scholarly journals Dual-labeling with 5–aminolevulinic acid and fluorescein for fluorescence-guided resection of high-grade gliomas: technical note

2018 ◽  
Vol 128 (2) ◽  
pp. 399-405 ◽  
Author(s):  
Eric Suero Molina ◽  
Johannes Wölfer ◽  
Christian Ewelt ◽  
André Ehrhardt ◽  
Benjamin Brokinkel ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Blue Light ◽  
Aminolevulinic Acid ◽  
Anesthesia Induction ◽  
Light Illumination ◽  
Normal Brain ◽  
High Grade ◽  
Cerebral Lesions ◽  
Tissue Fluorescence ◽  
Yellow 560

OBJECTIVEFluorescence guidance with 5–aminolevulinic acid (5-ALA) helps improve resections of malignant gliomas. However, one limitation is the low intensity of blue light for background illumination. Fluorescein has recently been reintroduced into neurosurgery, and novel microscope systems are available for visualizing this fluorochrome, which highlights all perfused tissues but has limited selectivity for tumor detection. Here, the authors investigate a combination of both fluorochromes: 5-ALA for distinguishing tumor and fluorescein for providing tissue fluorescence of adjacent brain tissue.METHODSThe authors evaluated 6 patients who harbored cerebral lesions suggestive of high-grade glioma. Patients received 5-ALA (20 mg/kg) orally 4 hours before induction of anesthesia. Low-dose fluorescein (3 mg/kg intravenous) was injected immediately after anesthesia induction. Pentero microscopes (equipped either with Yellow 560 or Blue 400 filters) were used to visualize fluorescence. To simultaneously visualize both fluorochromes, the Yellow 560 module was combined with external blue light illumination (D-light C System).RESULTSFluorescein-induced fluorescence created a useful background for protoporphyrin IX (PPIX) fluorescence, which appeared orange to red, surrounded by greenly fluorescent normal brain and edematous tissue. Green brain-tissue fluorescence was helpful in augmenting background. Levels of blue illumination that were too strong obscured PPIX fluorescence. Unspecific extravasation of fluorescein was noted at resection margins, which did not interfere with PPIX fluorescence detection.CONCLUSIONSDual labeling with both PPIX and fluorescein fluorescence is feasible and gives superior background information during fluorescence-guided resections. The authors believe that this technique carries potential as a next step in fluorescence-guided resections if it is completely integrated into the surgical microscope.

scholarly journals Multicellular ‘hotspots’ harbour high-grade potential in lower-grade gliomas

2021 ◽  
Author(s):  
Alastair J Kirby ◽  
José P Lavrador ◽  
Istvan Bodi ◽  
Francesco Vergani ◽  
Ranjeev Bhangoo ◽  
...  
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Aminolevulinic Acid ◽  
Ex Vivo ◽  
Brain Slices ◽  
Glioma Cells ◽  
Malignant Progression ◽  
Lower Grade ◽  
High Grade ◽  
Human Brain Tissue

Abstract Background Lower-grade gliomas may be indolent for many years before developing malignant behaviour. The reasons mechanisms underlying malignant progression remain unclear. Methods We collected blocks of live human brain tissue donated by people undergoing glioma resection. The tissue blocks extended through the peritumoral cortex and into the glioma. The living human brain tissue was cut into ex vivo brain slices and bathed in 5-aminolevulinic acid (5-ALA). High-grade glioma cells avidly take up 5-aminolevulinic acid (5-ALA) and accumulate high levels of the fluorescent metabolite, Protoporphyrin IX (PpIX). We exploited the PpIX fluorescence emitted by higher-grade glioma cells to investigate the earliest stages of malignant progression in lower-grade gliomas. Results We found sparsely-distributed ‘hot-spots’ of PpIX-positive cells in living lower-grade glioma tissue. Glioma cells and endothelial cells formed part of the PpIX hotspots. Glioma cells in PpIX hotspots were IDH1 mutant and expressed nestin suggesting they had acquired stem-like properties. Spatial analysis with 5-ALA conjugated quantum dots indicated that these glioma cells replicated adjacent to blood vessels. PpIX hotspots formed in the absence of angiogenesis. Conclusion Our data show that PpIX hotspots represent microdomains of cells with high-grade potential within lower-grade gliomas and identify locations where malignant progression could start.

