Fardad T. Afshari

Schwann cell migration is integrin-dependent and inhibited by astrocyte-produced aggrecan.

Schwann cells transplantation has considerable promise in spinal cord trauma to bridge the site of injury and for remyelination in demyelinating conditions. They support axonal regeneration and sprouting by secreting growth factors and providing a permissive surface and matrix molecules while shielding axons from the inhibitory environment of the central nervous system. However, following transplantation Schwann cells show limited migratory ability and they are unable to intermingle with the host astrocytes. This in turn leads to formation of a sharp boundary and an abrupt transition between the Schwann cell graft and the host tissue astrocytes, therefore preventing regenerating axons from exiting the graft. The objective of this study was to identify inhibitory elements on astrocytes involved in restricting Schwann cell migration. Using in vitro assays of cell migration, we show that aggrecan produced by astrocytes is involved in the inhibition of Schwann cell motility on astrocytic monolayers. Knockdown of this proteoglycan in astrocytes using RNAi or digestion of glycosaminglycan chains on aggrecan improves Schwann cell migration. We further show aggrecan mediates its effect by disruption of integrin function in Schwann cells, and that the inhibitory effects of aggrecan can overcome by activation of Schwann cell integrins.

Astrocyte-Produced Ephrins Inhibit Schwann Cell Migration via VAV2 Signaling

Schwann cells are a promising candidate for bridging spinal cord injuries and remyelinating axons. However, grafted Schwann cells show little intermingling with host astrocytes and therefore limited migration from transplant sites. This leads to the formation of a sharp border between host astrocytes and Schwann cells, which results in axons stalling at the graft–host interface and failing to exit the graft. We investigated the possibility that Eph/ephrin interactions are involved in the segregation of Schwann cells and astrocytes and in limiting Schwann cell migration. Using reverse transcription-PCR, we have characterized the ephrin and Eph profile in cultured Schwann cells and astrocytes, showing that astrocytes produce all the ephrinAs and Schwann cells produce the receptors EphA2, EphA4, and EphA7. Several ephrinAs inhibit Schwann cell migration on laminin, with ephrinA5 being the most effective. Blocking the EphA receptors with excess EphA4–Fc increases Schwann cell migration on astrocytes and improves Schwann–astrocyte intermingling. We show that the action of ephrinA5 on Schwann cells is mediated via VAV2. Both clustered ephrinA5 and astrocyte contact increases the phosphorylation of VAV2 in Schwann cells. Knockdown of VAV2 abrogates the inhibitory effect of clustered ephrinA5 on migration and increases the ability of Schwann cells to migrate on astrocytes. In addition, we found a role for ephrinA5 in inhibiting Schwann cell integrin signaling and function. Overall, we suggest that Eph/ephrin interactions inhibit Schwann cell migration and intermingling with astrocytes via VAV signaling affecting integrin function.

Extrinsic and intrinsic factors controlling axonal regeneration after spinal cord injury.

Spinal cord injury is one of the most devastating conditions that affects the central nervous system. It can lead to permanent disability and there are around two million people affected worldwide. After injury, accumulation of myelin debris and formation of an inhibitory glial scar at the site of injury leads to a physical and chemical barrier that blocks axonal growth and regeneration. The mammalian central nervous system thus has a limited intrinsic ability to repair itself after injury. To improve axonal outgrowth and promote functional recovery, it is essential to identify the various intrinsic and extrinsic factors controlling regeneration and navigation of axons within the inhibitory environment of the central nervous system. Recent advances in spinal cord research have opened new avenues for the exploration of potential targets for repairing the cord and improving functional recovery after trauma. Here, we discuss some of the important key molecules that could be harnessed for repairing spinal cord injury.

