A practical method for multimodal registration and assessment of whole-brain disease burden using PET, MRI, and optical imaging.

Many neurological diseases present with substantial genetic and phenotypic heterogeneity, making assessment of these diseases challenging. This has led to ineffective treatments, significant morbidity, and high mortality rates for patients with neurological diseases, including brain cancers and neurodegenerative disorders. Improved understanding of this heterogeneity is necessary if more effective treatments are to be developed. We describe a new method to measure phenotypic heterogeneity across the whole rodent brain at multiple spatial scales. The method involves co-registration and localized comparison of in vivo radiologic images (e.g. MRI, PET) with ex vivo optical reporter images (e.g. labeled cells, molecular targets, microvasculature) of optically cleared tissue slices. Ex vivo fluorescent images of optically cleared pathology slices are acquired with a preclinical in vivo optical imaging system across the entire rodent brain in under five minutes, making this methodology practical and feasible for most preclinical imaging labs. The methodology is applied in various examples demonstrating how it might be used to cross-validate and compare in vivo radiologic imaging with ex vivo optical imaging techniques for assessing hypoxia, microvasculature, and tumor growth.

Optical Characterization of Neurosurgical Operating Microscopes: Quantitative Fluorescence and Assessment of PpIX Photobleaching.

Protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (5-ALA) is increasingly used as a fluorescent marker for fluorescence-guided resection of malignant gliomas. Understanding how the properties of the excitation light source and PpIX fluorescence interact with the surgical microscope is critical for effective use of the fluorescence-guided tumor resection technique. In this study, we performed a detailed assessment of the intensity of the emitted blue light and white light and the light beam profile of clinical grade operating microscopes used for PpIX visualization. These measurements revealed both recognized fluorescence photobleaching limitations and unrecognized limitations that may alter quantitative observations of PpIX fluorescence obtained with the operating microscope with potential impact on research and clinical uses. We also evaluated the optical properties of a photostable fluorescent standard with an excitation-emission profile similar to PpIX. In addition, we measured the time-dependent dynamics of 5-ALA-induced PpIX fluorescence in an animal glioma model. Finally, we developed a ratiometric method for quantification of the PpIX fluorescence that uses the photostable fluorescent standard to normalize PpIX fluorescence intensity. This method increases accuracy and allows reproducible and direct comparability of the measurements from multiple samples.

Assessment of tumor cells in a mouse model of diffuse infiltrative glioma by Raman spectroscopy.

Glioma of infiltrative nature is challenging for surgeons to achieve tumor-specific and maximal resection. Raman spectroscopy provides structural information on the targeted materials as vibrational shifts. We utilized Raman spectroscopy to distinguish invasive tumors from normal tissues. Spectra obtained from replication-competent avian sarcoma-(RCAS-) based infiltrative glioma cells and glioma tissues (resembling low-grade human glioma) were compared with those obtained from normal mouse astrocytes and normal tissues. In cell analysis, the spectra at 950-1000, 1030, 1050-1100, 1120-1130, 1120-1200, 1200-1300, 1300-1350, and 1450 cm(-1) were significantly higher in infiltrative glioma cells than in normal astrocytes. In brain tissue analysis, the spectra at 1030, 1050-1100, and 1200-1300 cm(-1) were significantly higher in infiltrative glioma tissues than in normal brain tissues. These spectra reflect the structures of proteins, lipids, and DNA content. The sensitivity and specificity to predict glioma cells by distinguishing normal cells were 98.3% and 75.0%, respectively. Principal component analysis elucidated the significance of spectral difference between tumor tissues and normal tissues. It is possible to distinguish invasive tumors from normal tissues by using Raman spectroscopy.

In vivo assessment of high-grade glioma biochemistry using microdialysis: a study of energy-related molecules, growth factors and cytokines.

