Jaime J. Bravo

Neuron Morphology Influences Axon Initial Segment Plasticity

Visual Abstract In most vertebrate neurons, action potentials are initiated in the axon initial segment (AIS), a specialized region of the axon containing a high density of voltage-gated sodium and potassium channels. In most vertebrate neurons, action potentials are initiated in the axon initial segment (AIS), a specialized region of the axon containing a high density of voltage-gated sodium and potassium channels. It has recently been proposed that neurons use plasticity of AIS length and/or location to regulate their intrinsic excitability. Here we quantify the impact of neuron morphology on AIS plasticity using computational models of simplified and realistic somatodendritic morphologies. In small neurons (e.g., dentate granule neurons), excitability was highest when the AIS was of intermediate length and located adjacent to the soma. Conversely, neurons having larger dendritic trees (e.g., pyramidal neurons) were most excitable when the AIS was longer and/or located away from the soma. For any given somatodendritic morphology, increasing dendritic membrane capacitance and/or conductance favored a longer and more distally located AIS. Overall, changes to AIS length, with corresponding changes in total sodium conductance, were far more effective in regulating neuron excitability than were changes in AIS location, while dendritic capacitance had a larger impact on AIS performance than did dendritic conductance. The somatodendritic influence on AIS performance reflects modest soma-to-AIS voltage attenuation combined with neuron size-dependent changes in AIS input resistance, effective membrane time constant, and isolation from somatodendritic capacitance. We conclude that the impact of AIS plasticity on neuron excitability will depend largely on somatodendritic morphology, and that, in some neurons, a shorter or more distally located AIS may promote, rather than limit, action potential generation.

Sub-diffuse optical biomarkers characterize localized microstructure and function of cortex and malignant tumor.

This study uses a sub-diffusive light transport model to analyze fiber-optic measurements of reflectance spectra to recover endogenous tissue biomarkers and to correct raw fluorescence emissions for distortions from background optical properties. Measurements in tissue-simulating phantoms validated accurate recovery of the reduced scattering coefficient [(0.3-3.4  mm-1), error 10%], blood volume fraction [(1-3 vol%), error 7%], and a dimensionless metric of anisotropic scattering, γ, that is sensitive to submillimeter tissue ultrastructure [(1.29-2.06), error 11%]. In vivo sub-diffusive optical data acquired during clinical neurosurgeries characterize differences in microstructure (γ), perfusion (blood volume), and metabolism (PpIX fluorescence) between normal cortex and malignant tumor.

Hyperspectral data processing improves PpIX contrast during fluorescence guided surgery of human brain tumors

Fluorescence guided surgery (FGS) using aminolevulinic-acid (ALA) induced protoporphyrin IX (PpIX) provides intraoperative visual contrast between normal and malignant tissue during resection of high grade gliomas. However, maps of the PpIX biodistribution within the surgical field based on either visual perception or the raw fluorescence emissions can be masked by background signals or distorted by variations in tissue optical properties. This study evaluates the impact of algorithmic processing of hyperspectral imaging acquisitions on the sensitivity and contrast of PpIX maps. Measurements in tissue-simulating phantoms showed that (I) spectral fitting enhanced PpIX sensitivity compared with visible or integrated fluorescence, (II) confidence-filtering automatically determined the lower limit of detection based on the strength of the PpIX spectral signature in the collected emission spectrum (0.014–0.041 μg/ml in phantoms), and (III) optical-property corrected PpIX estimates were more highly correlated with independent probe measurements (r = 0.98) than with spectral fitting alone (r = 0.91) or integrated fluorescence (r = 0.82). Application to in vivo case examples from clinical neurosurgeries revealed changes to the localization and contrast of PpIX maps, making concentrations accessible that were not visually apparent. Adoption of these methods has the potential to maintain sensitive and accurate visualization of PpIX contrast over the course of surgery.

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

OBJECTIVE The 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. METHODS To 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. RESULTS Although, 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. CONCLUSIONS Red-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).

5-Aminolevulinic Acid-Induced Fluorescence in Focal Cortical Dysplasia: Report of 3 Cases

BACKGROUND Three patients enrolled in a clinical trial of 5-aminolevulinic-acid (5-ALA)-induced fluorescence-guidance, which has been demonstrated to facilitate intracranial tumor resection, were found on neuropathological examination to have focal cortical dysplasia (FCD). OBJECTIVE To evaluate in this case series visible fluorescence and quantitative levels of protoporphyrin IX (PpIX) during surgery and correlate these findings with preoperative magnetic resonance imaging (MRI) and histopathology. METHODS Patients were administered 5-ALA (20 mg/kg) approximately 3 h prior to surgery and underwent image-guided, microsurgical resection of their MRI- and electrophysiologically identified lesions. Intraoperative visible fluorescence was evaluated using an operating microscope adapted with a commercially available blue light module. Quantitative PpIX levels were assessed using a handheld fiber-optic probe and a wide-field imaging spectrometer. Sites of fluorescence measurements were co-registered with both preoperative MRI and histopathological analysis. RESULTS Three patients with a pathologically confirmed diagnosis of FCD (Types 1b, 2a, and 2b) underwent surgery. All patients demonstrated some degree of visible fluorescence (faint or moderate), and all patients had quantitatively elevated concentrations of PpIX. No evidence of neoplasia was identified on histopathology, and in 1 patient, the highest concentrations of PpIX were found at a tissue site with marked gliosis but no typical histological features of FCD. CONCLUSION FCD has been found to be associated with intraoperative 5-ALA-induced visible fluorescence and quantitatively confirmed elevated concentrations of the fluorophore PpIX in 3 patients. This finding suggests that there may be a role for fluorescence-guidance during surgical intervention for epilepsy-associated FCD.