Integrin activation or alpha9 expression allows retinal pigmented epithelial cell adhesion on Bruch’s membrane in wet age-related macular degeneration

Retinal pigment epithelial cell malfunction is a causative feature of age-related macular degeneration, and transplantation of new retinal pigment epithelial cells is an attractive strategy to prevent further progression and visual loss. However, transplants have shown limited efficacy, mainly because transplanted cells fail to adhere and migrate onto pathological Bruchs membrane. Adhesion to Bruchs membrane is integrin-mediated. Ageing of Bruchs membrane leads to a decline in integrin ligands and, added to this, wet age-related macular degeneration leads to upregulation of anti-adhesive molecules such as tenascin-C. We have therefore investigated whether manipulation of integrin function in retinal pigment epithelial cells can restore their adhesion and migration on wet age-related macular degeneration-damaged Bruchs membrane. Using spontaneously immortalized human retinal pigment epithelial cells (adult retinal pigment epithelium-19), we show that adhesion and migration on the Bruchs membrane components is integrin-dependent and enhanced by integrin-activating agents manganese and TS2/16. These allowed cells to adhere and migrate on low concentrations of ligand, as would be found in aged Bruchs membrane. We next developed a method for stripping cells from Bruchs membrane so that adhesion and migration assays can be performed on its surface. Integrin activation had a moderate effect on enhancing retinal pigmented epithelial cell adhesion and migration on normal human and rat Bruchs membrane. However, on Bruchs membrane prepared from human wet age-related macular degeneration-affected eyes, adhesion was lower and integrin activation had a much greater effect. A candidate molecule for preventing retinal pigmented epithelial interaction with age-related macular degeneration-affected Bruchs membrane is tenascin-C which we confirm is present at high levels in wet age-related macular degeneration membrane. We show that tenascin-C is anti-adhesive for retinal pigmented epithelial cells, but after integrin activation, they can adhere and migrate on it using alphaVbeta3 integrin. Alternatively, we find that transduction of retinal pigmented epithelial cells with alpha9 integrin, a tenascin-C-binding integrin, led to a large increase in alpha9beta1-mediated adhesion and migration on tenascin-C. Both expression of alpha9 integrin and integrin activation greatly enhanced the ability of retinal pigment epithelial cells to adhere to tenascin-rich wet age-related macular degeneration-affected Bruchs membranes. Our results suggest that manipulation of retinal pigment epithelial cell integrins through integrin activating strategies, or expression of new integrins such as alpha9, could be effective in improving the efficacy of retinal pigment epithelial cell transplantation in wet age-related macular degeneration-affected eyes.

Improving RPE adhesion to Bruch's membrane.

Age-related macular degeneration is the leading cause of blindness in the developing world. Retinal pigmented epithelium (RPE) transplantation in subretinal space, has been assessed in various animal models of age-related macular degeneration and in humans as a potential technique to preserve the visual function. However, the RPE cell survival posttransplantation is limited because of lack of attachment of the transplanted cells to the pathological Bruchs membrane and also partly because of iatrogenic removal of adhesive elements in the membrane during the removal of choroidal new vessels before transplantation procedure. Although pathological Bruchs membrane is well studied, there is still much debate as to why and how changes in the structure and components of this membrane leads to loss of RPE cells and disruption of their function and subsequent death of photoreceptors leading to visual loss. Integrins on RPE cells have been characterized and shown to be important for attachment of cells to Bruchs membrane. Considering the essential role of integrins in functions such as cell migration and adhesion, it is plausible that lack of attachment of RPE cells posttransplantation can be overcome by improving integrin function. Here, we have focused on some of the recent findings on the use of integrins and modulation of their function to improve the adhesion of RPE cells to normal and pathological Bruchs membrane. This work also aims at elucidating a potential mechanism by which accumulating inhibitory molecules in the Bruchs membrane in the pathological state, interferes with integrin function.

Proposal for establishment of the UK Cranial Reconstruction Registry (UKCRR).

