Alia Al-Ebraheem

Efficient and Rapid Labeling of Transplanted Cell Populations With Superparamagnetic Iron Oxide Nanoparticles Using Cell Surface Chemical Biotinylation for In Vivo Monitoring by MRI

Determination of the dynamics of specific cell populations in vivo is essential for the development of cell-based therapies. For cell tracking by magnetic resonance imaging (MRI), cells need to internalize, or be surface labeled with a MRI contrast agent, such as superparamagnetic iron oxide nanoparticles (SPIOs): SPIOs give rise to signal loss by gradient-echo and T2-weighted MRI techniques. In this study, cancer cells were chemically tagged with biotin and then magnetically labeled with anti-biotin SPIOs. No significant detrimental effects on cell viability or death were observed following cell biotinylation. SPIO-labeled cells exhibited signal loss compared to non-SPIO-labeled cells by MRI in vitro. Consistent with the in vitro MRI data, signal attenuation was observed in vivo from SPIO-labeled cells injected into the muscle of the hind legs, or implanted subcutaneously into the flanks of mice, correlating with iron detection by histochemical and X-ray fluorescence (XRF) methods. To further validate this approach, human mesenchymal stem cells (hMSCs) were also employed. Chemical biotinylation and SPIO labeling of hMSCs were confirmed by fluorescence microscopy and flow cytometry. The procedure did not affect proliferation and multipotentiality, or lead to increased cell death. The SPIO-labeled hMSCs were shown to exhibit MRI signal reduction in vitro and was detectable in an in vivo model. In this study, we demonstrate a rapid, robust, and generic methodology that may be a useful and practical adjuvant to existing methods of cell labeling for in vivo monitoring by MRI. Further, we have shown the first application of XRF to provide iron maps to validate MRI data in SPIO-labeled cell tracking studies.

Characterization of the depth distribution of Ca, Fe and Zn in skin samples, using synchrotron micro-x-ray fluorescence (SμXRF) to help quantify in-vivo measurements of elements in the skin.

In vivo monitoring of trace and biometals in skin is normally quantified using phantoms that assume a constant elemental distribution within the skin. Layered calibration skin phantoms could potentially improve the reliability of in vivo calibration skin phantoms by better representing the actual in vivo distribution. This work investigates the micro-distribution of iron, calcium and zinc in prepared human skin samples taken from a number of locations on the body. Slices (orientation running from the skin surface into the dermis) were extracted from 18 formalin-fixed necropsy samples and scanned using the micro-XRF setup at the VESPERS beamline (Canadian Light Source). Elemental surface maps were produced using a 6×6 μm(2) beam in steps of 10 μm. Microscope images of histology slides were obtained for comparison. Statistically significant differences (p<0.01) were noted between the epidermal and dermal layers of skin for the elements examined (Ca, Fe and Zn), demonstrating the ability to clearly distinguish elemental content in each layer. Iron was consistently noted at the epidermal/dermal boundary. These results would indicate that when using phantoms to quantify elemental levels measured in the skin, note should be taken of the appropriate depth distribution.

Altered transition metal homeostasis in the cuprizone model of demyelination.

In the cuprizone model of demyelination, the neurotoxin cuprizone is fed to mice to induce a reproducible pattern of demyelination in the brain. Cuprizone is a copper chelator and it has been hypothesized that it induces a copper deficiency in the brain, which leads to demyelination. To test this hypothesis and investigate the possible role of other transition metals in the model, we fed C57Bl/6 mice a standard dose of cuprizone (0.2% dry chemical to dry food weight) for 6 weeks then measured levels of copper, manganese, iron, and zinc in regions of the brain and visceral organs. As expected, this treatment induced demyelination in the mice. We found, however, that while the treatment significantly reduced copper concentrations in the blood and liver in treated animals, there was no significant difference in concentrations in brain regions relative to control. Interestingly, cuprizone disrupted concentrations of the other transition metals in the visceral organs, with the most notable changes being decreased manganese and increased iron in the liver. In the brain, manganese concentrations were also significantly reduced in the cerebellum and striatum. These data suggest a possible role of manganese deficiency in the brain in the cuprizone model.

Differences in iron and manganese concentration may confound the measurement of myelin from R1 and R2 relaxation rates in studies of dysmyelination

A model of dysmyelination, the Long Evans Shaker (les) rat, was used to study the contribution of myelin to MR tissue properties in white matter. A large region of white matter was identified in the deep cerebellum and was used for measurements of the MR relaxation rate constants, R1 = 1/T1 and R2 = 1/T2, at 7 T. In this study, R1 of the les deep cerebellar white matter was found to be 0.55 ± 0.08 s –1 and R2 was found to be 15 ± 1 s–1, revealing significantly lower R1 and R2 in les white matter relative to wild‐type (wt: R1 = 0.69 ± 0.05 s–1 and R2 = 18 ± 1 s–1). These deviated from the expected ΔR1 and ΔR2 values, given a complete lack of myelin in the les white matter, derived from the literature using values of myelin relaxivity, and we suspect that metals could play a significant role. The absolute concentrations of the paramagnetic transition metals iron (Fe) and manganese (Mn) were measured by a micro‐synchrotron radiation X‐ray fluorescence (μSRXRF) technique, with significantly greater Fe and Mn in les white matter than in wt (in units of μg [metal]/g [wet weight tissue]: les: Fe concentration,19 ± 1; Mn concentration, 0.71 ± 0.04; wt: Fe concentration,10 ± 1; Mn concentration, 0.47 ± 0.04). These changes in Fe and Mn could explain the deviations in R1 and R2 from the expected values in white matter. Although it was found that the influence of myelin still dominates R1 and R2 in wt rats, there were non‐negligible changes in the contribution of the metals to relaxation. Although there are already problems with the estimation of myelin from R1 and R2 changes in disease models with pathology that also affects the relaxation rate constants, this study points to a specific pitfall in the estimation of changes in myelin in diseases or models with disrupted concentrations of paramagnetic transition metals. Copyright

