Vincristine-Induced Peripheral Neuropathy: Assessing Preventable Strategies in Paediatric Acute Lymphoblastic Leukaemia

Background: Acute Lymphoblastic Leukaemia is the most common cancer experienced by children with overall survival rates now exceeding 90%. However, most children will experience vincristine-induced peripheral neuropathy (VIPN) during treatment resulting in sensory-motor abnormalities. To date, there are no approved preventative therapeutics or mitigation strategies for VIPN. This body of work set out to: (1) establish a high-throughput and high-content assay with the capacity to identify neuroprotective compounds, (2) test the feasibility of repurposing olesoxime as a neuroprotectant, and (3) compare traditional statistical methods with machine learning models to identify patients at risk of VIPN. Methods: (1) In vitro neuronal cultures were exposed to vincristine to recapitulate the VIPN phenotype and olesoxime assessed as a positive control. The neurotoxicity assay was miniaturised in 384-well microplates with automation steps to reduce manual handling. (2) Olesoxime and vincristine were applied to proliferating malignant cell lines to ensure the efficacy of vincristine was maintained. (3) Machine learning algorithms were developed using data from a local retrospective cohort to predict VIPN. Results: (1) Neurite length was reduced in a dose-responsive manner with vincristine. Assay miniaturisation and automation steps helped facilitate a high-throughput workflow. An optimised multiplexed dye solution enabled image acquisition and neurite quantification. Further, olesoxime was found to protect neurites and deemed suitable as a positive control (2) Cell viability assays confirmed olesoxime did not interfere with vincristine efficacy in leukemia cells. (3) Machine learning algorithms showed equivalency to traditional univariate analysis. The observation of severe class imbalance meant that patients who were least susceptible to VIPN could be identified. Conclusions: This body of work demonstrates the successful development of a neurotoxicity assay suitable for neuroprotectant drug discovery. Olesoxime was found suitable as a positive control in the assay. Further, viability studies indicated that vincristine retains it efficacy with olesoxime, opening the possibility of its use as an adjunctive therapy. Finally, this work developed machine learning models with the capacity to identify patients with VIPN-free survival. The utility of this model may mean that it can be used to stratify patients prospectively in the clinic based on favourable clinical features

Histone deacetylase inhibition-mediated cytotoxicity of oligodendrocytes.

Recent studies indicate post-translational deacetylation by members of the superfamily of histone deacetylase complexes (HDACs) is necessary for oligodendrocyte precursor cell (OPC) differentiation into mature oligodendrocytes (OLs). However, it remains unknown if transient HDAC inhibition may promote OPC survival as has been shown in other cell types and neurological disease models. Chapter 2 demonstrates the development of a novel primary culture of OPCs from mouse pup cortices enriching the O4+ OPC population using Magnetic Activated Cell Sorting (MACS) technology. Once plated, OPCs were glial fibrillary acidic protein- (GFAP-), A2B5+, NG2+, and O4+. When induced to differentiate, mouse OPCs became either more complex O4+ and O1 + oligodendrocytes (OLs) or GFAP+ astrocytes. Bi-potentiality is lost in co-culture, however, with rat embryonically derived dorsal root ganglia (DRG) as mature OLs aligned with DRG neurites. In Chapter 3, following OPC treatment with the FDA approved pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), approximately 65% reduction in cell survival relative to vehicle-treated OPCs was observed. SAHA-mediated death of OPCs induces apoptosis partly by caspase activation with improvement of OPC survival when SAHA is accompanied by treatment with the general caspase inhibitor q-vd-oph. SAHA treatment of CNP-EGFP transgenic mouse pups increased the number of caspase activated apoptotic cortical OPCs compared to vehicle treated pups (p \u3c 0.05). In Chapter 4, we extended this study to spinal cord-derived rat primary OPCs to determine putative mechanism(s). Rat OPCs were similarly susceptible to SAHA-mediated HDACi. SAHA treatment of rat OPCs increased phosphorylated histone H2A.X+ (yH2AX) indicating the presence of HDACimediated DNA double-strand breaks. SAHA treatment also increased phosphorylated p53 and cleaved caspase 3 levels suggesting HDACi P53 mediated, caspase-dependant rat OPC apoptosis. These results provide strong evidence of HDACi-mediated apoptosis of normal mouse pup and adult rat OPCs. SAHA is currently involved in more than 180 clinical trials, some of which include a pediatric patient population, and is in consideration for use in the treatment of psychiatric and neurodegenerative conditions. These results strongly suggest that such clinical use of SAHA may negatively impact OPC survival and potentially be detrimental to the myelinating brain and spinal cord

Neuroimaging - Clinical Applications

Modern neuroimaging tools allow unprecedented opportunities for understanding brain neuroanatomy and function in health and disease. Each available technique carries with it a particular balance of strengths and limitations, such that converging evidence based on multiple methods provides the most powerful approach for advancing our knowledge in the fields of clinical and cognitive neuroscience. The scope of this book is not to provide a comprehensive overview of methods and their clinical applications but to provide a "snapshot" of current approaches using well established and newly emerging techniques

A comparative study of the biological and molecular properties of mesenchymal stem cells isolated from bone marrow and the olfactory system

