Pathological and Biomedical Characteristics of Spinal Cord Injury Determined Using Diffusion Tensor Imaging

Traumatic spinal cord injury: SCI) is the most devastating injury that often causes the victim permanent paralysis and undergo a lifetime of therapy and care. It is caused by a mechanical impact that ultimately causes pathophysiological consequences which at this moment in time are an unresolved scientific challenge of great social impact. Scientists have long used animal contusion models to study the pathophysiology of SCI in the discovery of progressive secondary tissue degeneration, demyelination, and apoptosis. More importantly, most therapies that have gone to human clinical trial were first validated in spinal cord contusion models. Magnetic resonance imaging: MRI) is the modality of choice to noninvasively detect the soft tissue injury, particularly suitable for assessing the tissue integrity in SCI. However, the convention MRI lacks capability of detecting and evaluating the injury severity acutely, probably resulting in lost opportunities of effective prognostication or treatment stratification for SCI patients. Diffusion Tensor Magnetic Resonance Imaging: DTMRI, DTI) is an emerging technique known to provide dynamic contrast reflecting the progression of the underlying pathology in CNS tissues. In this study, we hypothesized that axial: ||) and radial: λ^) diffusivity derived from DTI is sensitive to the pathological alteration in spinal cord white matter: WM) tract and could be used as potential biomarkers detecting and characterizing the axonal and myelin damage in SCI. A mouse model of contusion SCI was examined using DTI, behavioral assessment, and histology to test our hypothesis. Techniques employed including the simplification of diffusion weighting scheme, the implementation of diffusion weighted multiple spin-echo sequence, and verified for setting up the experimental protocol and data processing procedures. Secondly, the hypothesis was test on the projects comparing the change of these biomarkers on both the myelinated and dysmyelinated shiverer mice cooperating with histological analysis, and behavioral assessment. Finally, a finite element analysis: FEA) of contusion SCI was deployed to provide evidences of injury mechanics correlated with the injury patterns detected by diffusion MRI for a better characterized animal model of contusion SCI

D-Aspartate treatment stimulates differentiation of oligodendrocyte precursors, prevents demyelination and accelerates remyelination in the cuprizone mouse model of Multiple Sclerosis

Emerging evidence support a role for some D-Aminoacids as neurotrasmitters and neuromodulators, since they are found in mammalian tissues and also in the central nervous system (CNS) (Hashimoto and Oka, 1997). They play important roles in some physiological processes, including dendritic morphology, synaptic plasticity and cognition (Wolosker et al., 2008; Billard, 2012). Among D-Aminoacids, recent studies suggest that D-Aspartic acid (D-Asp), a newly discovered agonist for NMDA receptors, play a role in NMDA receptor-dependent processes such as synaptic plasticity and memory (Errico et al., 2015). The D-Asp was described in the multi-lamellar membrane that insulate axons (Fisher et al., 1986) and its effects on the hormone biosynthesis and release have been largely explored in the years (Gold and Voskuhl 2009; Nuñez et al., 2000; Cerget et al., 2006). The exact mechanism of myelination process is still unknown, but emerging studies demonstrated the importance of intracellular changes in [Ca2+]i levels during myelination and remyelination processes (Soliven et al., 2001). Indeed, differentiation of oligodendrocyte precursors cells (OPC) and remyelination are associated with NMDARs-dependent [Ca2+]i changes (Martinez-Lozada et al., 2014). A recent work performed by our research group demonstrated that [Ca2+]i signaling mediated by the Na+/Ca2+ exchanger NCX3 plays an important role during oligodendrocytes differentiation and myelin formation (Boscia et al., 2012; Casamassa et al., 2016). In the present study, we investigated the effects of D-Asp during the OPC differentiation and remyelination by using both in vitro and in vivo techniques. In vitro, we evaluated the effects of D-Asp exposure both in human oligodendrocyte MO3.13 cell line and rat primary OPC, exposed to different concentrations of D-Aspartic acid (10-100-200 µM). Quantitative RT-PCR analyses showed that 10-200 μM D-Asp exposure for 3 days, upregulated, in a concentration-dependent manner, both the myelin markers CNPase and MBP and NCX3 transcripts in human oligodendrocytes M03.13 progenitors. The transcripts increase were significantly prevented by the NMDA receptor antagonist 10 µM MK-801 and the two NCX3 blockers, 30nM YM-244769 and 100nM BED. In accordance, microfluorimetric studies demonstrated that 100μM D-Asp administration induced an initial calcium peak of intracellular Ca2+ concentration [Ca2+]i followed by an oscillatory [Ca2+]i pattern both in oligodendrocyte MO3.13 progenitors and rat primary OPC. The NMDA antagonist 10µM MK-801 completly suppressed [Ca2+]i oscillations but only partially affected the first [Ca2+]i peak. Similar effects were observed in presence of the two selective blockers for NCX3, 30nM YM-244769 and 100nM BED. In addition, electrophysiological recordings performed in oligodendrocytes M03.13 progenitors showed that the current elicited by 100 µM D-Asp stimulation were dependent by AMPA activation, since the AMPA receptor inhibitor 10μM DNQX significantly prevented D-Asp induced inward currents. Our in vitro results suggest that D-Asp stimulates oligodendrocyte development through a mechanism involving calcium signaling through the glutamate receptors AMPA and NMDA and the Na+/Ca2+exchanger NCX3. Next, we investigated the effects of D-Asp administration in an in vivo model of demyelination/remyelination, the cuprizone mouse model. D-Asp was given during cuprizone feeding (demyelination), or after cuprizone withdrawal (remyelination). In both conditions, D-Asp treatment improved motor coordination performance in the beam balance and rotarod test. When given during demyelination D-Asp prevented MBP loss and reduced inflammation, as revealed by Western Blot analysis of MBP, Iba1 and GFAP proteins and quantitative coexpression analysis of MBP with the axonal marker NF200. Finally, electron microscopy performed on corpus callosum sections showed that D-Asp treatment accelerates remyelination in cuprizone mice, as demonstrated by the increased number in myelinated axons if compared to untreated cuprizone mice. Collectively, our results show that treatment with D-Aspartate, by influencing calcium signaling in oligodendrocytes, might produce beneficial effects during demyelination and remyelination processes

