Effects of starch/polycaprolactone-based blends for spinal cord injury regeneration in neurons/glial cells viability and proliferation

Spinal cord injury (SCI) leads to drastic alterations on the quality of life of afflicted individuals. With the advent of Tissue Engineering and Regenerative Medicine where approaches combining biomaterials, cells and growth factors are used, one can envisage novel strategies that can adequately tackle this problem. The objective of this study was to evaluate a blend of starch with poly(ε-caprolactone) (SPCL) aimed to be used for the development of scaffolds spinal cord injury (SCI) repair. SPCL linear parallel filaments were deposited on polystyrene coverslips and assays were carried out using primary cultures of hippocampal neurons and glial cells. Light and fluorescence microscopy observations revealed that both cell populations were not negatively affected by the SPCL-based biomaterial. MTS and total protein quantification indicated that both cell viability and proliferation rates were similar to controls. Both neurons and astrocytes occasionally contacted the surface of SPCL filaments through their dendrites and cytoplasmatic processes, respectively, while microglial cells were unable to do so. Using single cell [Ca2+ ]i imaging, hippocampal neurons were observed growing within the patterned channels and were functional as assessed by the response to a 30 mM KCl stimulus. The present data demonstrated that SPCL-based blends are potentially suitable for the development of scaffolds in SCI regenerative medicine.Portuguese Foundation for Science and Technology through funds from POCTI and/or FEDER programs (Funding to ICVS, 3B's Research Group and post doctoral fellowship to A.J. Salgado-SFRH/BPD/17595/2004)

The Infectivity of Naegleria fowleri Cysts in vivo and in vitro, and Mediation of Encystment by cAMP

The free-living amoeba and causative agent of Primary Amoebic Meningoencephalitis, Naegleria fowleri, has three life stages: the trophozoite, the flagellate, and the cyst. This study examined the ability of the cyst to attach to, excyst upon, and destroy cell cultures grown to confluent monolayers, and to cause Primary Amoebic Meningoencephalitis in a murine animal model. The co-culture of cysts with P388D.1, CHME3, Vero, human nasal epithelial, and rat primary mixed glial cells resulted in destruction of the monolayer of all cell types once the cysts attached and excysted. One day post exposure to cysts, the mixed glial cells exhibited a two-fold increase in lactate dehydrogenase (LDH) release compared to cells without cysts, and on day eight post exposure, showed a nearly four-fold increase in LDH. In this study, the cysts of N. fowleri were shown not to be infective in vivo in a murine model using B6C3F1 male mice. The mediation of the encystment process by the intracellular concentration of the secondary messenger cAMP, as described in other closely related genera and species of amoeba, was also investigated. Encystment of N. fowleri was shown to be mediated at least in part by the secondary messenger cAMP by treating cultures of the trophozoite with 100 uM dipyridamole, an inhibitor of cAMP-specific phosphodiesterases. Dipyridamole (100 μM) increased the rate of encystment by nearly two-fold compared to 0.1% DMSO by the end of a five day period of observation. This suggests that cAMP is an essential mediator of the encystment process within Naegleria fowleri

The Transcriptional Effects of Photobiomodulation in an In Vitro Model of Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of blindness. The pathophysiology of DR is complicated, involving inflammation, oxidative stress, retinal vascular proliferation, and vascular degeneration. Symptomatically, the growth and subsequent rupture of vessels within the frame of view leads to the development of vision loss and eventual blindness. Prior to the development of symptoms, oxidative stress involved in DR leads to the activation of the transcription factor, nuclear factor-kB (NF-kB), resulting in the excess production of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1), proteins involved in vascular development and immune dysregulation, respectively. The most common therapeutic approach for DR utilizes anti-VEGF agents to reduce vascular proliferation. These treatments are expensive, invasive, frequently ineffective, and have numerous adverse effects, such as retinal detachment, infection, and inflammation inside the eye. A non-invasive alternative therapy is clearly needed. Photobiomodulation (PBM) using far-red to near infrared (NIR) light has been shown to reduce oxidative stress and inflammation in vitro and in vivo and is an ideal candidate for an alternative therapy. Indeed, PBM slows the progression of DR in animal models via attenuation of oxidative stress and by reducing the relative level of ICAM-1. We hypothesize that PBM will reduce the activity of NF-kB and reduce the production of VEGF and ICAM-1 in an in vitro model of DR. To test this hypothesis, we used an in vitro model system of cultured retinal Müller glial cells grown in normal (5 mM) or high (25 mM) glucose conditions for either 3 or 6 days to simulate normoglycemia and hyperglycemia. Cultures were treated with 670 nm light emitting diode (LED) (180 seconds at 25 mW/cm2; 4.5 J/cm2) or no light (sham) for 3 or 5 days. NF-kB activity and ICAM-1 concentrations were significantly increased under high glucose conditions, as measured by a dual luciferase assay or western blot, respectively. Treatment with 670 nm LED significantly reduced NF-kB activity of high glucose culture cells to values comparable to transcriptional activity measured under normoglycemic condition and decreased the level of ICAM-1. VEGF concentrations were not affected by high glucose or PBM. These data are in partial support of our central hypothesis that in an in vitro model of DR, 670 nm light will reduce activation of NF-kB, and reduce the synthesis of ICAM-1 and VEGF. The lack of an observable effect of hyperglycemia or PBM on VEGF concentrations indicates that the stimulation of VEGF secretion requires the activation of additional signaling pathways not induced by high glucose alone

Biological response to structured and functionalized substrates for nerve tissue regeneration

