Current Fluorescence-Guided Neurosurgery and Moving Forward

1 Current Fluorescence-Guided Neurosurgery and Moving Forward

Remi A. Kessler, Frank J. Yuk, and Constantinos G. Hadjipanayis

Abstract
This introductory chapter provides a current overview of fluorescence-guided neurosurgery and includes future directions. The concepts of fluorescence and fluorescence-guided surgery (FGS) are introduced. Currently used fluorescent contrast agents in patients are summarized, including 5-aminolevulinic acid (5-ALA), fluorescein, and indocyanine green. Excitation light sources are discussed for each fluorescent contrast agent. Targeted fluorophores under clinical development for FGS are also introduced. Future directions in fluorescence-guided neurosurgery including handheld devices to better detect tumor fluorescence, dual fluorophore imaging, metabolic imaging in combination with FGS, and detection of the tumor margin will be discussed.

Keywords: fluorescence, near-infrared imaging, fluorescence-guided neurosurgery, fluorescence-guided surgery, fluorophore, fluorescein sodium, 5-ALA, indocyanine green, operative microscope

1.1 Fluorescence-Guided Surgery

The ability to delineate abnormal from normal structures is the hallmark of any surgical subspecialty. Enhanced visualization provides greater delineation of tissues, and as a result, the surgeon can perform better and safer surgery. While stains and dyes have long been used to identify biological structures and processes in tissue samples, real-time intraoperative detection of structures has relied on tissue magnification and illumination.1 Fluorescence of abnormal tumor tissues or vascular blood flow has been introduced as a method to enhance visualization in the field of neurosurgery over the past two decades. Optical imaging of fluorescent contrast agents can permit sensitive and specific detection of tissues in the brain. The principle of fluorescence-guided surgery (FGS) relies on the use of optical imaging agents that are administered to patients prior to or during surgery that selectively accumulate in tumor tissues or vascular compartments. FGS provides real-time image guidance and improved intraoperative visualization of brain tumor tissue and vascular blood flow, independent of neuronavigation and brain shift.^2,3,4,5 In this introductory chapter, we will briefly discuss the currently used fluorescent agents in neurosurgery for neuro-oncology and cerebrovascular applications. We will also discuss the currently used operative microscopes that permit the visualization of fluorescence within the operative field and new technologies that will push FGS forward.

1.2 What Is Fluorescence?

Certain molecules have the capacity to absorb light energy, and this absorption causes an elevation to a higher energy state, termed the excited state. Once at this excited state, the energy absorbed decays over time, resulting in the emission of light energy known as fluorescence. A fluorophore is a molecule capable of fluorescence. In its low-energy ground state configuration, fluorophores do not fluoresce. When light from an external source illuminates the fluorophore, it can absorb the energy to reach an excited state; multiple excited states exist depending on the energy and wavelength of the light source. Since the fluorophore is unstable at this higher energy state, it will revert back to a lower energy state, and during this process emits light. This infinitesimally small amount of elapsed time is known as the excited lifetime. Each fluorescent agent has a maximal fluorescence, or a specific wavelength of light at which the largest number of fluorophores is excited ( Fig. 1.1). Nevertheless, there exists a spectrum of fluorescence excitation through which fluorophores can absorb and emit a wide range of wavelengths.⁶ Photobleaching is the process by which fluorescence intensity can decay over time with continuous excitation of the fluorophore.⁷