Microdialysis enables measurement of the chemistry of the cerebral extracellular fluid. This study's objective was to utilise microdialysis to monitor levels of glucose, lactate, pyruvate, glutamate and glycerol in patients following surgery for intrinsic brain tumours, and to assess the concentration of growth factors, cytokines and other proteins involved in the pathogenesis of high-grade gliomas in vivo. Eight patients with suspected high-grade gliomas were studied. Seven of these underwent resection with one microdialysis catheter placed at the tumour resection margin and, in six of these seven cases, a second microdialysis catheter in macroscopically normal peritumour tissue. The remaining glioma patient had an image-guided biopsy with a single catheter inserted stereotactically at the tumour margin. Histology demonstrated WHO IV glioblastoma in five cases, WHO III anaplastic astrocytoma in two cases, and one cerebral lymphoma. In the high-grade gliomas (WHO IV and III), tumour margin microdialysates consistently showed significantly lower glucose, higher lactate/pyruvate (L/P) ratio, higher glutamate and higher glycerol, relative to peritumour microdialysates (P < 0.05). These results indicate that malignant glioma margin tissue is metabolically extremely active. There was great variability in the microdialysate concentrations of growth factors (TGFalpha, EGF, VEGF), cytokines (IL-1alpha, IL-1beta, IL-1ra, IL-6, IL-8), matrix metalloproteinases (MMP-2, MMP-9) and their endogenous inhibitors (TIMP-1, TIMP-2). Notably, microdialysates from the glioma resection margin demonstrated significantly higher IL-8 concentration and higher MMP-2/TIMP-1 ratio when compared to peritumour microdialysates (P < 0.05), suggesting an environment favouring invasion and angiogenesis at the tumour margin. Microdialysis is a promising technique to study in vivo glioma metabolism, and may assist in the development of new therapies.

Multiparametric 3T MR approach to the assessment of cerebral gliomas: tumor extent and malignancy.

INTRODUCTION: Contrast-enhanced MR imaging is the method of choice for routine assessment of brain tumors, but it has limited sensitivity and specificity. We verified if the addition of metabolic, diffusion and hemodynamic information improved the definition of glioma extent and grade. METHODS: Thirty-one patients with cerebral gliomas (21 high- and 10 low-grade) underwent conventional MR imaging, proton MR spectroscopic imaging ((1)H-MRSI), diffusion weighted imaging (DWI) and perfusion weighted imaging (PWI) at 3 Tesla, before undergoing surgery and histological confirmation. Normalized metabolite signals, including choline (Cho), N-acetylaspartate (NAA), creatine and lactate/lipids, were obtained by (1)H-MRSI; apparent diffusion coefficient (ADC) by DWI; and relative cerebral blood volume (rCBV) by PWI. RESULTS: Perienhancing areas with abnormal MR signal showed 3 multiparametric patterns: "tumor", with abnormal Cho/NAA ratio, lower ADC and higher rCBV; "edema", with normal Cho/NAA ratio, higher ADC and lower rCBV; and "tumor/edema", with abnormal Cho/NAA ratio and intermediate ADC and rCBV. Perienhancing areas with normal MR signal showed 2 multiparametric patterns: "infiltrated", with high Cho and/or abnormal Cho/NAA ratio; and "normal", with normal spectra. Stepwise discriminant analysis showed that the better classification accuracy of perienhancing areas was achieved when regarding all MR variables, while (1)H-MRSI variables and rCBV better differentiated high- from low-grade gliomas. CONCLUSION: Multiparametric MR assessment of gliomas, based on (1)H-MRSI, PWI and DWI, discriminates infiltrating tumor from surrounding vasogenic edema or normal tissues, and high- from low-grade gliomas. This approach may provide useful information for guiding stereotactic biopsies, surgical resection and radiation treatment.

The contribution of magnetic resonance spectroscopy and echoplanar perfusion-weighted MRI in the initial assessment of brain tumours.

Conventional Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) are the cornerstone in the initial evaluation of brain tumours. The purpose of this study is to evaluate the contribution of Magnetic Resonance Spectroscopy (MRS) and Perfusion-weighted MRI to distinguish malignant from benign tumours. We included 55 patients diagnosed with single brain tumour by CT and MRI, and final histopathological verification of the tumour type: 25 were low-grade gliomas, 8 anaplastic gliomas, 11 glioblastomas, and 11 solitary metastases. We carried out brain MRS and dynamic perfusion-weighted echoplanar MRI in all cases. Perfusion was assessed in the centre of the lesion and in the area of maximum contrast-enhancement. In MRS, we found significant differences in Choline/Creatine ratios in relation to the tumour type with the highest values in high-grade gliomas and metastases. A Ch/Cr ratio equal or higher than 1.78 predicted malignancy at 80% sensitivity and 73% specificity. We found no significant differences in the relative cerebral blood volume (rCBV) for every type of tumour. The mean rCBV was 1.24 for benign tumours and 1.5 for the malignant ones(1.24 for low-grade gliomas, 1.91 for anaplastic gliomas, 1.03 for glioblastomas, and 1.57 for metastases). We conclude that, individually considered, MRS is superior to Perfusion-weighted MRI in the initial assessment of brain tumours. Perfusion MRI has not demonstrated predictive power to distinguish malignant from benign tumours.