Abstract Background. The increasing utilisation of decompressive craniectomy for traumatic brain injury and stroke has led to an increase in the number of cranioplasties undertaken. Cranioplasty is also undertaken following excision of tumours originating from or invading the skull vault, removal of bone flaps due to post-operative infection, and decompressive craniectomy for the management of rarer causes of brain oedema and/or refractory intracranial hypertension. The existing literature which mainly consists of single-centre, retrospective studies, shows a significant variation in practice patterns and a wide range of morbidity. There also exists a need to measure the outcome as perceived by the patients themselves with patient reported outcome measures (PROMs; functional outcome, quality of life, satisfaction with cosmesis). In the UK, the concept of long-term surveillance of neurosurgical implants is well established with the UK shunt registry. Based on this background, we propose to establish the UK Cranial Reconstruction Registry (UKCRR). Aim. The overarching aim of the UKCRR is to collect high-quality data about cranioplasties undertaken across the UK and Ireland in order to improve outcomes for patients. Methods. Any patient undergoing reconstruction of the skull vault with autologous bone, titanium, or synthetic material in participating units will be eligible for inclusion. Data will be submitted directly by participating units to the Outcome Registry Intervention and Operation Network secure platform. A Steering Committee will be responsible for overseeing the strategic direction and running of the UKCRR. Outcome measures. These will include re-operation due to a cranioplasty-related issue, surgical site infection, re-admission due to a cranioplasty-related issue, unplanned post-operative escalation of care, adverse events, length of stay in admitting unit, destination at discharge from admitting unit, mortality at discharge from admitting unit, neurological status and PROMs during routine follow-up. Conclusion. The UKCRR will be an important pillar in the ongoing efforts to optimise the outcomes of patients undergoing cranioplasty.

A report from the inaugural meeting of the British Neurosurgical Trainee Research Collaborative held in the Royal College of Surgeons of England, 19 October 2012.

Abstract Clinical research, which is essential for improving patient outcomes, is increasingly carried out in the context of networks established between multiple institutions. Research is also considered an important component of training curricula. The recent successful completion of a randomised trial (ROSSINI), which was led by general surgical trainees of the West Midlands Research Collaborative, has established the feasibility of trainee collaborative research networks. A research network for neurosurgical trainees in the UK and Ireland was, therefore, established following the meeting of the British Neurosurgical Trainee Association (BNTA) in Aberdeen on 19 April 2012. This BNTA initiative quickly gained the full support from the Society of British Neurological Surgeons and the UK Neurosurgical Research Network. The inaugural meeting of the British Neurosurgical Trainee Research Collaborative took place at the Royal College of Surgeons of England, London, on 19 October 2012. The purpose of this report is both to record progress to date and to promote this concept.

Astrocyte–Schwann-Cell Coculture Systems

Schwann cells are one of the cellular candidates used in repair strategies following trauma and demyelination of the spinal cord. One of the major obstacles in the use of Schwann cells is their limited migratory ability within the astrocytic environment of the CNS and boundary formation between the Schwann cells of the graft and the host astrocytes. This boundary creates an abrupt obstacle for regenerating axons attempting to exit the Schwann cell graft back to the CNS. To facilitate the study of mechanisms underlying these interactions, in vitro coculture assays of Schwann-Astrocytes have been developed. In this chapter, we have described the methodology for two commonly used coculture systems known as the Schwann-Astrocyte boundary assay and the inverted coverslip migration assay.

Analysis of Schwann-astrocyte interactions using in vitro assays.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors (1,2) and providing growth promoting adhesion molecules (3) and extracellular matrix molecules (4). In addition they myelinate the demyelinated axons at the site of injury (5). However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes (6,7). This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules (8-13). In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour. These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries. The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer (14,15). This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip. Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) (16,17), FGF/Heparin (18), Eph/Ephrins(19). This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Diffuse cerebral vasospasm following resection of a hypoglossal schwannoma in a child

Abstract Diffuse cerebral vasospasm is a rare complication following tumour resection. This phenomenon seems to be even rarer in the paediatric population and more so following resections of posterior fossa tumours. Here we report diffuse cerebral vasospasm in a child with hypoglossal nerve Schwannoma eight days following resection of the tumour.