Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.     

Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin

Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500–750 mm³) and measurements of tumor pO₂ and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO₂ levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO₂<10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.     

Automated Tracking of Quantitative Assessments of Tumor Burden in Clinical Trials

There are two key challenges hindering effective use of quantitative assessment of imaging in cancer response assessment: 1) Radiologists usually describe the cancer lesions in imaging studies subjectively and sometimes ambiguously, and 2) it is difficult to repurpose imaging data, because lesion measurements are not recorded in a format that permits machine interpretation and interoperability. We have developed a freely available software platform on the basis of open standards, the electronic Physician Annotation Device (ePAD), to tackle these challenges in two ways. First, ePAD facilitates the radiologist in carrying out cancer lesion measurements as part of routine clinical trial image interpretation workflow. Second, ePAD records all image measurements and annotations in a data format that permits repurposing image data for analyses of alternative imaging biomarkers of treatment response. To determine the impact of ePAD on radiologist efficiency in quantitative assessment of imaging studies, a radiologist evaluated computed tomography (CT) imaging studies from 20 subjects having one baseline and three consecutive follow-up imaging studies with and without ePAD. The radiologist made measurements of target lesions in each imaging study using Response Evaluation Criteria in Solid Tumors 1.1 criteria, initially with the aid of ePAD, and then after a 30-day washout period, the exams were reread without ePAD. The mean total time required to review the images and summarize measurements of target lesions was 15% (P < .039) shorter using ePAD than without using this tool. In addition, it was possible to rapidly reanalyze the images to explore lesion cross-sectional area as an alternative imaging biomarker to linear measure.We conclude that ePAD appears promising to potentially improve reader efficiency for quantitative assessment of CT examinations, and it may enable discovery of future novel image-based biomarkers of cancer treatment response.     

Multispectral optoacoustic tomography of muscle perfusion and oxygenation under arterial and venous occlusion: A human pilot study

Perfusion and oxygenation are critical parameters of muscle metabolism in health and disease. They have been both the target of many studies, in particular using near‐infrared spectroscopy (NIRS). However, difficulties with quantifying NIRS signals have limited a wide dissemination of the method to the clinics. Our aim was to investigate whether clinical multispectral optoacoustic tomography (MSOT) could enable the label‐free imaging of muscle perfusion and oxygenation under clinically relevant challenges: the arterial and venous occlusion. We employed a hybrid clinical MSOT/ultrasound system equipped with a hand‐held scanning probe to visualize hemodynamic and oxygenation changes in skeletal muscle under arterial and venous occlusions. Four (N = 4) healthy volunteers were scanned over the forearm for both 3‐minute occlusion challenges. MSOT‐recorded pathophysiologically expected results during tests of disturbed blood flow with high resolution and without the need for contrast agents. During arterial occlusion, MSOT‐extracted Hb‐values showed an increase, while HbO₂‐ and total blood volume (TBV)‐values remained roughly steady, followed by a discrete increase during the hyperemic period after cuff deflation. During venous occlusion, results showed a clear increase in intramuscular HbO₂, Hb and TBV within the segmented muscle area. MSOT was found to be capable of label‐free non‐invasive imaging of muscle hemodynamics and oxygenation under arterial and venous occlusion. We introduce herein MSOT as a novel modality for the assessment of vascular disorders characterized by disturbed blood flow, such as acute limb ischemia and venous thrombosis.     

Advanced 3D-Sonographic Imaging as a Precise Technique to Evaluate Tumor Volume

Determination of tumor volume in subcutaneously inoculated xenograft models is a standard procedure for clinical and preclinical evaluation of tumor response to treatment. Practitioners frequently use a hands-on caliper method in conjunction with a simplified formula to assess tumor volume. Non-invasive and more precise techniques as investigation by MR or (μ)CT exist but come with various adverse effects in terms of radiation, complex setup or elevated cost of investigations. Therefore, we propose an advanced three-dimensional sonographic imaging technique to determine small tumor volumes in xenografts with high precision and minimized observer variability. We present a study on xenograft carcinoma tumors from which volumes and shapes were calculated with the standard caliper method as well as with a clinically available three-dimensional ultrasound scanner and subsequent processing software. Statistical analysis reveals the suitability of this non-invasive approach for the purpose of a quick and precise calculation of tumor volume in small rodents.     

