region extending into the ventricle. Figure is available in color online only.

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