scholarly journals 5-aminolevulinic acid-induced accumulation of protoporphyrin IX in rat brain tissue

2021 ◽  
Vol 67 (6) ◽  
pp. 849-854
Author(s):  
Arina Kokorina ◽  
Artem Rafaelyan ◽  
Ksenia Chemodakova ◽  
Natalia Pak ◽  
Viktor Aleksandrov ◽  
...  
Keyword(s):  
Rat Brain ◽  
Brain Tissue ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Laboratory Animals ◽  
Normal Brain ◽  
Rat Glioma ◽  
The Brain ◽  
Rat Brain Tissue ◽  
Intact Rats

The aim of the study was to compare the level of accumulation of protoporphyrin IX (ППIX) in the brain of rats in normal conditions and in experimental C6 glioma. Materials and methods. In an experiment on 15 rats, one group of animals (n=5) was intracranially implanted with rat glioma of the C6 line. 14 days after tumor implantation, the animals were injected into the lateral vein of the tail with a photosensitizer — a preparation of 5-aminolevulinic acid (5-ALA) Alasens at a dose of 100 mg / kg. Another group consisted of 5 intact rats, which were also injected with Alasens. The rats were euthanized 4–5 hours after the injection of the photosensitizer, and fluorescent metabolic navigation was performed with illumination of the brain with light with wavelengths of 417 and 435 nm. For objectification, fluorescence biospectroscopy was performed. Similar manipulations were performed with animals of another group (n=5) — intact rats that did not receive Alasens. Results. In contrast to humans, in rats, the 5-ALA metabolite — PPIX accumulates in healthy brain tissue, while the fluorescence intensity does not differ from that visualized in the tumor area. It was also noted that the light of the blue spectrum promotes weak fluorescence of the white matter of the rat brain in the absence of exogenous 5-ALA, which can potentially be explained by the activation of endogenous PPIX or other fluorophores. Conclusion. After the administration of Alasens (5-ALA preparation), the accumulation of PPIX by the rat brain tissue occurs not only by malignant cells, but also by normal brain tissue without signs of malignancy or other pathological changes. A more thorough study of this phenomenon is required, since significant differences in the metabolism of 5-ALA in humans and laboratory animals will call into question the correctness of translation of experimental results into clinical practice.

scholarly journals New Incoherent Autofluorescence/Fluorescence System for Early Detection of Lung Cancer

1999 ◽  
Vol 5 (2) ◽  
pp. 71-75 ◽  
Author(s):  
Martin Leonhard
Keyword(s):  
Lung Cancer ◽  
Early Detection ◽  
Blue Light ◽  
White Light ◽  
High Intensity ◽  
Aminolevulinic Acid ◽  
Broad Band ◽  
Light Illumination ◽  
Auto Fluorescence ◽  
White Light Illumination

A new autofluorescence (AF) system for bronchoscopy that operates as compact as a conventional white light bronchoscopy system is described. The system is also capable of white light illumination and excitation of aminolevulinic acid (ALA) induced fluorescence. Changing between white light and (auto-) fluorescence mode is easy and always possible. Broad band excitation with blue light (AF: 380–460 nm; ALA 380–440 nm) delivers high intensity illumination at the distal end of the bronchoscope (AF typically 50 mW). A special optical observation technique makes the AF directly visible to the eye instead of indirect techniques used in other AF systems. A compact (160 g)and sensitive (typically 0.2 lux) camera can be used for documentation.

scholarly journals Fluorescence-Guided High-Grade Glioma Surgery More Than Four Hours After 5-Aminolevulinic Acid Administration

2021 ◽  
Vol 12 ◽  
Author(s):  
Georgios A. Maragkos ◽  
Alexander J. Schüpper ◽  
Nikita Lakomkin ◽  
Panagiotis Sideras ◽  
Gabrielle Price ◽  
...  
Keyword(s):  
Aminolevulinic Acid ◽  
Peak Plasma ◽  
Progression Free Survival ◽  
High Grade Glioma ◽  
Clinical Results ◽  
Anesthesia Induction ◽  
High Grade ◽  
Karnofsky Score ◽  
Glioma Surgery ◽  
Intraoperative Fluorescence

