Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology

Noise-free scattering of the quantized electromagnetic field from a dispersive linear dielectric

We study the scattering of the quantized electromagnetic field from a linear, dispersive dielectric using the scattering formalism for quantum fields. The medium is modeled as a collection of harmonic oscillators with a number of distinct resonance frequencies. This model corresponds to the Sellmeir expansion, which is widely used to describe experimental data for real dispersive media. The integral equation for the interpolating field in terms of the in field is solved and the solution used to find the out field. The relation between the in and out creation and annihilation operators is found which allows one to calculate the S-matrix for this system. In this model, we find that there are absorption bands, but the input-output relations are completely unitary. No additional quantum noise terms are required.Comment: Revtex, submitted to Physical Review

Clinical indicators for common paediatric conditions: processes, provenance and products of the CareTrack Kids study

BACKGROUND:In order to determine the extent to which care delivered to children is appropriate (in line with evidence-based care and/or clinical practice guidelines (CPGs)) in Australia, we developed a set of clinical indicators for 21 common paediatric medical conditions for use across a range of primary, secondary and tertiary healthcare practice facilities. METHODS:Clinical indicators were extracted from recommendations found through systematic searches of national and international guidelines, and formatted with explicit criteria for inclusion, exclusion, time frame and setting. Experts reviewed the indicators using a multi-round modified Delphi process and collaborative online wiki to develop consensus on what constituted appropriate care. RESULTS:From 121 clinical practice guidelines, 1098 recommendations were used to draft 451 proposed appropriateness indicators. In total, 61 experts (n = 24 internal reviewers, n = 37 external reviewers) reviewed these indicators over 40 weeks. A final set of 234 indicators resulted, from which 597 indicator items were derived suitable for medical record audit. Most indicator items were geared towards capturing information about under-use in healthcare (n = 551, 92%) across emergency department (n = 457, 77%), hospital (n = 450, 75%) and general practice (n = 434, 73%) healthcare facilities, and based on consensus level recommendations (n = 451, 76%). The main reason for rejecting indicators was 'feasibility' (likely to be able to be used for determining compliance with 'appropriate care' from medical record audit). CONCLUSION:A set of indicators was developed for the appropriateness of care for 21 paediatric conditions. We describe the processes (methods), provenance (origins and evolution of indicators) and products (indicator characteristics) of creating clinical indicators within the context of Australian healthcare settings. Developing consensus on clinical appropriateness indicators using a Delphi approach and collaborative online wiki has methodological utility. The final indicator set can be used by clinicians and organisations to measure and reflect on their own practice.Louise K. Wiles, Tamara D. Hooper, Peter D. Hibbert, Charlotte Molloy, Les White ... Helena Williams ... et al

Electrospinning with dual collection rings

The formation of electrospun poly(var epsilon-caprolactone) (PCL) fibres between two collection rings is described, and the conversion of these fibres into a multi-filament yarn of is demonstrated. During electrospinning, when two grounded rings are placed equidistantly from the spinneret, an array of fibres is formed between the collection rings. These rapidly produced fibres are three-dimensionally suspended in air, and demonstrate low incidence of fibre splitting when lower voltages are applied. Such electrospun fibres, with a diameter of 1.26±0.19 ?m and a length between 40 and 100 mm, were oriented and continuous. Furthermore, manufacture of the fibre array was easily achievable, reproducible and not subject to relatively small changes in spinneret-ground distance or applied voltage. Rotation of one of the collection rings results in a wound multi-filament yarn with a diameter below 5 ?m, and a length of 50 mm

Hydrophilic sponges based on 2-hydroxyethyl methacrylate - Part VII: Modulation of sponge characteristics by changes in reactivity and hydrophilicity of crosslinking agents

