Characterization of an Orthotopic Rat Model of Glioblastoma Using Multiparametric Magnetic Resonance Imaging and Bioluminescence Imaging

Glioblastoma multiforme (GBM) is a lethal and incurable disease. The C6 rat model of GBM shares several similarities to human GBM and longitudinal non-invasive imaging may allow tumour features to be studied. In this thesis, a multimodality imaging framework, consisting of bioluminescence imaging (BLI) and multiparametric magnetic resonance imaging (mpMRI), was applied to the C6 rat model to characterize the growth of orthotopic tumours. BLI signal, a measure of cell viability, tended to increase and then decrease in the majority of animals, whereas tumour volume (from MRI) continually increased. Cellular viability and tumour volume did not correlate across all days, highlighting the value of using complimentary imaging modalities. Apparent diffusion coefficient maps and immunohistochemistry suggests decreases in BLI signal are in part due to decreased tumour cellularity (i.e. necrosis). This is the first use of BLI and mpMRI to characterize this model, and highlights the inter-subject variability in tumour growth

Investigating tumor perfusion, glycolysis and pH environments with multimodal in vivo imaging

Background: Cancer cells have a complex microenvironment that helps create optimal conditions for cancer proliferation. Nutrients such as glucose will pass through a tortuous and leaky vascular structure developed by cancer cells, and are subsequently distributed and transported inside the cancer cells to meet their metabolic demands. This leaky and poorly organized vasculature leads to a buildup in the interstitial fluid pressure surrounding the tumor, subsequently resulting in tumor hypoxia. Due to an overreliance on glycolysis, more acid and protons are produced by cancer cells, leading to a more acidic environment which, in combination with tumor hypoxia, often leads to a poor patient outcome. This thesis aims to explore the intrinsic relationship between tumor perfusion, glycolysis and its pH environment using a C6 rat model of glioma. Methods: All subjects were implanted with 1 million C6 glioma cells using stereotactic surgery. The growth of tumor cells was monitored either with computed tomography perfusion (CTP) or magnetic resonance imaging (MRI). Once tumors had reached the optimal size, tumor perfusion was measured using CTP. Tumor glycolytic metabolism was measured using positron emission tomography (PET) with 18F-flurodeoxyglucose (FDG) and MR spectroscopy imaging (MRSI) using hyperpolarized [1-13C]pyruvate. Chemical exchange saturation (CEST) MRI was also used to investigate tumor glucose distribution (glucose contrast enhancement, or ∆CEST) and pH environments (intra-/extracellular pH, pHi and pHe respectively and simultaneously) during/after a glucose infusion/injection. All experimental procedures were completed within 24 hours. Measurements of tumor perfusion, glycolysis, ∆CEST and pH environments were correlated using Pearson’s correlation. Results: Tumor perfusion measurement of permeability surface-area product (PS) was significantly correlated with tumor glycolysis measurement of Lac:Pyr from hyperpolarized MRSI as well as ∆CEST. Tumor Lac:Pry was also significantly correlated with tumor pHi. Tumor metabolic rate of glucose derived from dynamic PET was significantly correlated with tumor pHi and pHe. Conclusion: This research showed the possibility of measuring the intracellular and extracellular pH environment simultaneously. Multimodal imaging approaches provided a more complete picture of the tumor microenvironment and helped elucidate the intrinsic relationship between tumor perfusion, glycolysis and the pH environment

Combined Effects of Focused Ultrasound and Photodynamic Treatment for Malignant Brain Tumors Using C6 Glioma Rat Model

Purpose: Glioblastoma (GBM) is an intractable disease for which various treatments have been attempted, but with little effect. This study aimed to measure the effect of photodynamic therapy (PDT) and sonodynamic therapy (SDT), which are currently be ing used to treat brain tumors, as well as sono-photodynamic therapy (SPDT), which is the combination of these two. Materials and Methods: Four groups of Sprague-Dawley rats were injected with C6 glioma cells in a cortical region and treated with PDT, SDT, and SPDT. Gd-MRI was monitored weekly and 18F-FDG-PET the day before and 1 week after the treatment. The acoustic power used during sonication was 5.5 W/cm2 using a 0.5-MHz single-element transducer. The 633-nm laser was illumi nated at 100 J/cm2. Oxidative stress and apoptosis markers were evaluated 3 days after treatment using immunohistochemistry (IHC): 4-HNE, 8-OhdG, and Caspase-3. Results: A decrease in tumor volume was observed in MRI imaging 12 days after the treatment in the PDT group (p<0.05), but the SDT group showed a slight increase compared to the 5-Ala group. The high expression rates of reactive oxygen species-related factors, such as 8-OhdG (p<0.001) and Caspase-3 (p<0.001), were observed in the SPDT group compared to other groups in IHC. Conclusion: Our findings show that light with sensitizers can inhibit GBM growth, but not ultrasound. Although SPDT did not show the combined effect in MRI, high oxidative stress was observed in IHC. Further studies are needed to investigate the safety parameters to apply ultrasound in GBM.ope