Ongoing advances in quantitative PpIX fluorescence guided intracranial tumor resection(Conference Presentation)

Aminolevulinc-acid induced protoporphyrin IX (ALA-PpIX) is being investigated as a biomarker to guide neurosurgical resection of brain tumors. ALA-PpIX fluorescence can be observed visually in the surgical field; however, raw fluorescence emissions can be distorted by factors other than the fluorophore concentration. Specifically, fluorescence emissions are mixed with autofluorescence and attenuated by background absorption and scattering properties of the tissue. Recent work at Dartmouth has developed advanced fluorescence detection approaches that return quantitative assessments of PpIX concentration, which are independent of background optical properties. The quantitative fluorescence imaging (qFI) approach has increased sensitivity to residual disease within the resection cavity at the end of surgery that was not visible to the naked eye through the operating microscope. This presentation outlines clinical observations made during an ongoing investigation of ALA-PpIX based guidance of tumor resection. PpIX fluorescence measurements made in a wide-field hyperspectral imaging approach are co-registered with point-assessment using a fiber optic probe. Data show variations in the measured PpIX accumulation among different clinical tumor grades (i.e. high grade glioma, low grade glioma), types (i.e. primary tumors. metastases) and normal structures of interest (e.g. normal cortex, hippocampus). These results highlight the contrast enhancement and underscore the potential clinical benefit offered from quantitative measurements of PpIX concentration during resection of intracranial tumors.

Mathematical model to interpret localized reflectance spectra measured in the presence of a strong fluorescence marker

Abstract. Quantification of multiple fluorescence markers during neurosurgery has the potential to provide complementary contrast mechanisms between normal and malignant tissues, and one potential combination involves fluorescein sodium (FS) and aminolevulinic acid-induced protoporphyrin IX (PpIX). We focus on the interpretation of reflectance spectra containing contributions from elastically scattered (reflected) photons as well as fluorescence emissions from a strong fluorophore (i.e., FS). A model-based approach to extract μa and μs′ in the presence of FS emission is validated in optical phantoms constructed with Intralipid (1% to 2% lipid) and whole blood (1% to 3% volume fraction), over a wide range of FS concentrations (0 to 1000  μg/ml). The results show that modeling reflectance as a combination of elastically scattered light and attenuation-corrected FS-based emission yielded more accurate tissue parameter estimates when compared with a nonmodified reflectance model, with reduced maximum errors for blood volume (22% versus 90%), microvascular saturation (21% versus 100%), and μs′ (13% versus 207%). Additionally, quantitative PpIX fluorescence sampled in the same phantom as FS showed significant differences depending on the reflectance model used to estimate optical properties (i.e., maximum error 29% versus 86%). These data represent a first step toward using quantitative optical spectroscopy to guide surgeries through simultaneous assessment of FS and PpIX.

Quantitative Multi-plexed Fluorescence Spectroscopy in the Presence of a Strong Fluorescent Marker

Localized reflectance spectra containing fluorescence emissions from a strong marker (fluorescein) are accurately modeled. The model is used to estimate metrics of vascular physiology and correct PpIX for absorption and scattering-based distortions.

Method for accurate quantitation of background tissue optical properties in the presence of emission from a strong fluorescence marker

Quantification of targeted fluorescence markers during neurosurgery has the potential to improve and standardize surgical distinction between normal and cancerous tissues. However, quantitative analysis of marker fluorescence is complicated by tissue background absorption and scattering properties. Correction algorithms that transform raw fluorescence intensity into quantitative units, independent of absorption and scattering, require a paired measurement of localized white light reflectance to provide estimates of the optical properties. This study focuses on the unique problem of developing a spectral analysis algorithm to extract tissue absorption and scattering properties from white light spectra that contain contributions from both elastically scattered photons and fluorescence emission from a strong fluorophore (i.e. fluorescein). A fiber-optic reflectance device was used to perform measurements in a small set of optical phantoms, constructed with Intralipid (1% lipid), whole blood (1% volume fraction) and fluorescein (0.16-10 μg/mL). Results show that the novel spectral analysis algorithm yields accurate estimates of tissue parameters independent of fluorescein concentration, with relative errors of blood volume fraction, blood oxygenation fraction (BOF), and the reduced scattering coefficient (at 521 nm) of <7%, <1%, and <22%, respectively. These data represent a first step towards quantification of fluorescein in tissue in vivo.