Time-dependent spatial specificity of high-resolution fMRI: insights into mesoscopic neurovascular coupling

High-resolution functional magnetic resonance imaging (fMRI) is becoming increasingly popular because of the growing availability of ultra-high magnetic fields which are capable of improving sensitivity and spatial resolution. However, it is debatable whether increased spatial resolutions for haemodynamic-based techniques, like fMRI, can accurately detect the true location of neuronal activity. We have addressed this issue in functional columns and layers of animals with haemoglobin-based optical imaging and different fMRI contrasts, such as blood oxygenation level-dependent, cerebral blood flow and cerebral blood volume fMRI. In this review, we describe empirical evidence primarily from our own studies on how well these fMRI signals are spatially specific to the neuronally active site and discuss insights into neurovascular coupling at the mesoscale.This article is part of the theme issue ‘Key relationships between non-invasive functional neuroimaging and the underlying neuronal activity’.     

Clinical Report on the First Prototype of a Photoacoustic Tomography System with Dual Illumination for Breast Cancer Imaging

Photoacoustic tomography is a recently developed imaging modality that can provide high spatial-resolution images of hemoglobin distribution in tissues such as the breast. Because breast cancer is an angiogenesis-dependent type of malignancy, we evaluated the clinical acceptability of breast tissue images produced using our first prototype photoacoustic mammography (PAM) system in patients with known cancer. Post-excisionally, histological sections of the tumors were stained immunohistochemically (IHC) for CD31 (an endothelial marker) and carbonic anhydrase IX (CAIX) (a marker of hypoxia). Whole-slide scanning and image analyses were used to evaluate the tumor microvessel distribution pattern and to calculate the total vascular perimeter (TVP)/area for each lesion. In this clinical study, 42 lesions were primarily scanned using PAM preoperatively, three of which were reported to be benign and were excluded from statistical analysis. Images were produced for 29 out of 39 cancers (visibility rate = 74.4%) at the median depth of 26.5 (3.25–51.2) mm. Age, menopausal status, body mass index, history of neoadjuvant treatment, clinical stage and histological tumor angiogenesis markers did not seem to affect the visibility. The oxygen saturation level in all of the measured lesions was lower than in the subcutaneous counterpart vessels (Wilcoxon test, p value<0.001), as well as in the counterpart contralateral normal breast region of interest (ROI) (Wilcoxon test, p value = 0.001). Although the oxygen saturation level was not statistically significant between CAIX-positive vs. -negative cases, lesional TVP/area showed a positive correlation with the oxygen saturation level only in the group that had received therapy before PAM. In conclusion, the vascular and oxygenation data obtained by PAM have great potential for identifying functional features of breast tumors.     

Functional deficits of central sensory and motor pathways in patients with cervical spinal stenosis: a study of SEPs and EMG responses to non-invasive brain stimulation

The central conduction time of the descending and ascending fibers of the spinal cord were examined in patients with radiologically defined cervical spinal stenosis (antero-posterior diameter of the spinal canal less than 13 mm). Nineteen patients were examined, only 4 of whom showed clinical signs of spastic weakness or ataxia. The electromyographic response after non-invasive stimulation of the leg area of the motor cortex was delayed in13 of the 15 clinically unaffected patients. The central latency (N21-P39) of the somatosensory evoked response after stimulation of the tibial nerve (tibialis SEP) was increased in 12 of the 15 individuals. The 4 patients with clinical signs showed abnormal latencies with both methods.The use of both techniques for the examination of the function of the spinal cord revealed increased latencies in the central motor and/or sensory pathways in all patients. The technique of non-invasive stimulation of the corticospinal system therefore provides an additional tool to detect and quantity subclinical and clinically apparent lesions in patients with defined cervical spinal stenosis.     

Physiological basis of muscle functional MRI

Muscle functional magnetic resonance imaging (MRI) refers to changes in the contrast properties of certain MR images that occur in exercising muscles. In part, these changes result indirectly from increased rates of cellular energy metabolism, which alter the image contrast properties by increasing the water content and by decreasing the intracellular pH. Also, increases in blood oxygen extraction cause a rapidly evolving, small, and negative contribution to signal. Together, these changes produce a complex time course of contrast changes during exercise. Analysis of this time course may provide insight into the physiology of exercising muscles. These contrast changes also provide a non-invasive method for determining the spatial pattern of muscle activation.     