After a lesion in the CNS, glial cells play a fundamental role, being the mediators of both the inhibitory and the beneficial response for neural regeneration. The tissue engineering approach consists in the use of biomaterials to help the regeneration and guide the regenerative capable cells to create a permissive environment. The main working hypothesis of this thesis is that we can promote a favourable environment for CNS regeneration identifying material properties which can modulate neuronal cells behaviour. In a first place we analyzed glial and neuronal response to two very different biopolymers, PMMA and chitosan. Wettability, surface and mechanical properties were characterized for both materials. Then line pattern of different dimensions in the micrometrical range were introduced. The response of glial cell and neurons were analyzed in terms of cell adhesion, morphology and differentiation state. Finally, we studied the behaviour of glial cells on glass model surfaces functionalized by self assembling monolayers with different wettability (OH, COOH, NH2, CH3), in order to identify the specific role that wettability plays in determining cell response. The dates suggest that the adhesion, the morphology and the differentiation state of neuron and glial cells can be controlled by choosing the proper combination of material properties and physical patterns. Overall, line patterns resulted to be a suitable tool to use in biomaterial design for nerve regeneration. However, the performance of each material must be analyzed with attention, since the combination of material properties, which most of the time is not predictable, play important roles in the biological activity

Developing Experimental Models of Non-Traumatic Spinal Cord Injury

Over 50% of non-traumatic spinal cord injuries (NTSCI) are caused by mechanical compression either due to osteophytes in degenerative disease, or tumours (New et al., 2014). The pathophysiology of NTSCI is poorly understood, with no distinct injury cascade (Karadimas et al., 2013). The aim of this project was to evaluate cellular responses to mechanical insults in the context of NTSCI. In-vitro, a model was developed to apply high and low velocity compression to astrocyte-seeded collagen hydrogels. Outcomes included hydrogel contraction, GFAP expression, cellular shape, and cytokine release. In-vivo a balloon lesion model was modified to induce a non-traumatic ventral lesion, by developing an injection port and inflating over 3 days. Functional deficits and histological outcomes were assessed. In-vitro, 100 mm.s-1 compression elicited an astrogliotic and inflammatory response from day 11, indicative of TSCI. This comprised a significant increase in GFAP area per cell, astrocyte ramification, and IL-6 expression. Conversely, at <100 mm.s-1, no differences were observed. The findings of this study suggest slow compression of astrocytes alone does not induce NTSCI. In-vivo, surgery was undertaken on 10 animals (including 3 shams). In injury groups, functional deficits were observed , which increased with each inflation. Animals were grouped into mild and severe based on their motor function (severe animals exhibited paraplegia). Minimum motor function correlated with minimum cross-sectional area, and greater parenchyma disruption. In the severe group only, there was a trend of mild astrogliosis, demyelination and vasculature narrowing at the epicentre. This corresponds with the wider literature, where demyelination and disruption to the vasculature are hypothesised to be involved in NTSCI pathology. Overall, in-vitro and in-vivo models of NTSCI have been successfully developed. Physiological changes were observed in both models, with differences to TSCI. Further investigations can be undertaken to understand the pathology of NTSCI

A novel polymeric microelectrode array for highly parallel, long-term neuronal culture and stimulation

Thesis (M. Eng.)--Harvard-MIT Division of Health Sciences and Technology, 2008.Includes bibliographical references (leaves 51-56).Cell-based high-throughput screening is emerging as a disruptive technology in drug discovery; however, massively parallel electrical assaying of neurons and cardiomyocites has until now been prohibitively expensive. To address this limitation, we developed a scalable, all-organic 3D microelectrode array technology. The cheap, disposable arrays would be integrated into a fixed stimulation and imaging setup, potentially amenable to automated handling and data analysis. A combination of activity-dependent plasticity, made possible by independent control of up to 64 stimulating electrodes, and, eventually, of substrate chemical patterning would be employed to constrain the neuronal culture network connectivity. In order to ensure longterm survival of the cultures, a bottom feeder layer of glial cells would be grown. In addition to high-throughput screening application, the polymeric microelectrode arrays and integrated stimulation systems were designed to allow the long-term study of synaptic plasticity, combining excellent long-term culture capabilities with a unique ability to independently control each electrode stimulation pattern. The resulting activity could be monitored optically, e,g, with calcium or voltage sensitive dyes, and the images could be stored and processed (possibly even in real time) within the same environment (LabView) as the stimulator. To fabricate the polymeric microelectrode array, we prepare a multilayered mask substrate, by reversibly bonding together two sheets of implant-grade polydimethylsiloxane (PDMS) sheets, with or without a glass coverslip between them. Thanks to PDMS self-adhesive properties the various layers are held together stably but reversibly. The mask is then laser-patterned, using either a standard CO2 laser or a 193 nm excimer laser.(cont.) The mask can then be adhered onto a glassy carbon or ITO electrode, and polypyrrole, doped with either hyaluronic acid or sodium dodecylbenzesulfonic acid, can be electrodeposited through it. Finally, the construct is removed from the deposition bath and the upper, sacrificial mask layer carefully peeled away. This fabrication method allows exquisite control overall 3D electrode geometry, is suitable to produce structures between one and several hundred micrometers in diameter, either filled or tubular, and scales extremely well, so that, for example, 384 by 64 electrodes arrays can be patterned in just a few minutes and grown in the same time as a single array.by Giovanni Talei Franzesi.M.Eng

Novel CNS gene delivery systems

Ph.DDOCTOR OF PHILOSOPH