1.2.1 Near-Infrared Fluorescence

Optical imaging technologies that utilize fluorescence can be further specialized to absorb and emit light in an optical spectrum of near-infrared (NIR), consisting of a wavelength range of 700 to 900 nm.^8,9 This is particularly useful when maximizing depth of tissue penetration is important. Fluorescent agents (e.g., protoporphyrin IX [PpIX], the fluorescent intracellular metabolite of 5-aminolevulinic acid [5-ALA], and fluorescein sodium) with excitation wavelengths below 700 nm typically possess a penetration depth of less than 1 cm and produce low tissue absorption. The light absorption of hemoglobin, lipid, melanin, and other tissues can result in autofluorescence in the visible light spectrum range up to 700 nm. An increase in wavelength causes a decrease in light scattering and light absorption. In the NIR spectrum, the interference of biomolecules and solvents is negligible and light can penetrate deeper into tissues. This provides for the detection of imaging signals at a depth of several centimeters. Of particular importance, NIR signals with emission of 700 nm and above maintain low-fluorescence background from blood absorption and tissue scattering, which can allow for optical detection of the exposed tumor.^10,11 A currently used NIR agent in neurosurgery is indocyanine green (ICG).

1.3 Fluorescent Contrast Agents Currently Used in Patients

1.3.1 5-Aminolevulinic Acid

5-ALA (brand name Gliolan or Gleolan) is a natural metabolite produced in the hemoglobin metabolic pathway. It is an orally administered pro-agent that can rapidly penetrate the blood–brain barrier (BBB) and accumulate within brain tumors.⁴ After uptake by glioma cells, 5-ALA is metabolized intracellularly into PpIX, its fluorescent metabolite ( Fig. 1.2).¹² Due to elevated PpIX levels within brain tumor cells, malignant tumor tissue can be visualized by violet–red fluorescence at 635 nm (smaller secondary peak at 704 nm) following excitation with 405 nm wavelength blue light.^13,14 The majority of high-grade glioma tumors will reveal solid red fluorescence, while a pink fluorescence will be visible at the tumor margin in which cancer cells infiltrate normal brain tissue. Tumor tissue fluorescence can persist for greater than 8 hours after oral administration. The mechanism for the accumulation of 5-ALA and its metabolite, PpIX, within glioma cells involves reduced levels of the enzyme, ferrochelatase, and impaired cellular clearance by an adenosine triphosphate (ATP)-binding cassette subfamily B member 6 (ABCB6) transporter.¹⁵ Ferrochelatase is an enzyme that produces heme with the addition of iron (Fe) to PpIX. 5-ALA-induced fluorescence is also influenced by the vascularity of the tumor, BBB permeability, tumor cell proliferative activity, and cellular density.⁴ 5-ALA is the only optical imaging agent approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the real-time detection and visualization of malignant tissue during glioma surgery. 5-ALA is also the most extensively studied of all fluorescent agents available for FGS of central nervous system (CNS) tumors worldwide. Tissue fluorescence due to 5-ALA is correlated with high sensitivity, specificity, and positive predictive values (values over 90%) for the identification of malignant tumor tissue.^{16,17,18,19,20,21,22,23} In particular, 5-ALA allows for intraoperative recognition of anaplastic foci and histopathologic diagnosis of gliomas with nonsignificant MRI contrast enhancement.²⁴ 5-ALA is essentially nontoxic, and can be used in resections of newly diagnosed (Chapter 3) and recurrent high-grade gliomas (Chapter 4), diffuse infiltrating gliomas with anaplastic foci (Chapter 5), low-grade gliomas (Chapter 5), meningiomas (Chapter 6), brain metastases (Chapter 7), ependymomas (Chapter 8), and other tumor types including pediatric tumors (Chapter 9).^2,25 A landmark randomized, controlled trial proved that the use of 5-ALA FGS results in a more complete resection of high-grade gliomas and better progression-free survival (PFS) in patients.²⁶

Fig. 1.1 Fluorescence emission wavelengths of fluorophores used in neurosurgery (5-aminolevulinic acid, indocyanine green, and fluorescein sodium).

Fig. 1.2 Use of the operative microscope for excitation of the 5-aminolevulinic acid metabolite, PpIX, for intraoperative tumor fluorescence and real-time image-guided fluorescence-guided surgery.