[F-18]-Fluoro-2-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose positron emission tomography as a tool for early detection of immunotherapy response in a murine B cell lymphoma model

[F-18]-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) is a non-invasive imaging technique which has recently been validated for the assessment of therapy response in patients with aggressive non-Hodgkin’s lymphoma. Our objective was to determine its value for the evaluation of immunotherapy efficacy in immunocompetent Balb/c mice injected with the A20 syngeneic B lymphoma cell line. The high level of in vitro FDG uptake by A20 cells validated the model for further imaging studies. When injected intravenously, the tumour developed as nodular lesions mostly in liver and spleen, thus mimicking the natural course of an aggressive human lymphoma. FDG-PET provided three-dimensionnal images of tumour extension including non-palpable lesions, in good correlation with ex vivo macroscopic examination. When mice were pre-immunized with an A20 cell lysate in adjuvant before tumour challenge, their significantly longer survival, compared to control mice, were associated with a lower incidence of lymphoma visualized by PET at different time points. Estimation of tumour growth and metabolism using the calculated tumour volumes and maximum standardized uptake values, respectively, also demonstrated delayed lymphoma development and lower activity in the vaccinated mice. Thus, FDG-PET is a sensitive tool relevant for early detection and follow-up of internal tumours, allowing discrimination between treated and non-treated small animal cohorts without invasive intervention. C. Chaise and E. Itti contributed equally to the work.     

Cerebral Hemodynamic Changes of Mild Traumatic Brain Injury at the Acute Stage

Mild traumatic brain injury (mTBI) is a significant public health care burden in the United States. However, we lack a detailed understanding of the pathophysiology following mTBI and its relation to symptoms and recovery. With advanced magnetic resonance imaging (MRI), we can investigate brain perfusion and oxygenation in regions known to be implicated in symptoms, including cortical gray matter and subcortical structures. In this study, we assessed 14 mTBI patients and 18 controls with susceptibility weighted imaging and mapping (SWIM) for blood oxygenation quantification. In addition to SWIM, 7 patients and 12 controls had cerebral perfusion measured with arterial spin labeling (ASL). We found increases in regional cerebral blood flow (CBF) in the left striatum, and in frontal and occipital lobes in patients as compared to controls (p = 0.01, 0.03, 0.03 respectively). We also found decreases in venous susceptibility, indicating increases in venous oxygenation, in the left thalamostriate vein and right basal vein of Rosenthal (p = 0.04 in both). mTBI patients had significantly lower delayed recall scores on the standardized assessment of concussion, but neither susceptibility nor CBF measures were found to correlate with symptoms as assessed by neuropsychological testing. The increased CBF combined with increased venous oxygenation suggests an increase in cerebral blood flow that exceeds the oxygen demand of the tissue, in contrast to the regional hypoxia seen in more severe TBI. This may represent a neuroprotective response following mTBI, which warrants further investigation.     

Imagerie moléculaire de la plaque d’athérome vulnérable. Évaluation préclinique et clinique de traceurs radioactifs

Atherosclerotic cardiovascular diseases (CVD) are the leading cause of mortality worldwide, accounting for greater than 19.10⁶ deaths annually. Despite major advances in the treatment of CVD, a high proportion of CVD victims die suddenly while being apparently healthy, the great majority of these accidents being due to the rupture or erosion of a vulnerable coronary atherosclerotic plaque. Indeed, an acute heart attack is the first symptom of atherosclerosis in as much as 50% of individuals with severe disease. A non-invasive imaging methodology allowing the early detection of vulnerable atherosclerosis in selected individuals prior to the occurrence of any symptom would therefore be of great public health benefit. Nuclear imaging could potentially allow the identification of vulnerable patients by non-invasive scintigraphic imaging following administration of a radiolabeled tracer. The development of radiolabeled probes that specifically bind to and allow the in vivo imaging of vulnerable atherosclerotic plaques is therefore the subject of intense ongoing experimental and clinical research. Radiotracers targeted at the inflammatory process seem particularly relevant and promising. Recently, macrophage targeting allowed the experimental in vivo detection of atherosclerosis using either SPECT or PET imaging. A few tracers have also been evaluated clinically. Targeting of apoptosis and macrophage metabolism both allowed the imaging of vulnerable atherosclerotic plaques in the carotid vessels of patients. However, nuclear imaging of vulnerable plaques at the level of the coronary arteries remains a challenging issue because of the small size of atherosclerotic lesions and of their vicinity with blood and the circulating tracer activity.     

