Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

The variation in transparency of amniotic membrane used in ocular surface regeneration

Background/aims: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. Methods: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. Results: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. Conclusion: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency.

Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release

-Aminobutyric acid type A (GABAA) receptors, a family of Cl-permeable ion channels, mediate fast synaptic inhibition as postsynaptically enriched receptors for -aminobutyric acid at GABAergic synapses. Here we describe an alternative type of inhibition mediated byGABAA receptors present on neocortical glutamatergic nerve terminals and examine the underlying signaling mechanism(s). By monitoring the activity of the presynaptic CaM kinase II/synapsin I signaling pathway in isolated nerve terminals, we demonstrate that GABAA receptor activation correlated with an increase in basal intraterminal [Ca2]i. Interestingly, this activation of GABAA receptors resulted in a reduction of subsequent depolarization-evoked Ca2 influx, which thereby led to an inhibition of glutamate release. To investigate how the observed GABAA receptor-mediated modulation operates, we determined the sensitivity of this process to the Na-K-2Cl cotransporter 1 antagonist bumetanide, as well as substitution of Ca2 with Ba2, or Ca2/calmodulin inhibition by W7. All of these treatments abolished the modulation by GABAA receptors. Application of selective antagonists of voltage-gated Ca2 channels (VGCCs) revealed that the GABAA receptor-mediated modulation of glutamate release required the specific activity of L- and R-type VGCCs. Crucially, the inhibition of release by these receptors was abolished in terminals isolated from R-type VGCC knock-out mice. Together, our results indicate that a functional coupling between nerve terminal GABAA receptors and L- or R-type VGCCs is mediated by Ca2/calmodulin-dependent signaling. This mechanism provides a GABA-mediated control of glutamatergic synaptic activity by a direct inhibition of glutamate release.

Refashioning local government and inner-city regeneration: The Salford experience

This paper explores the role of local government in urban regeneration in England. The first part describes local-central government relations during recent decades. It concludes that 'actually occurring' regeneration fuses top-down and bottom-up priorities and preferences, as well as path dependencies created by past decisions and local relations. The second part illustrates this contention by examining the regeneration of inner-city Salford over a 25-year period. It describes Salford City Council's approach in achieving the redevelopment of the former Salford Docks and how this created the confidence for the council to embark on further regeneration projects. Yet the top-down decision-making model has failed to satisfy local expectations, creating apathy which threatens the Labour government's desire for active citizens in regeneration projects.

Remaking place and securitising space: Urban regeneration and the strategies, tactics and practices of policing in the UK

Urban regeneration programmes in the UK over the past 20 years have increasingly focused on attracting investors, middle-class shoppers and visitors by transforming places and creating new consumption spaces. Ensuring that places are safe and are seen to be safe has taken on greater salience as these flows of income are easily disrupted by changing perceptions of fear and the threat of crime. At the same time, new technologies and policing strategies and tactics have been adopted in a number of regeneration areas which seek to establish control over these new urban spaces. Policing space is increasingly about controlling human actions through design, surveillance technologies and codes of conduct and enforcement. Regeneration agencies and the police now work in partnerships to develop their strategies. At its most extreme, this can lead to the creation of zero-tolerance, or what Smith terms 'revanchist', measures aimed at particular social groups in an effort to sanitise space in the interests of capital accumulation. This paper, drawing on an examination of regeneration practices and processes in one of the UK's fastest-growing urban areas, Reading in Berkshire, assesses policing strategies and tactics in the wake of a major regeneration programme. It documents and discusses the discourses of regeneration that have developed in the town and the ways in which new urban spaces have been secured. It argues that, whilst security concerns have become embedded in institutional discourses and practices, the implementation of security measures has been mediated, in part, by the local socio-political relations in and through which they have been developed.

Assessing the discourses and practices of urban regeneration in a growing region

Most research on the discourses and practices of urban regeneration in-the UK has examined case studies located in areas of relative socio-economic distress. Less research has been undertaken on regeneration projects and agendas in areas characterise by strong economic growth. Yet, it is in such places that some of the best examples of the discourses, practices and impacts of contemporary urban regeneration can be. found. In some areas of high demand regeneration projects have used inner urban brownfield sites as locations for new investment. With the New Labour government's urban policy agendas targeting similar forms of regeneration, an examination of completed or on-going schemes is timely and relevant to debates over the direction that policy should take. This paper, drawing on a study of urban regeneration in one of England's, fastest growing towns, Reading in Berkshire, examines the discourses, practices and impacts of redevelopment schemes during the 1990s and 2000s. Reading's experiences have received national attention and have been hailed as a model for other urban areas to follow. The research documents the discursive and concrete aspects of local regeneration and examines the ways in which specific priorities and defined problems have come to dominate agendas. Collectively, the study argues that market-driven objectives come to dominate regeneration agendas, even in areas of strong demand where development agencies wield a relatively high degree of influence. Such regeneration plays a symbolic and practical role in creating new forms of exclusion and interpretations of place. (C) 2003 Elsevier Science Ltd. All rights reserved.