Assessment of malignancy in gliomas by 3T 1H MR spectroscopy .

The purpose of this study was to assess clinical 1H MR spectroscopy (MRS) as a noninvasive method for evaluating brain tumor malignancy at 3T high-field system. Using 3T MRI/MRS system, localized water-suppressed single-voxel technique in patients with brain tumor (i.e., gliomas) was employed to evaluate spectra with peaks of N-acetyl aspartate (NAA), choline-containing compounds (Cho), creatine/phosphocreatine (Cr) and lactate. On the basis of Cr, these peak areas were quantitated as a relative ratio. The variation of metabolite measurements of the designated region in 10 normal volunteers was less than 10%. Normal ranges of NAA/Cr and Cho/Cr ratios were 1.67+/-018 and 1.16+/-0.15, respectively. NAA/Cr ratio of gliomas was significantly lower than that of the normal tissues (P= .005), but Cho/Cr ratio of gliomas was significantly higher (P= .001). Cho/Cr ratio of high-grade gliomas was significantly higher than that of low-grade gliomas. The present study demonstrated that the neuronal degradation or loss was observed in all gliomas. Higher-grade glioma was correlated with higher Cho/Cr ratio, indicating a significant dependence of Cho levels on malignancy of gliomas. Our results suggest that clinical 1H MR spectroscopy could be useful to predict tumor malignancy.

Assessment of the peripheral benzodiazepine receptors in human gliomas by two methods.

This study was designed to evaluate the density of peripheral benzodiazepine receptor (PBR) sites as a function of tumor malignancy in human gliomas, and to compare the results obtained with autoradiographic and liquid scintillation measurements performed on the same tissue specimens. In vitro binding of [3H]PK-11195[1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoguinol ine carboxamide] to human gliomas in radioligand binding studies revealed a significantly higher level (about 3 fold) of PBR binding sites in both low grade and high grade gliomas as compared to normal cortex. The Bmax (mean +/- SD) of high and low grade gliomas, when entire tissue sections were measured by autoradiography, was 5.5 +/- 0.3 pmol/mg-tissue (n = 5) and 1.8 +/- 0.9 pmol/mg-tissue (n = 6), respectively, although it was evident that there was area of hot spots in the high grade tumors. This difference was significant (p < 0.05; two-tailed t-test). Similarly, the KD values (dissociation constant; nM) between the high (KD = 20.4 +/- 1.3 nM) and low (KD = 14.3 +/- 2.1 nM) grade gliomas were significantly different. A significant difference in binding site density (Bmax) between the two types of gliomas was also obtained in liquid scintillation measurements. The hot spot areas which showed the most intense binding of [3H]PK-11195 had KD of 24.5 +/- 1.0 nM and Bmax of 6.2 +/- 0.42 pmol/mg-tissue, values significantly higher (p < 0.05, two-tailed t-test) than those obtained when the entire tissue section was measured. The data on the Bmax/KD ratios presented here suggest that it might be possible to differentiate high from low grade gliomas in human by in vivo imaging with 11C-labelled PK-11195.

Methodologic aspects of nuclear DNA assessment of gliomas with astrocytic and/or oligodendrocytic differentiation. Correlation of image and flow cytometric studies on paraffin-embedded specimens.

A total of 239 samples from paraffin-embedded, formalin-fixed astrocytic and/or oligodendrocytic gliomas from 111 patients were deparaffinized and disaggregated for image cytometric (ICM) and flow cytometric (FCM) DNA assessments. Each measurement technique produced evaluable histograms in about 85% of the samples analyzed. In the 10% that could not be analyzed by FCM, the background counts were too high and the coefficients of variation were too broad for precise evaluation. The failures with ICM were due to a shortage of Feulgen-stained tumor cell nuclei after the deparaffinization and disaggregation procedures. The results obtained were identical in 77% of the samples evaluable by both methods and practically identical (i.e., euploid versus aneuploid) in an additional 18%. The reasons for completely divergent DNA ploidy patterns in 5% of the samples could not be clarified. About 80% of the histopathologically highly malignant gliomas were found to consist of neoplastic cells with an aneuploid or tetraploid nuclear DNA distribution pattern. The results show that cytometric DNA assessments can be reliably performed on paraffin-embedded specimens of gliomas with astrocytic and/or oligodendrocytic differentiation by means of FCM and ICM on deparaffinized and disaggregated specimens.