Variability of Microcirculation Detected by Blood Pulsation Imaging

The non-invasive assessment of blood flow is invaluable for the diagnostic and monitoring treatment of numerous vascular and neurological diseases. We developed a non-invasive and non-contact method of blood pulsation imaging capable of visualizing and monitoring of the two-dimensional distribution of two key parameters of peripheral blood flow: the blood pulsation amplitude and blood pulsation phase. The method is based on the photoplethysmographic imaging in the reflection mode. In contrast with previous imaging systems we use new algorithm for data processing which allows two dimensional mapping of blood pulsations in large object''s areas after every cardiac cycle. In our study we carried out the occlusion test of the arm and found (i) the extensive variability of 2D-distribution of blood pulsation amplitude from one cardiac cycle to another, and (ii) existence of the adjacent spots to which the blood is asynchronously supplied. These observations show that the method can be used for studying of the multicomponent regulation of peripheral blood circulation. The proposed technique is technologically simple and cost-effective, which makes it applicable for monitoring the peripheral microcirculation in clinical settings for example, in diagnostics or testing the efficiency of new medicines.     

In vivo diagnostic imaging using micro-CT: sequential and comparative evaluation of rodent models for hepatic/brain ischemia and stroke

Background

There is an increasing need for animal disease models for pathophysiological research and efficient drug screening. However, one of the technical barriers to the effective use of the models is the difficulty of non-invasive and sequential monitoring of the same animals. Micro-CT is a powerful tool for serial diagnostic imaging of animal models. However, soft tissue contrast resolution, particularly in the brain, is insufficient for detailed analysis, unlike the current applications of CT in the clinical arena. We address the soft tissue contrast resolution issue in this report.

Methodology

We performed contrast-enhanced CT (CECT) on mouse models of experimental cerebral infarction and hepatic ischemia. Pathological changes in each lesion were quantified for two weeks by measuring the lesion volume or the ratio of high attenuation area (%HAA), indicative of increased vascular permeability. We also compared brain images of stroke rats and ischemic mice acquired with micro-CT to those acquired with 11.7-T micro-MRI. Histopathological analysis was performed to confirm the diagnosis by CECT.

Principal Findings

In the models of cerebral infarction, vascular permeability was increased from three days through one week after surgical initiation, which was also confirmed by Evans blue dye leakage. Measurement of volume and %HAA of the liver lesions demonstrated differences in the recovery process between mice with distinct genetic backgrounds. Comparison of CT and MR images acquired from the same stroke rats or ischemic mice indicated that accuracy of volumetric measurement, as well as spatial and contrast resolutions of CT images, was comparable to that obtained with MRI. The imaging results were also consistent with the histological data.

Conclusions

This study demonstrates that the CECT scanning method is useful in rodents for both quantitative and qualitative evaluations of pathologic lesions in tissues/organs including the brain, and is also suitable for longitudinal observation of the same animals.     

A Two-Stage Model for In Vivo Assessment of Brain Tumor Perfusion and Abnormal Vascular Structure Using Arterial Spin Labeling

The ability to assess brain tumor perfusion and abnormalities in the vascular structure in vivo could provide significant benefits in terms of lesion diagnosis and assessment of treatment response. Arterial spin labeling (ASL) has emerged as an increasingly viable methodology for non-invasive assessment of perfusion. Although kinetic models have been developed to describe perfusion in healthy tissue, the dynamic behaviour of the ASL signal in the brain tumor environment has not been extensively studied. We show here that dynamic ASL data acquired in brain tumors displays an increased level of ‘biphasic’ behaviour, compared to that seen in healthy tissue. A new two-stage model is presented which more accurately describes this behaviour, and provides measurements of perfusion, pre-capillary blood volume fraction and transit time, and capillary bolus arrival time. These biomarkers offer a novel contrast in the tumor and surrounding tissue, and provide a means for measuring tumor perfusion and vascular structural abnormalities in a fully non-invasive manner.     

Multi-Parametric MRI at 14T for Muscular Dystrophy Mice Treated with AAV Vector-Mediated Gene Therapy

The objective of this study was to investigate the efficacy of using quantitative magnetic resonance imaging (MRI) as a non-invasive tool for the monitoring of gene therapy for muscular dystrophy. The clinical investigations for this family of diseases often involve surgical biopsy which limits the amount of information that can be obtained due to the invasive nature of the procedure. Thus, other non-invasive tools may provide more opportunities for disease assessment and treatment responses. In order to explore this, dystrophic mdx^4cv mice were systemically treated with a recombinant adeno-associated viral (AAV) vector containing a codon-optimized micro-dystrophin gene. Multi-parametric MRI of T2, magnetization transfer, and diffusion effects alongside 3-D volume measurements were then utilized to monitor disease/treatment progression. Mice were imaged at 10 weeks of age for pre-treatment, then again post-treatment at 8, 16, and 24 week time points. The efficacy of treatment was assessed by physiological assays for improvements in function and quantification of expression. Tissues from the hindlimbs were collected for histological analysis after the final time point for comparison with MRI results. We found that introduction of the micro-dystrophin gene restored some aspects of normal muscle histology and pathology such as decreased necrosis and resistance to contraction-induced injury. T2 relaxation values showed percentage decreases across all muscle types measured (tibialis anterior, gastrocnemius, and soleus) when treated groups were compared to untreated groups. Additionally, the differences between groups were statistically significant for the tibialis anterior as well. The diffusion measurements showed a wider range of percentage changes and less statistical significance while the magnetization transfer effect measurements showed minimal change. MR images displayed hyper-intense regions of muscle that correlated with muscle pathology in histological sections. T2 relaxation, alongside diffusion and magnetization transfer effects provides useful data towards the goal of non-invasively monitoring the treatment of muscular dystrophy.     