Despite previous unsuccessful attempts to use hydrated poly(2-hydroxyethyl methacrylate) sponges as implantable biomaterials, recently these materials became important as peripheral components in an artificial cornea of the core-and-skirt design. The low mechanical strength of sponges prompted this study on possible improvement of tensile properties by the use of a variety of crosslinking agents. Three vinylic (dimethacrylates) and two allylic compounds were used at different concentrations (0.1 to 2% (mol)) as crosslinking agents in the production of sponges. Their influence on the mechanical properties, porous morphology and swelling behavior of resulting sponges was evaluated. The onset of phase separation during polymerization was also measured by visible spectrophotometry. The results suggested an inherent heterogeneity of sponges, i.e. pores of non-uniform size and structural inhomogeneities. While the effects of changes in the nature and concentration of crosslinking agents on the equilibrium water content of sponges were ambiguous, some of the mechanical properties, such as toughness and elasticity, were improved by crosslinking with allylic agents. Scanning electron microscopic examination suggested that the mechanical effect is related to the variation of size of the polymer particles constituting the sponge structure, which was proved to be dependent upon the onset of phase separation during polymerization

Synthetic hydrogel guidance channels facilitate regeneration of adult rat brainstem motor axons after complete spinal cord transection

Synthetic guidance channels or tubes have been shown to promote axonal regeneration within the spinal cord from brainstem motor nuclei with the inclusion of agents such as matrices, cells, or growth factors to the tube. We examined the biocompatibility and regenerative capacity of synthetic hydrogel tubular devices that were composed of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (PHEMA-MMA). Two PHEMA-MMA channels, having a mean elastic modulus of either 177 or 311 kPa were implanted into T8-transected spinal cords of adult Sprague Dawley rats. The cord stumps were inserted into the channels and fibrin glue was applied to the cord-channel interface. An expanded polytetrafluoroethylene (ePTFE) membrane was used for duraplasty. Controls underwent cord transection alone. Gross and microscopic examination of the spinal cords showed continuity of tissue within the synthetic guidance channels between the cord stumps at 4 and 8 weeks. There was a trend towards an increased area and width of bridging neural tissue in the 311-kPa guidance channels compared to the 177-kPa channels. Neurofilament stained axons were visualized within the bridging tissue, and serotonergic axons were found to enter the 311-kPa channel. Retrograde axonal tracing revealed regeneration of axons from reticular, vestibular, and raphe brainstem motor nuclei. For both channels, there was minimal scarring at the channel-cord interface, and less scarring at the channel-dura interface compared to that observed next to the ePTFE. The present study is the first to show that axons from brainstem motor nuclei regenerated in unfilled synthetic hydrogel guidance channels after complete spinal cord transection

Design, fabrication and characterization of PCL electrospun\ud scaffolds—a review

The expanding interest in electrospinning fibers for bioengineering includes a significant use of polyesters, including poly(3-caprolactone) (PCL). This review summarizes literature on PCL and selected blends, and provides extensive descriptions of the broad range of parameters used in manufacturing such electrospun fibers. Furthermore the chemical, physical and biological approaches for characterizing the electrospun material are described and opinions offered on important information to include in future publications with this electrospun material

Growth factor enhancement of peripheral nerve regeneration through a novel synthetic hydrogel tube

Object. The authors’ long-term goal is repair of peripheral nerve injuries by using synthetic nerve guidance devices that improve both regeneration and functional outcome relative to an autograft. They report the in vitro processing and in vivo application of synthetic hydrogel tubes that are filled with collagen gel impregnated with growth factors.Methods. Poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (PHEMA-MMA) porous 12-mm-long tubes with an inner diameter of 1.3 mm and an outer diameter of 1.8 mm were used to repair surgically created 10-mm gaps in the rat sciatic nerve. The inner lumen of the tubes was filled with collagen matrix alone or matrix supplemented with either neurotropin-3 at 1 g/ml, brain-derived neurotrophic factor at 1 g/ml, or acidic fibroblast growth factor (FGF-1) at 1 or 10 g/ml. Nerve regeneration through the growth factor–enhanced tubes was assessed at 8 weeks after repair by histomorphometric analysis at the midgraft level and in the nerve distal to the tube repair. The tubes were biostable and biocompatible, and supported nerve regeneration in more than 90% of cases. Nerve regeneration was improved in tubes in which growth factors were added, compared with empty tubes and those containing collagen gel alone (negative controls). Tubes filled with 10 g/ml of FGF-1 dispersed in collagen demonstrated regeneration comparable to autografts (positive controls) and showed significantly better regeneration than the other groups.Conclusions. The PHEMA-MMA tubes augmented with FGF-1 in their lumens appear to be a promising alternative to autografts for repair of nerve injuries. Studies are in progress to assess the long-term biocompatibility of these implants and to enhance regeneration further