Santiago Costantino

Semi-automated quantification of filopodial dynamics.

Cellular motility underlies critical physiological processes including embryogenesis, metastasis and wound healing. Nerve cells undergo cellular migration during development and also extend neuronal processes for long distances through a complex microenvironment to appropriately wire the nervous system. The growth cone is a highly dynamic structure that responds to extracellular cues by extending and retracting filopodia and lamellipodia to explore the microenvironment and to dictate the path and speed of process extension. Neuronal responses to a myriad of guidance cues have been studied biochemically, however, these approaches fail to capture critical spatio-temporal elements of growth cone dynamics. Live imaging of growth cones in culture has emerged as a powerful tool to study growth cone responses to guidance cues but the dynamic nature of the growth cone requires careful quantitative analysis. Space time kymographs have been developed as a tool to quantify lamellipodia dynamics in a semi-automated fashion but no such tools exist to analyze filopodial dynamics. In this work we present an algorithm to quantify filopodial dynamics from cultured neurons imaged by time-lapse fluorescence microscopy. The method is based on locating the end tips of filopodia and tracking their locations as if they were free-moving particles. The algorithm is a useful tool and should be broadly applicable to filopodial tracking from multiple cell types.

Innate Immune-Mediated Neuronal Injury Consequent to Loss of Astrocytes

Neuronal injury and loss are recognized features of neuroinflammatory disorders, including acute and chronic encephalitides and multiple sclerosis; destruction of astrocytes has been demonstrated in cases of Rasmussen encephalitis. Here, we show that innate immune cells (i.e. natural killer [NK] and γδ T cells) cause loss of neurons from primary human neuron-enriched cultures by destroying the supporting astrocytes. Interleukin 2-activated NK cells caused loss of astrocytes within 1 hour, whereas neurons were lost at 4 hours. Time-lapse imaging indicated that delayed neuron loss was due to early destruction of supporting astrocytes. Selective blocking of astrocyte death with anti-NKG2D antibodies reduced neuron loss, as did blocking of CD54 on astrocytes. γδ T cells also induced astrocyte cytotoxicity, leading to subsequent neuronal displacement. In astrocytes, NK cells caused caspase-dependent fragmentation of the intermediate filament proteins glial fibrillary acidic protein and vimentin, whereas anti-CD3-activated T cells produced fragmentation to a lesser extent and without measurable cytotoxicity. Glial fibrillary acidic protein fragmentation was also demonstrated in lysates from chronic multiple sclerosis plaques but not from normal control white matter. These data suggest that non-major histocompatibility complex-restricted immune effector cells may contribute to neuron loss in neuroinflammatory disorders indirectly through injury of glia.

Second harmonic generation microscopy to investigate collagen configuration: a pericarditis case study

We have used second-harmonic-generation (SHG) to image collagen fibers in pericardial tissue removed from a patient with constrictive pericarditis and compared this to healthy pericardium. SHG imaging allowed for the visualization of collagen fibers without the need for staining or pretreatment. Images were compared to stained histology slides. Collagen fibers in SHG and histology images displayed the same structure and morphology. The mature collagen of the parietal pericardium was easily distinguishable from the new collagen accumulation due to the pericarditis. SHG imaging can provide a convenient and valuable architectural profile of collagen organization.