Local development companies and the regeneration of Britain's cities

Efforts to decentralise the pursuit of economic and social development have increased in recent years. The authors examine the rationale for establishing local development companies in areas of high unemployment and deprivation. The broad purpose is to establish a new style of organisation that combines attributes of the public and private sectors-to adapt and integrate economic and social services to meet local needs, to champion local interests in external arenas, and to act as enabling agents to promote local investment and development. These arguments are elaborated and illustrated with reference to one of Britain's most successful local development companies, Govan Initiative. The analysis reveals important strengths of the Initiative, including its action orientation, commitment to quality, and a local leadership role, but also certain weaknesses including its limited leverage over wider policies and resource flows. Local development companies need meaningful commitment from regional and national public organisations to fulfil their potential.

Characterisation and regulation of E2F-6 and E2F-6b in the rat heart: a potential target for myocardial regeneration?

The E2F transcription factors are instrumental in regulating cell cycle progression and growth, including that in cardiomyocytes, which exit the cell cycle shortly after birth. E2F-6 has been demonstrated to act as a transcriptional repressor; however, its potential role in normal cardiomyocyte proliferation and hypertrophy has not previously been investigated. Here we report the isolation and characterisation of E2F-6 and E2F-6b in rat cardiomyocytes and consider its potential as a target for myocardial regeneration following injury. At the mRNA level, both rat E2F-6 and the alternatively spliced variant, E2F-6b, were expressed in E18 myocytes and levels were maintained throughout development into adulthood. Interestingly, E2F-6 protein expression was down-regulated during myocyte development suggesting that it is regulated post-transcriptionally in these cells. During myocyte hypertrophy, the mRNA expressions of E2F-6 and E2F-6b were not regulated whereas E2F-6 protein was up-regulated significantly. Indeed, E2F-6 protein expression levels closely parallel the developmental withdrawal of myocytes from the cell cycle and the subsequent reactivation of their cell cycle machinery during hypertrophic growth. Furthermore, depletion of E2F-6, using anti-sense technology, results in death of cultured neonatal myocytes. Taken together, abrogation of E2F-6 expression in neonatal cardiomyocytes leads to a significant decrease in their viability, consistent with the notion that E2F-6 might be required for maintaining normal myocyte growth.

Molecular aluminum hydrides identified by inelastic neutron scattering during H-2 regeneration of catalyst-doped NaAlH4

Catalyst-doped sodium aluminum hydrides have been intensively studied as solid hydrogen carriers for onboard proton-exchange membrane (PEM) fuel cells. Although the importance of catalyst choice in enhancing kinetics for both hydrogen uptake and release of this hydride material has long been recognized, the nature of the active species and the mechanism of catalytic action are unclear. We have shown by inelastic neutron scattering (INS) spectroscopy that a volatile molecular aluminum hydride is formed during the early stage of H-2 re-eneration of a depleted, catalyst-doped sodium aluminum hydride. Computational modeling of the INS spectra suggested the formation of AlH3 and oligomers (AlH3)(n) (Al2H6, Al3H9, and Al4H12 clusters), which are pertinent to the mechanism of hydrogen storage. This paper demonstrates, for the first time, the existence of these volatile species.

Rat neurosphere cells protect axotomized rat retinal ganglion cells and facilitate their regeneration

We investigated the ability of a population of rat neural stem and precursor cells derived from rat embryonic spinal cord to protect injured neurons in the rat central nervous system (CNS). The neonatal rat optic pathway was used as a model of CNS injury, whereby retinal ganglion cells (RGCs) were axotomized by lesion of the lateral geniculate nucleus one day after birth. Neural stem and precursor cells derived from expanded neurospheres (NS) were transplanted into the lesion site at the time of injury. Application of Fast Blue tracer dye to the lesion site demonstrated that significant numbers of RGCs survived at 4 and 8 weeks in animals that received a transplant, with an average of 28% survival, though in some individual cases survival was greater than 50%. No RGCs survived in animals that received a lesion alone. Furthermore, labeled RGCs were also observed when Fast Blue was applied to the superior colliculus (SC) at 4 weeks, suggesting that neurosphere cells also facilitated RGC to regenerate to their normal target. Transplanted cells did not migrate or express neural markers after transplantation, and secreted several neurotrophic factors in vitro. We conclude that NS cells can protect injured CNS neurons and promote their regeneration. These effects are not attributable to cell replacement, and may be mediated via secretion of neurotrophic factors. Thus, neuroprotection by stem cell populations may be a more viable approach for treatment of CNS disorders than cell replacement therapy.