SignificanceALA-PpIX and second-window indocyanine green (ICG) have been studied widely for guiding the resection of high-grade gliomas. These agents have different mechanisms of action and uptake characteristics, which can affect their performance as surgical guidance agents. Elucidating these differences in animal models that approach the size and anatomy of the human brain would help guide the use of these agents. Herein, we report on the use of a new pig glioma model and fluorescence cryotomography to evaluate the 3D distributions of both agents throughout the whole brain.AimWe aim to assess and compare the 3D spatial distributions of ALA-PpIX and second-window ICG in a glioma-bearing pig brain using fluorescence cryotomography.ApproachA glioma was induced in the brain of a transgenic Oncopig via adeno-associated virus delivery of Cre-recombinase plasmids. After tumor induction, the pro-drug 5-ALA and ICG were administered to the animal 3 and 24 h prior to brain harvest, respectively. The harvested brain was imaged using fluorescence cryotomography. The fluorescence distributions of both agents were evaluated in 3D in the whole brain using various spatial distribution and contrast performance metrics.ResultsSignificant differences in the spatial distributions of both agents were observed. Indocyanine green accumulated within the tumor core, whereas ALA-PpIX appeared more toward the tumor periphery. Both ALA-PpIX and second-window ICG provided elevated tumor-to-background contrast (13 and 23, respectively).ConclusionsThis study is the first to demonstrate the use of a new glioma model and large-specimen fluorescence cryotomography to evaluate and compare imaging agent distribution at high resolution in 3D.
Gross total resection of low grade gliomas is associated with prolonged time to malignant transformation and ultimately overall survival. Unfortunately, rates of gross total resection are reported to be as low as 20-40%. Current strategies in fluorescence guidance are lacking for low grade gliomas and represent an unmet need. We have developed a novel fluorescence exploiting the highly conserved IDH mutant protein that is present in >80% of these tumors. To date there are well described small molecule IDH mutant inhibitors in clinical trial. We describe the development of small molecule fluorescent inhibitors of the IDH mutant protein and their potential applications in surgery for LGG.
The delivery of genetic payloads to cells using genetic medicines is challenging to predict and the existing tools to assess delivery in animal models, although critical for discovering and advancing these drugs to the clinic, do not readily facilitate assessments of functional delivery throughout the entire body. To complement these existing techniques, our lab has used whole-body 3-D hyperspectral fluorescence cryo-imaging for the evaluation of functional delivery in whole animal specimens at high resolution. This instrument acquires hyperspectral fluorescence images of whole animal specimen while they are sectioned at micron-level resolution. In this study, mice were administered AAV9, a common adeno-associated viral delivery vehicle to express and image a fluorescent reporter. As the specimen is sectioned the acquisition can be paused to collect whole-body tissue samples which are then stained for immunofluorescence (IF) analysis. Herein, we describe a technique to reconstruct IF images into a single whole-body tissue specimen to be assessed alongside the co-registered cryo-images.
Pre-operative MRI with gadolinium-based contrast agents (Gd-MRI) is a central feature in surgical planning and intra-surgical navigation of glioma, yet brain movement during the surgical procedure can degrade the accuracy of these pre-operative images. Fluorescence guided neurosurgery is a technique which can complement MRI guidance by providing direct visualization of the tumor during surgery, and several agents either used routinely or under clinical development have shown effective tumor discrimination and impact on surgical outcomes. We have built a multi-spectral kinetic imaging system to acquire behavior of fluorophores overtime in animal models. Here, we exhibit this fluorescence kinetic imaging system and report its performance with tissue-simulating phantoms with multiple fluorophores. Also reported is our first experience with multiple fluorescent contrast agents in a novel oncopig model.
Fluorescence cryo-imaging is a high-resolution optical imaging technique that produces 3-D whole-body biodistributions of fluorescent molecules within an animal specimen. To accomplish this, animal specimens are administered a fluorescent molecule or reporter and are frozen to be autonomously sectioned and imaged at a temperature of -20°C or below. Thus, to apply this technique effectively, administered fluorescent molecules should be relatively invariant to low temperature conditions for cryo-imaging and ideally the fluorescence intensity should be stable and consistent in both physiological and cryo-imaging conditions. Herein, we assessed the mean fluorescence intensity of 11 fluorescent contrast agents as they are frozen in a tissue-simulating phantom experiment and show an example of a tested fluorescent contrast agent in a cryo-imaged whole pig brain. Most fluorescent contrast agents were stable within ~25% except for FITC and PEGylated FITC derivatives, which showed a dramatic decrease in fluorescence intensity when frozen.
Neurosurgical fluorescence guidance relies on contrast agents to identify tumor regions to aid in increasing the extent of resection. Existing contrast agents for this indication each have their own limitation: unpredictable contrast from tumor heterogeneity, significant extravasation into the background brain and long incubation times. An ideal contrast agent should have high and rapid contrast that persists well into the surgical procedure. By using a whole animal hyperspectral cryo-imaging system several CAs were screened for these favorable properties and compared to the gold standard of gadolinium enhanced MR. Herein, we briefly report on the leading candidate Rd-PEG1k, which shows high contrast within minutes of administration that persists for at least 90 minutes.
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