Background: Fluorescence-guided surgery (FGS) using 5-aminolevulic acid (5-ALA) is a widely used strategy for delineating tumor tissue from surrounding brain intraoperatively during high-grade glioma (HGG) resection. 5-ALA reaches peak plasma levels ~4 h after oral administration and is currently approved by the FDA for use 2–4 h prior to induction to anesthesia.Objective: To demonstrate that there is adequate intraoperative fluorescence in cases undergoing surgery more than 4 h after 5-ALA administration and compare survival and radiological recurrence to previous data.Methods: Retrospective analysis of HGG patients undergoing FGS more than 4 h after 5-ALA administration was performed at two institutions. Clinical, operative, and radiographic pre- and post-operative characteristics are presented.Results: Sixteen patients were identified, 6 of them female (37.5%), with mean (SD) age of 59.3 ± 11.5 years. Preoperative mean modified Rankin score (mRS) was 2 ± 1. All patients were dosed with 20 mg/kg 5-ALA the morning of surgery. Mean time to anesthesia induction was 425 ± 334 min. All cases had adequate intraoperative fluorescence. Eloquent cortex was involved in 12 cases (75%), and 13 cases (81.3%) had residual contrast enhancement on postoperative MRI. Mean progression-free survival was 5 ± 3 months. In the study period, 6 patients died (37.5%), mean mRS was 2.3 ± 1.3, Karnofsky score 71.9 ± 22.1, and NIHSS 3.9 ± 2.4.Conclusion: Here we demonstrate that 5-ALA-guided HGG resection can be performed safely more than 4 h after administration, with clinical results largely similar to previous reports. Relaxation of timing restrictions could improve procedure workflow in busy neurosurgical centers, without additional risk to patients.

scholarly journals Seeing the light: Improving the usability of fluorescence-guided surgery by adding an optimized secondary light source

2020 ◽  
Author(s):  
Jonathan T. Elliott ◽  
Dennis J. Wirth ◽  
Scott C. Davis ◽  
Jonathan D. Olson ◽  
Nathan E. Simmons ◽  
...  
Keyword(s):  
Blue Light ◽  
Aminolevulinic Acid ◽  
Color Contrast ◽  
Light Illumination ◽  
Background Color ◽  
Bright Pink ◽  
Color Rendering Index ◽  
Light Mode ◽  
Opposing Effects ◽  
Color Rendering

AbstractBackgroundTumors that take up and metabolize 5-aminolevulinic acid (5-AlA) emit bright pink fluorescence when illuminated with blue light, aiding surgeons in identifying the margin of resection. The adoption of this method is hindered by the blue light illumination, which is too dim to safely operate under, and therefore, necessitates switching back and forth from white-light mode. This paper examines the addition of an optimized secondary illuminant adapter (SIA) to improve usability of blue-light mode without degrading tumor contrast.MethodsWe used color science methods to evaluate the color of the secondary illuminant and its impact on color rendering index (CRI) as well as the tumor-to-background color contrast (TBCC). A secondary illuminant adapter was built to provide 475-600 nm light the intensity of which can be controlled by the surgeon and was evaluated in two patients.ResultsSecondary illuminant color had opposing effects on color rendering index (CRI) and tumor to background color contrast (TBCC); providing surgeon control of intensity allows this trade-off to be balanced in real-time. Experience in two cases suggests additional visibility adds value.ConclusionThe addition of a secondary illuminant may mitigate surgeon complaints that the operative field is too dark under the blue light illumination required for 5-ALA fluorescence guidance by providing improved CRI without completely sacrificing TBCC.

P16.11 Vitamin C levels and the hypoxic pathway in human glioma tissues

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii58
Author(s):  
E R Burgess ◽  
R L I Crake ◽  
E Phillips ◽  
H R Morrin ◽  
J A Royds ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Tissue Bank ◽  
The Body ◽  
Low Grade ◽  
Normal Brain ◽  
High Grade ◽  
Human Glioma ◽  
Cancer Society ◽  
Ca Ix ◽  
Hif Pathway