TSPO PET Detects Acute Neuroinflammation but not Diffuse Chronically Activated MHCII Microglia in the Rat

Abstract Background Accurate and sensitive imaging biomarkers are required to study the progression of white matter (WM) microglial activation in neurological diseases in vivo. The translocator protein (TSPO) is considered a sensitive target for imaging microglial activation with positron emission tomography (PET). This study aimed to test the ability of TSPO to detect WM microglial activation marked by major histocompatibility complex class II (MHCII) molecules in rat models of prodromal Alzheimer’s disease and acute subcortical stroke. Methods Fischer 344 wild-type (n = 12) and TgAPP21 (n = 11) rats were imaged with [18F]FEPPA PET and MRI to investigate TSPO tracer uptake in the corpus callosum. Wild-type rats subsequently received an endothelin-1 (ET1)-induced subcortical stroke and were imaged at days 7 and 28 post-stroke before immunohistochemistry of TSPO, GFAP for astrocytes, iNOS for microglia releasing toxic nitrous oxide, and the MHCII rat antigen, OX6. Results [18F]FEPPA-PET findings that TSPO expression was not increased in WM of TgAPP21 rats and was only increased in the infarct and proximal WM were confirmed by immunohistochemistry (infarct TSPO cells/mm2: day 7 = 555 ± 181; day 28 = 307 ± 153; proximal WMTSPO cells/mm2: day 7 = 113 ± 93; day 28 = 5 ± 7). TSPO and iNOS were not able to detect the chronic WM microglial activation that was detected with MHCII in the contralateral corpus callosum (day 28 OX6 % area: saline = 0.62 ± 0.38; stroke = 4.30 ± 2.83; P = .029). Conclusion Within the regions and groups investigated, TSPO was only expressed in the stroke-induced insult and proximal tissue, and therefore was unable to detect remote and non-insult-related chronically activated microglia overexpressing MHCII in WM

Developing sodium MRI for longitudinal assessment of chemotherapy of glioma in a rat model of cancer

Glioblastoma (GBM) is an aggressive cancer which presents significant challenges for disease management and treatment. Tissue sodium concentration (TSC) is a sensitive indicator of disease and changes in TSC can be used as a potential biomarker for treatment response. A longitudinal orthotopic C6 GBM model was used to study changes in TSC due to cancer. Animals were imaged with contrast enhanced T1-weighted, T2-weighted and sodium MRI and were either un-treated or received temozolomide (TMZ) treatment. Accurate assessment of TSC was possible for tumour volumes greater than 150mm3 using 3mm isotropic imaging voxels. TSC increased with tumour growth for all animals. However, there were no differences in TSC changes between treated and non-treated animals. Ultimately TSC at endpoint was lower for treated animals, however this difference was not significant. This study demonstrates the feasibility of preclinical sodium MRI at 3T and highlights future potential challenges to overcome for this modality

Measuring inflammation in the entire myocardium in a canine model of myocardial infarction with hybrid PET/MRI

Background: After myocardial infarction (MI), fibrosis and an ongoing dysregulated inflammatory response are associated with adverse cardiac remodeling. Fluorodeoxyglucose (FDG) positron emission tomography (PET) is sensitive to inflammation provided suppression protocols are implemented to restrict the uptake of glucose in myocytes. Magnetic resonance imaging can be used to determine extracellular volume, a surrogate measure of fibrosis. In some cases, patients present with markedly reduced flow in the setting of a large infarct, i.e. microvascular obstruction, restricting the delivery of FDG and contrast agents. To overcome this problem, a constant infusion was explored as an alternative to the clinical standard bolus injection. This led to three objectives: a) comparison of the constant infusion to the bolus injection in healthy canines, b) investigation of the potential of the constant infusion to discriminate post-MI tissue types, and c) determination of the efficacy of the suppression protocol and its effect on extracellular volume. Methods: All imaging was done with a hybrid PET/MRI scanner. MRI images were used to determine the regions of interest: remote, injured and obstructed myocardium. PET images were used to determine inflammation. To compare the injection strategies, five healthy canines were examined with all three. Subsequently, eight animals were imaged at baseline and days 3, 7, 14, 21, and 42 post-MI using a 60-min infusion. Lastly, seven animals were imaged at baseline and day 5 post-MI using a 150-min infusion. Forty minutes into the infusion, suppression of glucose uptake in myocytes was started. Results: No significant differences in terms of glucose metabolism and extracellular volume were seen in healthy myocardium between the three injection strategies: bolus injection, constant infusion and bolus followed by constant infusion, showing that a strategy involving the constant infusion produced similar results as to those obtained with a bolus injection. Following MI, a significant increase in extracellular volume was seen in remote tissue on days 14 and 21, suggesting an inflammatory response. During the 150-min infusion, suppression of myocardial glucose uptake had the unexpected result of reducing FDG uptake in inflammatory cells within the infarcted area. Conclusion: This research showed the possibility of using a constant infusion of Gd-DTPA and FDG to investigate inflammation within the entire myocardium. The finding that suppression affected inflammatory cells highlights the need for tracers which do not rely on myocardial glucose suppression