Neurodegenerative conditions such as Multiple Sclerosis (MS) and spinal cord injury (SCI) affect hundreds of thousands of people each year worldwide, and numerous cell transplant-based therapeutic strategies are being investigated to aid in the repair and regeneration of the central nervous system. Of particular interest are mesenchymal stem cells (MSCs), due to their differentiation potential, their immunomodulatory effects, and their ability to stimulate various biological properties due to the substantial variety of growth factors, chemokines, and other signalling molecules secreted by these cells. MSCs taken from the bone marrow (BM-MSCs) have demonstrated significant reparative potential in animal models of both MS and SCI. The question I address throughout this thesis however, is whether MSCs from another niche; the olfactory mucosa (OM-MSCs), are a preferable or at least alternative candidate for such therapies, compared to BM-MSCs, and if they are, why are they? Previous studies have shown that OM-MSCs can be purified and grown from human olfactory mucosa and when incubated with rat glial/neuronal co-cultures are capable of increasing axonal myelination, an effect not elicited by BM-MSCs. This potentially has great therapeutic benefit for a range of neurodegenerative conditions, as a significant part of the regenerative process involves replacing the protective myelin membrane which ensheaths axons. A comparative study of the two types of MSCs shows a number of similarities, including the expression of the same panel of MSC markers, a 64% homology in miRNA expression, an ability to differentiate towards bone and fat, and a propensity for bone formation when cultured on osteogenic nanotographies. This thesis also outlines a number of differences between each phenotype which suggest that OM-MSCs could even be a preferred alternative, especially in neuroregenerative therapies. OM-MSCs were shown to express significantly more Nestin than BM-MSCs, and to proliferate at a significantly higher rate, two observations which may be related. This increased proliferation would have enormous benefit for their use, as BM-MSCs are mitotically quite slow, and any MSC-based therapies would require very large numbers of cells. Twenty six different miRNA were shown to be differentially expressed between BM-MSCs and OM-MSCs. Three of these; miR-140-5p, miR-146a-5p, and miR-335-5p were linked to three important biological functions; myelination, cell survival, and cell proliferation respectively. These three biological functions, importantly, are ones which were observed as being behavioural differences between OM-MSCs and BM-MSCs. OM-MSCs were also shown to secrete significantly more of the pro-myelinating chemokine, CXCL12, which was confirmed as being regulated by the microRNA, miR-140-5p. This offered a potential mechanism for the pro-myelinating effect of OM-MSCs, and also opens up new research potential for investigating therapeutic targets to regulate myelination. The data presented in this thesis shows many similarities between BM-MSCs and OM-MSCs, but it also highlights some profound differences which suggest that either they originate from a different lineage entirely, or that the cellular niche that they reside in does indeed affect the differentiation and behaviour of mesenchymal stem cells

Case series of breast fillers and how things may go wrong: radiology point of view

INTRODUCTION: Breast augmentation is a procedure opted by women to overcome sagging breast due to breastfeeding or aging as well as small breast size. Recent years have shown the emergence of a variety of injectable materials on market as breast fillers. These injectable breast fillers have swiftly gained popularity among women, considering the minimal invasiveness of the procedure, nullifying the need for terrifying surgery. Little do they know that the procedure may pose detrimental complications, while visualization of breast parenchyma infiltrated by these fillers is also deemed substandard; posing diagnostic challenges. We present a case series of three patients with prior history of hyaluronic acid and collagen breast injections. REPORT: The first patient is a 37-year-old lady who presented to casualty with worsening shortness of breath, non-productive cough, central chest pain; associated with fever and chills for 2-weeks duration. The second patient is a 34-year-old lady who complained of cough, fever and haemoptysis; associated with shortness of breath for 1-week duration. CT in these cases revealed non thrombotic wedge-shaped peripheral air-space densities. The third patient is a 37‐year‐old female with right breast pain, swelling and redness for 2- weeks duration. Previous collagen breast injection performed 1 year ago had impeded sonographic visualization of the breast parenchyma. MRI breasts showed multiple non- enhancing round and oval shaped lesions exhibiting fat intensity. CONCLUSION: Radiologists should be familiar with the potential risks and hazards as well as limitations of imaging posed by breast fillers such that MRI is required as problem-solving tool

Characterization of alar ligament on 3.0T MRI: a cross-sectional study in IIUM Medical Centre, Kuantan

INTRODUCTION: The main purpose of the study is to compare the normal anatomy of alar ligament on MRI between male and female. The specific objectives are to assess the prevalence of alar ligament visualized on MRI, to describe its characteristics in term of its course, shape and signal homogeneity and to find differences in alar ligament signal intensity between male and female. This study also aims to determine the association between the heights of respondents with alar ligament signal intensity and dimensions. MATERIALS & METHODS: 50 healthy volunteers were studied on 3.0T MR scanner Siemens Magnetom Spectra using 2-mm proton density, T2 and fat-suppression sequences. Alar ligament is depicted in 3 planes and the visualization and variability of the ligament courses, shapes and signal intensity characteristics were determined. The alar ligament dimensions were also measured. RESULTS: Alar ligament was best depicted in coronal plane, followed by sagittal and axial planes. The orientations were laterally ascending in most of the subjects (60%), predominantly oval in shaped (54%) and 67% showed inhomogenous signal. No significant difference of alar ligament signal intensity between male and female respondents. No significant association was found between the heights of the respondents with alar ligament signal intensity and dimensions. CONCLUSION: Employing a 3.0T MR scanner, the alar ligament is best portrayed on coronal plane, followed by sagittal and axial planes. However, tremendous variability of alar ligament as depicted in our data shows that caution needs to be exercised when evaluating alar ligament, especially during circumstances of injury