Monitoring Alzheimer's disease in transgenic mice with ultra high field magnetic resonance imaging

While aging remains one of the most significant risk factors for development of Alzheimer disease (AD), increasing evidence strongly points to the potential roles of cerebrovascular and white matter abnormalities in the disease development. A better understanding of the manner in which these abnormalities contribute to disease progression can be achieved by in vivo characterization of AD related pathologies. To this end, MR based techniques serve as effective non-invasive tools to longitudinally monitor changes in AD brain. In this thesis, a variety of MR based techniques were optimized and employed to longitudinally monitor the AD progression in transgenic mouse models of the disease at 9.4T and 17.6T. In Chapter 2, age-dependent blood flow alterations were examined in a Tg2576 mouse model of Alzheimer's disease using MR angiography at 17.6T. AD is linked to abnormalities in the vascular system. In Chapter 3, in vivo T2 changes were longitudinally monitored in the corpus callosum, of the Tg2576 mice. In Chapter 4, age-dependent regional brain T1 and T2 changes in healty mice were established at 17.6T. In vivo imaging of these mouse models at ultra-high magnetic field strengths can permit a better understanding of the underlying cellular mechanism of AD.The Centre for Medical Systems Biology (CMSB), Internationale Stichting Alzheimer Onderzoek and Alzheimer NederlandSolid state NMR/Biophysical Organic Chemistr

Maternal High Fat Diet and Myelination within the Basolateral Amygdala and Hippocampus

Maternal obesity has significant effects on the neurodevelopmental outcomes of the offspring, increasing their propensity to neurodevelopmental disorders (NDDs) such as Autism Spectrum Disorders (ASDs). ASDs emerge from disturbances in the developing ‘social brain’. This includes the amygdala (BLA) and hippocampus (HP) which contribute to the regulation of emotions, social behaviours, and memories. Disturbances to myelination of these regions during development are thought to be central to the origins of ASDs. Oligodendrocytes (OLs) are responsible for myelination within the central nervous system, providing means to regulate the timings of signals propagating throughout the brain. This is essential during neurodevelopment, through mechanisms of spike timing dependent plasticity. If these mechanisms were undermined and the ability to regulate the temporal propagation of information lessened, such as by myelinogenic disturbances, circuits would have a greater propensity to develop improperly. Disturbances in the timing of myelination during development may be one of the mechanisms linking maternal obesity to the offspring’s increased risk to ASDs. It was hypothesised that OL development may be disrupted in the BLA and HP, in the offspring of obese mothers. To investigate this, OLs were analysed in offspring of control and maternal high fat diet (mHFD) fed dams, as a mouse model of maternal obesity. At postnatal day 16 (P16), the BLA and HP were labelled for Olig2, a pan OL marker, and MBP, a mature OL marker. At P30, the HP was labelled for Olig2 and MBP. The number and density of Olig2-immunoreactive (IR) cells were calculated, reflecting the size of the total OL lineage. MBP-IR integrated pixel densities (IntDen) were calculated, as a proxy measure for levels of myelination. The number and density of mature OLs, identified through the colocalization of Olig2 and MBP, were also calculated at P16. The number of OLs within the BLA was unchanged between the control and mHFD offspring at P16. Likewise, MBP-IR IntDen, a measure of myelination, was unchanged between the maternal diet groups. Thus, the OL lineage in the BLA appears unaffected by maternal nutritional adversity in utero. By contrast, MBP-IR IntDen was significantly reduced within the HP of the mHFD offspring at P16. While the total number of OLs was unchanged, there were fewer mature OLs in the HP of the mHFD offspring. Myelination however appeared to normalize with age, as no differences in the number of OLs or MBP-IR IntDen were observed in the HP between the control and mHFD offspring at P30. Thus, myelination of the HP is delayed in the mHFD offspring, likely due to maturational impediments, but normalizes to reach completion. These data provide insight into the regional effects of maternal obesity on myelination in the offspring. Disturbances during hippocampal myelination may contribute to neural circuit dysfunctions that are seen at an increased incidence in the offspring of obese mothers. Further work is necessary to gain understanding of the mechanisms underpinning these changes in development, and the predisposition to poor health and NDDs, in the offspring of obese mothers