Potential application of a handheld confocal endomicroscope imaging system using a variety of fluorophores in experimental gliomas and normal brain

Abstract

<jats:sec> <jats:title>Object</jats:title> <jats:p>The authors sought to assess the feasibility of a handheld visible-wavelength confocal endomicroscope imaging system (Optiscan 5.1, Optiscan Pty., Ltd.) using a variety of rapid-acting fluorophores to provide histological information on gliomas, tumor margins, and normal brain in animal models.</jats:p></jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>Mice (n = 25) implanted with GL261 cells were used to image fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA), acridine orange (AO), acriflavine (AF), and cresyl violet (CV). A U251 glioma xenograft model in rats (n = 5) was used to image sulforhodamine 101 (SR101). A swine (n = 3) model with AO was used to identify confocal features of normal brain. Images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope. Histological samples were acquired through biopsies from matched imaging areas. Samples were visualized with a benchtop confocal microscope. Histopathological features in corresponding confocal images and photomicrographs of H &amp; E–stained tissues were reviewed.</jats:p></jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Fluorescence induced by FNa, 5-ALA, AO, AF, CV, and SR101 and detected with the confocal endomicroscope allowed interpretation of histological features. Confocal endomicroscopy revealed satellite tumor cells within peritumoral tissue, a definitive tumor border, and striking fluorescent cellular and subcellular structures. Fluorescence in various tumor regions correlated with standard histology and known tissue architecture. Characteristic features of different areas of normal brain were identified as well.</jats:p></jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Confocal endomicroscopy provided rapid histological information precisely related to the site of microscopic imaging with imaging characteristics of cells related to the unique labeling features of the fluorophores. Although experimental with further clinical trial validation required, these data suggest that intraoperative confocal imaging can help to distinguish normal brain from tumor and tumor margin and may have application in improving intraoperative decisions during resection of brain tumors.</jats:p></jats:sec>

Prospective evaluation of the utility of intraoperative confocal laser endomicroscopy in patients with brain neoplasms using fluorescein sodium: experience with 74 cases

“…5,[15][16][17][18] We have investigated the feasibility of CLE in a murine malignant glioma model to distinguish normal brain, microvasculature, and tumor margins. 4,8,9,14 Furthermore, we have demonstrated its potential application as an intraoperative tool in human brain tumor surgery. 3,13 In the current study, we sought to evaluate the utility, specificity, and sensitivity of CLE using fluorescein sodium (FNa) as a fluorophore to detect brain tumor tissue.…”

Fluorescence and Glioma Surgery

“…Because of its greater imaging depth, near infrared systems can be superior to visible wavelength fluorescent methods for confocal microscopic identification of tumor margins. On one hand, sodium fluorescein is impermeable to cells, provides a fluorescent background immediately, and its fluorescence diminishes as it is exposed to lasers and lights [3]. On the other hand, indocyanine green provides a greater ability to identify malignant margins than fluorescein, is less toxic than fluorescein, is permeable to cells, and continues to fluoresce after prolonged exposure to a near infrared laser, but takes roughly 15 minutes to reach optimal fluorescence [14].…”

“…In order to use indocyanine green as a fluorescing agent, neurosurgeons must use endoscopes, microscopes, or other imaging techniques that are tailored to view near-infrared fluorescence. Also, PpIX fluorescence from 5-ALA can be detected in individual cells using a confocal endomicroscope, which allow tumor margins to be identified more easily; however, photobleaching occurs quickly (36% with 25 minutes of excitement) [3]. Overall, using fluorescein sodium, especially in conjunction with endomicroscopes equipped with the appropriate filters, to guide glioma resection surgeries could offer a robust strategy to maximize the amount of tumor tissue removed; however, Phase III clinical trials must be conducted to appropriately evaluate the viability of using this technique.…”

“…One way to counteract brain shift is through intraoperative imaging, especially by utilizing MRI and ultrasound images. Unfortunately, MRI is expensive and time consuming relative to the surgery [2][3]. Utilizing ultrasound imaging during surgery is cheaper, but one must be exceptionally skilled in ultrasound technique to obtain robust images during surgery, and the instruments can interfere greatly with surgery [3].…”

“…Unfortunately, MRI is expensive and time consuming relative to the surgery [2][3]. Utilizing ultrasound imaging during surgery is cheaper, but one must be exceptionally skilled in ultrasound technique to obtain robust images during surgery, and the instruments can interfere greatly with surgery [3]. In order to aid neurosurgeons to delineate tumor margins in real-time surgery, fluorescenceguided surgery, especially by 5-aminolevulinic acid (5-ALA)-induced fluorescence, offers a robust solution to maximizing the efficacy of tumor resection surgery.…”