Abstract BACKGROUND Gliomas are the most common brain cancer and survival is poor, with 11–15 months for high-grade glioblastoma patients, despite treatment. Gliomas are hypoxic tumours, which increases with tumour grade. Under hypoxia, the transcription factor hypoxia inducible factor-1 (HIF) accumulates and upregulates expression of genes involved in tumour development and progression. HIF-1 levels and activity are controlled by HIF hydroxylases which target HIF-1α for degradation and prevent co-activation. HIF hydroxylases are part of the 2-oxoglutarate (2-OG)-dependent dioxygenase enzyme family, that require 2-OG and oxygen as substrates and ascorbate and iron as co-factors. The role of ascorbate in regulating the hypoxic pathway in cancer is of interest, with previous research showing reduced HIF pathway activity with increasing tumour ascorbate levels. Brain tissue has one of the highest ascorbate levels in the body, and is one of the last to become depleted under deficiency, indicating an important role for ascorbate in this tissue. One previous study has analysed ascorbate levels in 11 human glioblastoma patients, and showed lower ascorbate in tumour tissue compared to normal brain tissue. There have been no studies investigating the relationship between ascorbate levels and the hypoxic pathway in human glioma tissues. MATERIAL AND METHODS Human glioma tissues (n = 39), obtained from the Cancer Society Tissue Bank Christchurch (ethics approval H19/163), were processed for ascorbate and hypoxic pathway proteins (HIF-1α, CA-IX, BNIP3, HKII, GLUT1 and VEGF). Ascorbate levels were quantified by HPLC-ED, and proteins were measured by Western blotting and ELISA. Spearman’s correlations were used to identify relationships between ascorbate and HIF pathway proteins. RESULTS Of the samples, 64% were GBM. Ascorbate was significantly lower in GBM compared to low-grade gliomas (p = 0.04). VEGF was significantly higher in GBM compared to astrocytomas (p = 0.01). Increased tumour ascorbate was associated with lower VEGF and CA-IX proteins. HIF-1α and BNIP3 protein were positively associated, and VEGF was positively associated with HKII and CA-IX. VEGF inversely associated with BNIP3, and CA-IX inversely associated with HKII. The hypoxic pathway score (calculated from protein levels of members of the hypoxic pathway) was reduced in tumours with higher ascorbate but this did not reach significance (p = 0.2). CONCLUSION This is the first study to show that ascorbate levels were reduced in high-grade gliomas compared to low-grade. Some members of the hypoxic pathway were associated with ascorbate levels. The overall hypoxic pathway score did not significantly correlate with ascorbate and increased numbers of samples are required to confirm any associations. Other variables, such as IDH-1 mutation status of the tumours may affect the correlation and will be analysed next.

scholarly journals Fibrin As a Target for Glioblastoma Detection and Treatment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3630-3630 ◽  
Author(s):  
Lynn M Knowles ◽  
Carolin Wolter ◽  
Ralf Ketter ◽  
Steffi Urbschat ◽  
Stefan Linsler ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Brain Tissue ◽  
Ex Vivo ◽  
Normal Brain ◽  
High Grade ◽  
Green Fluorescence ◽  
Normal Brain Tissue ◽  
Integrin Β3 ◽  
3D Matrix