Early regional cuprizone‐induced demyelination in a rat model revealed with MRI

The cuprizone model of demyelination is well established in the mouse as a tool for the study of the mechanisms of both demyelination and remyelination. It is often desirable, however, to have a larger model, such as the rat, especially for imaging‐based studies, yet initial work has failed to show demyelination in cuprizone‐fed rats. Several recent studies have demonstrated demyelination in the rat, but only in the corpus callosum. In this study, we acquired high‐resolution, three‐dimensional images of the whole brain every 2 weeks, using a T1‐weighted magnetization‐prepared rapid acquisition gradient echo imaging sequence, optimized for myelin contrast, in order to assess myelination across the entire rat brain over a period of 8 weeks on a 1% cuprizone diet. We observed a consistent pattern of demyelination, beginning in the cerebellum by 4 weeks and involving more rostral regions of the brain by 8 weeks on the cuprizone diet, with validation using Luxol fast blue histology. This imaging technique permits the effects of cuprizone‐induced demyelination to be followed longitudinally in a single animal, over the entire brain. In turn, this may facilitate the establishment of the cuprizone model of demyelination in the rat.

Emerging Patterns in the Distribution of Trace Elements in Ovarian, Invasive and In-Situ Breast Cancer

Breast cancer is the most common cancer and ovarian cancer is the 8th most common cancer affecting women worldwide. This study highlights the changes of trace element levels accompanied by the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast, using micro probe Synchrotron Radiation X-ray Fluorescence (μSRXRF). The average values for the increase in Ca, Fe and Zn in tumour regions with respect to surrounding regions for the DCIS samples were significantly higher compared to the increase in the IDC samples (P <0.01).This study was also carried out to find a connection between ovarian cancer and breast cancer with respect to the cellular distribution of Ca, Cu, Fe, and Zn. For IDC, DCIS and ovarian cases, the statistical analysis reveals a significant increase in the levels of Ca, Cu and Zn concentrations in cancer tissue when compared to the normal surrounding tissue. For Fe, the differences between tumour regions with respect to surrounding regions were found to be not significant in IDC and ovarian cases. In DCIS cases, the results reveal a significant increase in the levels of Fe in cancer tissue when compared to the surrounding normal breast tissue (P <0.01).

The Identification and Differentiation of Secondary Colorectal Cancer in Human Liver Tissue Using X-ray Fluorescence, Coherent Scatter Spectroscopy, and Multivariate Analysis

Secondary colorectal liver cancer is the most widespread malignancy in patients with colorectal cancer. The aim of this study is to identify and differentiate between normal liver tissue and malignant secondary colorectal liver cancer tissue using X-ray scattering and X-ray fluorescence spectroscopy to investigate the best combination of data that can be used to enable classification of these two tissue types. X-ray fluorescence (XRF) and coherent scatter data were collected for 24 normal and 24 tumor matched pair tissue samples. The levels of 12 elements (P, S, K, Ca, Cr, Fe, Cu, Zn, As, Se, Br, and Rb) were measured in all samples. When comparisons were made between normal and tumor tissues, statistically significant differences were determined for K (p = 0.046), Ca (p = 0.040), Cr (p = 0.011), Fe, Cu, Zn, Br, and Rb (p < 0.01). However, for P, S, As, and Se, no statistically significant differences were found (p > 0.05). For the coherent scatter spectra collected, three peaks due to adipose, fibrous content, and water content of tissue were observed. The amplitude, full width half-maximum, and area under both fibrous content and water content peaks were found to be significantly higher in secondary colorectal liver tumors compared with surrounding normal liver tissue (p < 0.05). However, no significant differences were found for the adipose peak parameters (p > 0.05). Soft independent modeling of class analogy was performed using the XRF, coherent scatter, and elemental ratio data separately, and the accuracy of the classification of 20 unknown samples was found to be 50, 30, and 80%, respectively. Further analysis has shown that using a combination of the XRF and coherent scatter data in a single combined model gave improved normal and tumor liver tissue classification, with an accuracy that was found to be 85%.

Time-resolved fluorescence (TRF) and diffuse reflectance spectroscopy (DRS) for margin analysis in breast cancer: TRF AND DRS FOR MARGIN ANALYSIS IN BREAST CANCER

One of the major problems in breast cancer surgery is defining surgical margins and establishing complete tumor excision within a single surgical procedure. The goal of this work is to establish instrumentation that can differentiate between tumor and normal breast tissue with the potential to be implemented in vivo during a surgical procedure.

Transition metal distribution in the brain and spinal cord of a dysmyelinated rodent model

Transition metal concentrations in the central nervous system (CNS) are altered in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and multiple sclerosis. A common symptom of these diseases is demyelination, which is the degradation of the myelin sheath that encapsulates the neurons in vertebrates. Transition metal concentrations were measured in Long Evans Shaker (LES) rodent model and compared to healthy age-matched controls to investigate the relationship between transition metals and myelination. Micro probe Synchrotron Radiation X-ray Fluorescence (µSRXRF) was used to measure concentrations of manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) in regions of grey matter and white matter in Shaker rodents and their age-matched Long Evans (LE) controls in the cerebellum and spinal cord. In the cerebellum, the concentrations of all elements were significantly increased in the white matter of the Shaker model, and decreased in the gray matter of the Shaker model in comparison to their age a...