title: 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.
abstract: OBJECTIVE
  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.
authors.affiliation: 1The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumour Research Centre;|3Division of Neurosurgery, Department of Surgery, University of Toronto;
authors.forename: Roberto Jose
authors.initials: RJ
authors.lastname: Diaz
authors.affiliation: 5Department of Neurosurgery, University of Mississippi Medical Center, Jackson, Mississippi;
authors.forename: Roberto Rey
authors.initials: RR
authors.lastname: Dios
authors.affiliation: 6Department of Pathology and Laboratory Medicine, Indiana University; and.
authors.forename: Eyas M
authors.initials: EM
authors.lastname: Hattab
authors.affiliation: 1The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumour Research Centre;
authors.forename: Kelly
authors.initials: K
authors.lastname: Burrell
authors.affiliation: 1The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumour Research Centre;
authors.forename: Patricia
authors.initials: P
authors.lastname: Rakopoulos
authors.affiliation: 1The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumour Research Centre;
authors.forename: Nesrin
authors.initials: N
authors.lastname: Sabha
authors.affiliation: 1The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumour Research Centre;
authors.forename: Cynthia
authors.initials: C
authors.lastname: Hawkins
authors.affiliation: 3Division of Neurosurgery, Department of Surgery, University of Toronto;|4Toronto Western Hospital University Health Network, Toronto, Ontario, Canada;
authors.forename: Gelareh
authors.initials: G
authors.lastname: Zadeh
authors.affiliation: 1The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumour Research Centre;|3Division of Neurosurgery, Department of Surgery, University of Toronto;
authors.forename: James T
authors.initials: JT
authors.lastname: Rutka
authors.affiliation: 7Goodman Campbell Brain and Spine, Department of Neurological Surgery, Indiana University, Indianapolis, Indiana.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Cohen-Gadol
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D001921:Brain / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D019793:Fluorescein / Q000493:pharmacokinetics*; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D014018:Tissue Distribution
chemical_list: D019793:Fluorescein
keywords: BBB = 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; distribution; fluorescein; glioma cells; marker; oncology; resection; tva = tumor virus A; uptake