Characterization of an Orthotopic Rat Model of Glioblastoma Using Multiparametric Magnetic Resonance Imaging and Bioluminescence Imaging

Glioblastoma multiforme (GBM) is the most common primary brain tumor, with most patients dying within 15–18 months of diagnosis despite aggressive therapy. Preclinical GBM models are valuable for exploring GBM progression and for evaluating new therapeutics or imaging approaches. The rat C6 glioma model shares similarities with human GBM, and application of noninvasive imaging enables better study of disease progression. Here, multiparametric magnetic resonance imaging (mpMRI) and bioluminescence imaging (BLI) were applied to characterize longitudinal development of orthotopic luciferase-expressing C6 tumors. Across all rats (n = 11), a large variability was seen for BLI signal, a relative measure of C6 cell viability, but in most individuals, BLI signal peaked at day 11 and decreased thereafter. T2 and contrast-enhanced T1 tumor volumes significantly increased over time (P &lt; .05), and volume measures did not correlate with BLI signal. After day 11, tumor regions of noncontrast enhancement appeared in postcontrast T1-weighted magnetic resonance imaging, and had significantly higher apparent diffusion coefficient values compared with contrast-enhanced regions (P &lt; .05). This suggests formation of ill-perfused, necrotic regions beyond day 11, which were apparent at end-point–matched tissue sections. Our study represents the first combined use of BLI and mpMRI to characterize the progression of disease in the orthotopic C6 rat model, and it highlights the variability in tumor growth, the complementary information from BLI and mpMRI, and the value of multimodality imaging to better characterize tumor development. Future application of these imaging tools will be useful for evaluation of treatment response, and should be pertinent for other preclinical models

Multimodality In Vivo Imaging of Perfusion and Glycolysis in a Rat Model of C6 Glioma

Purpose: Chemical exchange saturation transfer MRI using an infusion of glucose (glucoCEST) is sensitive to the distribution of glucose in vivo; however, whether glucoCEST is more related to perfusion or glycolysis is still debatable. We compared glucoCEST to computed tomography perfusion (CTP), [18F] fluorodeoxyglucose positron emission tomography (FDG-PET), and hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy imaging (MRSI) in a C6 rat model of glioma to determine if glucoCEST is more strongly correlated with measurements of perfusion or glycolysis. Methods: 106 C6 glioma cells were implanted in Wistar rat brains (n = 11). CTP (including blood volume, BV; blood flow, BF; and permeability surface area product, PS) and FDG-PET standardized uptake value (SUV) were acquired at 11 to 13 days post-surgery. GlucoCEST measurements (∆CEST) were acquired the following day on a 9.4 T MRI before and after an infusion of glucose solution. This was followed by MRSI on a 3.0 T MRI after the injection of hyperpolarized [1-13C] pyruvate to generate regional maps of the lactate:pyruvate ratio (Lac:Pyr). Pearson’s correlations between glucoCEST, CTP, FDG-PET, and Lac:Pyr ratio were evaluated. Results: Tumors had significantly higher SUV, BV, and PS than the contralateral brain. Tumor ∆CEST was most strongly correlated with CTP measurements of BV (ρ = 0.74, P = 0.01) and PS (ρ = 0.55, P = 0.04). No significant correlation was found between glycolysis measurements of SUV or Lac:Pyr with tumor ∆CEST. PS significantly correlated with SUV (ρ = 0.58, P = 0.005) and Lac:Pyr (ρ = 0.75, P = 0.005). BV significantly correlated with Lac:Pyr (ρ = 0.57, P = 0.02), and BF significantly correlated with SUV (ρ = 0.49, P = 0.02). Conclusion: This study determined that glucoCEST is more strongly correlated to measurements of perfusion than glycolysis. GlucoCEST measurements have additional confounds, such as sensitivity to changing pH, that merit additional investigation