Glioblastoma is a highly aggressive brain tumor characterized by diffuse growth and resistance to therapy. Angiogenesis in glioblastoma is poorly organized and therefore, tends to be associated with tumor cell necrosis, hemorrhage and thrombosis. This leads to the formation of a fibrin-rich extracellular matrix, which could provide important adhesive cues for glioblastoma growth and proliferation. To establish that blood clotting takes place in the extracellular matrix of malignant brain tumors, we assessed fibrin formation in tumor samples from patients with astrocytoma and glioblastoma using immunohistochemistry. Compared to normal brain tissue, which is essentially fibrin-free, this analysis revealed a marked upregulation of clot formation in the interstitial spaces of patients with high-grade tumors (i.e. astrocytoma 3 and GBM). The low-grade astrocytoma 2, however, expressed 3-3.5 fold less fibrin than was found in tissues from patients with astrocytoma 3 and GBM indicating that the degree of clot formation positively correlates with tumor grade. Paralleling these data, we found that primary GBM cells, that were freshly isolated from patients after tumor surgery, infiltrated and proliferated strongly after embedding in a three dimensional (3D) matrix of clotted plasma ex vivo. Primary tumor cells from patients with astrocytoma 2 and 3, on the other hand, infiltrated clot but were unable to proliferate in 3D. GBM proliferation in 3D depended on fibrin, which mediated upregulation of the stem cell marker nestin, whereas culturing glioblastoma cells in a 3D matrix of matrigel™ failed to promote nestin expression as well as glioblastoma proliferation. Therefore, these data suggest that the presence of clotted plasma in the tumor extracellular matrix represents a niche for glioblastoma stem cells and, as such, contributes to GBM progression. To determine the interaction of GBM cells with fibrin on a molecular basis, we transfected GBM cells with siRNA against integrin β3, which completely abolished invadopodia formation and, at the same time, caused a sustained growth inhibition. GBM cell proliferation in 3D fibrin also depended on the formation of a fibronectin matrix as knockdown of fibronectin led to complete growth arrest. These findings appear to be clinically relevant since freshly isolated tumor cells from patients with glioblastoma colonized 3D fibrin most efficiently when they express fibronectin in combination with integrin β3. This suggests that fibrin stimulates adhesive interactions between integrin β3 and fibronectin and that these interactions in turn support glioblastoma stemness. To assess fibrin formation in glioblastoma in vivo, mice with orthotopic U87MG xenografts were injected intravenously with the fluorescein-coupled dekapeptide CGLKIQKNEC, which is a derivative of the clot-binding peptide CLT1. Using a fluorescence endoscope in situ, we detected strong green fluorescence over the parietal lobe of the right cerebral hemisphere, where tumor growth had been established by MRI beforehand. Subsequently, we confirmed tumor binding of the peptide in isolated brain tissue by fluorescence microscopy ex vivo, which demonstrated specific green fluorescence in the tumor xenograft while adjacent normal brain tissue as well as tissues from distant organs only exhibited background fluorescence. Together, our data demonstrate a specific upregulation of fibrin in high-grade astrocytoma, which promotes infiltration and proliferation of glioblastoma stem cells via integrin β3 and fibronectin. Moreover, we present a strategy to identify fibrin in the tumor extracellular matrix as a possible means to identify astrocytoma progression in vivo. Disclosures Eichler: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Pilch:CSL Behring: Other: Grants (investigator initiated), Speakers Bureau; ASPIRE Award/Pfizer: Other: Grants (investigator initiated); Bayer: Consultancy, Speakers Bureau; Roche: Consultancy.

scholarly journals Red-light excitation of protoporphyrin IX fluorescence for subsurface tumor detection

2018 ◽  
Vol 128 (6) ◽  
pp. 1690-1697 ◽  
Author(s):  
David W. Roberts ◽  
Jonathan D. Olson ◽  
Linton T. Evans ◽  
Kolbein K. Kolste ◽  
Stephen C. Kanick ◽  
...  
Keyword(s):  
Blue Light ◽  
Aminolevulinic Acid ◽  
Red Light ◽  
Protoporphyrin Ix ◽  
Fluorescence Emission ◽  
Low Grade ◽  
Light Illumination ◽  
Wide Field ◽  
Light Excitation ◽  
Surgical Field

OBJECTIVEThe objective of this study was to detect 5-aminolevulinic acid (ALA)-induced tumor fluorescence from glioma below the surface of the surgical field by using red-light illumination.METHODSTo overcome the shallow tissue penetration of blue light, which maximally excites the ALA-induced fluorophore protoporphyrin IX (PpIX) but is also strongly absorbed by hemoglobin and oxyhemoglobin, a system was developed to illuminate the surgical field with red light (620–640 nm) matching a secondary, smaller absorption peak of PpIX and detecting the fluorescence emission through a 650-nm longpass filter. This wide-field spectroscopic imaging system was used in conjunction with conventional blue-light fluorescence for comparison in 29 patients undergoing craniotomy for resection of high-grade glioma, low-grade glioma, meningioma, or metastasis.RESULTSAlthough, as expected, red-light excitation is less sensitive to PpIX in exposed tumor, it did reveal tumor at a depth up to 5 mm below the resection bed in 22 of 24 patients who also exhibited PpIX fluorescence under blue-light excitation during the course of surgery.CONCLUSIONSRed-light excitation of tumor-associated PpIX fluorescence below the surface of the surgical field can be achieved intraoperatively and enables detection of subsurface tumor that is not visualized under conventional blue-light excitation.Clinical trial registration no.: NCT02191488 (clinicaltrials.gov)

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