FDG-PET imaging for radiotherapy target volume definition in lung cancer

Multimodality imaging plays a key role in the management of malignant tumours by radiation oncologists. The tumour extension at diagnosis identifies the patients in whom curative-intent radiotherapy is indicated. The target volumes and organs at risk are delineated on the relevant image modality (CT, MRI, PET), co-registered on a CT scan in treatment position for dose calculation purpose. The treatment machines are nowadays able to achieve heterogeneous dose distributions, possibly modulated within the target volume according to variations in the risk of failure. For instance, areas with high initial FDG uptake, low oxygenation, or insufficient response during radiotherapy could become the targets for selective dose increase. The scientific challenge includes registration and iterative segmentation of poorly contrasted images acquired on living bodies (variation in acquisition position, physiological movements and hollow organs filling, signal alteration induced by treatment…). The clinical validation of these innovative approaches and their diffusion in daily routine require a very close collaboration between many disciplines, based on a common language and understanding of radiotherapy target volumes, as illustrated in the present paper with FDG-PET imaging.     

Impact of Perfusion Map Analysis on Early Survival Prediction Accuracy in Glioma Patients

Studies investigating dynamic susceptibility contrast magnetic resonance imaging-determined relative cerebral blood volume (rCBV) maps as a metric of treatment response assessment have generated conflicting results. We evaluated the potential of various analytical techniques to predict survival of patients with glioma treated with chemoradiation. rCBV maps were acquired in patients with high-grade gliomas at 0, 1, and 3 weeks into chemoradiation therapy. Various analytical techniques were applied to the same cohort of serial rCBV data for early assessment of survival. Three different methodologies were investigated: 1) percentage change of whole tumor statistics (i.e., mean, median, and percentiles), 2) physiological segmentation (low rCBV, medium rCBV, or high rCBV), and 3) a voxel-based approach, parametric response mapping (PRM). All analyses were performed using the same tumor contours, which were determined using contrast-enhanced T1-weighted and fluid attenuated inversion recovery images. The predictive potential of each response metric was assessed at 1-year and overall survival. PRM was the only analytical approach found to generate a response metric significantly predictive of patient 1-year survival. Time of acquisition and contour volume were not found to alter the sensitivity of the PRM approach for predicting overall survival. We have demonstrated the importance of the analytical approach in early response assessment using serial rCBV maps. The PRM analysis shows promise as a unified early and robust imaging biomarker of treatment response in patients diagnosed with high-grade gliomas.     

Clinical significance of circulating tumor cells after chemotherapy in unresectable pancreatic ductal adenocarcinoma

Circulating tumor cells (CTCs) have emerged as liquid biopsy biomarker providing non-invasive assessment of cancer progression and biology. We investigated whether longitudinal analysis of CTCs could monitor disease progression, response to chemotherapy, and survival in patients with unresectable pancreatic ductal adenocarcinoma (PDAC). A total of 52 patients with PDAC were prospectively enrolled in this study. Peripheral blood samples were serially collected at the time of diagnosis and after chemotherapy with clinical assessments. CTCs were isolated through a centrifugal microfluidic disc, enumerated with immunostaining against Epithelial cell adhesion molecule (EpCAM), Cytokeratin (CK), Plectin-1 and CD45, and identified by an automated imaging system. One or more CTCs were detected in 84.62% patients with unresectable PDAC at the time of diagnosis. CTC numbers were not statistically different across tumor sizes, location and metastatic sites. The absolute number of CTCs after chemotherapy was inversely related to overall survival (OS), and the decreased number of CTCs after chemotherapy was significantly associated with longer OS in patients with PDAC. Identifying CTCs and monitoring CTC changes after chemotherapy could be a useful prognostic marker for survival in patients with unresectable PDACs.