5-aminolevulinic acid, fluorescein sodium, and indocyanine green for glioma margin detection: analysis of operating wide-field and confocal microscopy in glioma models of various grades

Abstract

IntroductionSurgical resection remains the first-line treatment for gliomas. Several fluorescent dyes are currently in use to augment intraoperative tumor visualization, but information on their comparative effectiveness is lacking. We performed systematic assessment of fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA)–induced protoporphyrin IX (PpIX), and indocyanine green (ICG) fluorescence in various glioma models using advanced fluorescence imaging techniques.MethodsFour glioma models were used: GL261 (high-grade model), GB3 (low-grade model), and an in utero electroporation model with and without red fluorescence protein (IUE +RFP and IUE -RFP, respectively) (intermediate-to-low-grade model). Animals underwent 5-ALA, FNa, and ICG injections and craniectomy. Brain tissue samples underwent fluorescent imaging using a wide-field operative microscope and a benchtop confocal microscope and were submitted for histologic analysis.ResultsOur systematic analysis showed that wide-field imaging of highly malignant gliomas is equally efficient with 5-ALA, FNa, and ICG, although FNa is associated with more false-positive staining of the normal brain. In low-grade gliomas, wide-field imaging cannot detect ICG staining, can detect FNa in only 50% of specimens, and is not sensitive enough for PpIX detection. With confocal imaging of low-intermediate grade glioma models, PpIX outperformed FNa.DiscussionOverall, compared to wide-field imaging, confocal microscopy significantly improved diagnostic accuracy and was better at detecting low concentrations of PpIX and FNa, resulting in improved tumor delineation. Neither PpIX, FNa, nor ICG delineated all tumor boundaries in studied tumor models, which emphasizes the need for novel visualization technologies and molecular probes to guide glioma resection. Simultaneous administration of 5-ALA and FNa with use of cellular-resolution imaging modalities may provide additional information for margin detection and may facilitate maximal glioma resection.

Advances in Intraoperative Glioma Tissue Sampling and Infiltration Assessment

“…However, the mechanism of its extravasation is not specific to alternative methods, leading to BBB disruption (e.g., injury, edema, etc.) [26]. Fluorescein emits an intense yellow color at the site of blood-brain barrier disruption affected by intracranial tumors.…”

“…In combination with fluorescence, recent studies have demonstrated promising results when compared to those of standard pathological diagnosis [80]. Moreover, compared to wide-field imaging, CLE demonstrated the superior detection of 5-ALA and fluorescein sodium [26].…”

Automated online safety margin (GLIOVIS) for glioma surgery model

“…Fluorescence-guided surgery has emerged as a sensitive and effective method to define tumor location and delineate its margins during the procedure, maximizing the extent of resection ( 9 – 11 ). Several fluorescent agents have been assessed in clinical trials over the past few years for malignant glioma including 5-aminolevulinic acid (5-ALA), fluorescein, indocyanine green, hypericin, 5-aminofluorescein bound to human serum albumin, and endogenous spectroscopy ( 9 , 12 ).…”

“…In addition to these limitations, when the normal brain overlaps the pathological tissue or the orientation of the microscope view is not appropriate (“non-orthogonal working corridors” or “dark corridors”), no fluorescence can be seen under the blue light filter ( 11 ). Similarly, blood, CSF, cottonoids, or other hemostatic agents can hide fluorescent tissue and limit the extent of resection.…”