1.3.2 Fluorescein Sodium

Fluorescein sodium is a small organic molecular salt that acts as a nonspecific extracellular fluorophore. It is currently FDA approved for retinal angiography and used off-label for brain tumor resections. Fluorescein is injected intravenously after intubation and prior to incision and relies on disruption of the BBB for accumulation in high-grade gliomas. Fluorescein sodium fluorescence of brain tumors appears as a yellow-green color under white light.14 Fluorescein’s peak excitation occurs at 480 nm (range 465–490 nm) with a fluorescence emission peak at 525 nm (range 500–530 nm).^14,27 Leakage and nonspecific tissue fluorescence can occur with fluorescein as the dura fluoresces in addition to the surrounding normal brain tissue if perturbed during surgery. The duration of fluorescein fluorescence is approximately 2 to 3 hours after systemic administration. Additionally, careful precautions should be taken when administering fluorescein as harmful side effects can result if injected too rapidly or in an excessive concentration.^28,29 Its utilization has primarily been described in high-grade gliomas (Chapters 10 and 11), but has been reported in other brain tumors (Chapter 10).^30,31,32,33

1.3.3 Indocyanine Green

ICG is a small, water-soluble organic molecule that acts as a nonspecific extracellular fluorophore. ICG is an NIR fluorophore, which was approved by the U.S. FDA in 1956 for diagnostic use in disorders of cardiovascular and liver function. Supplemental U. S. FDA approval for ophthalmic angiography was granted in 1975. After intravenous bolus injection, ICG is bound within 1 to 2 seconds, mainly to globulins (α1-lipoproteins and albumin), and remains intravascular with normal vascular permeability. ICG flow lasts only approximately 15 seconds. Disruption of the BBB can permit accumulation of ICG in brain tumors. ICG maximum absorption or excitation occurs at 805 nm (range 700–850 nm) and fluorescence at 835 nm (range 780–950 nm). The fluorescence is recorded by a nonintensified video camera. An optical filter blocks both ambient and excitation light so that only ICG-induced fluorescence is visualized. Thus, arterial, capillary, and venous angiographic images can be observed on the video screen in real time. ICG videoangiography has become a useful way to allow real-time assessment of intraoperative vascular anatomy and analysis of flow dynamics. ICG is primarily used for intraoperative videoangiography during cerebrovascular surgery, specifically during surgical treatment for aneurysm ligation (Chapter 18), arteriovenous malformation (AVM) (Chapter 19), and extracranial intracranial (EC-IC) bypass (Chapter 20).^34,35,36 Recently, ICG has been used to detect brain tumors based on disruption of the BBB. Coined “second window” ICG (SWIG) in order to distinguish it from traditional videoangiography procedures that visualize the molecule within minutes of injection, this method relies on visualization 24 hours following high-dose intravenous infusion (Chapter 12).³⁷

1.4 Excitation Light Source

Currently, the most commonly used excitation light source in neurosurgery procedures is the surgical operative microscope adapted with a filter set that permits visualization of fluorescence excitation in a specific wavelength range ( Fig. 1.3). The adaptation includes a standard white light emission source and a combination of excitation and observation low- and high-pass optical filters, with slightly overlapping transmission integrated into their optical configuration. The proposed filter set is commercially available. For PpIX fluorescence visualization, two xenon light sources are utilized with the operative microscope. Blue light (405 nm) is emitted from the microscope, which excites the PpIX, emitting fluorescence in the 480 to 730 nm range. Since blue light has a short wavelength, brain and tumor tissue depth penetration is limited. The red fluorescence emitted does not contain as much energy as the blue excitation light. Therefore, in order for the surgeon to visualize 100% of the red tumor fluorescence, a filter aimed at blocking 90% of the blue light must be applied. Only 90% of the blue light is blocked as the surgeon still requires some light to complete the resection. Fluorescein imaging is possible with an operative microscope that is equipped with dedicated filter sets for fluorescein imaging. The microscope offers blue-light excitation and 540 to 690 nm emission filters for visualizing fluorescein.