The first patients treated with MR-CBCT soft-tissue matching in a MR-only prostate radiotherapy pathway.

INTRODUCTION: Magnetic Resonance (MR)-only radiotherapy for prostate cancer has previously been reported using fiducial markers for on-treatment verification. MR-Cone Beam Computed Tomography (CBCT) soft-tissue matching does not require invasive fiducial markers and enables MR-only treatments to other pelvic cancers. This study evaluated the first clinical implementation of MR-only prostate radiotherapy using MR-CBCT soft-tissue matching. METHODS: Twenty prostate patients were treated with MR-only radiotherapy using a synthetic (s)CT-optimised plan with MR-CBCT soft-tissue matching. Two MR sequences were acquired: small Field Of View (FOV) for target delineation and large FOV for organs at risk delineation, sCT generation and on-treatment verification. Patients also received a CT for validation. The prostate was independently contoured on the small FOV MR, copied to the registered CT and modified if there were MR-CT soft-tissue alignment differences (MR-CT volume). This was compared to the MR-only volume with a paired t-test. The treatment plan was recalculated on CT and the doses compared. Independent offline CT-CBCT matches for 5/20 fractions were performed by three therapeutic radiographers using the MR-only contours and compared to the online MR-CBCT matches using two one-sided paired t-tests for equivalence within ±1 mm. RESULTS: The MR-only volumes were significantly smaller than MR-CT (p = 0.003), with a volume ratio 0.92 ± 0.02 (mean ± standard error). The sCT isocentre dose difference to CT was 0.2 ± 0.1%. MR-CBCT soft-tissue matching was equivalent to CT-CBCT (p < 0.001), with differences of 0.1 ± 0.2 mm (vertical), -0.1 ± 0.2 mm (longitudinal) and 0.0 ± 0.1 mm (lateral). CONCLUSIONS: MR-only radiotherapy with soft-tissue matching has been successfully clinically implemented. It produced significantly smaller target volumes with high dosimetric and on-treatment matching accuracy. IMPLICATIONS FOR PRACTICE: MR-only prostate radiotherapy can be safely delivered without using invasive fiducial markers. This enables MR-only radiotherapy to be extended to other pelvic cancers where fiducial markers cannot be used.

A comprehensive atlas of Aggrecan, Versican, Neurocan and Phosphacan expression across time in wildtype retina and in retinal degeneration.

As photoreceptor cells die during retinal degeneration, the surrounding microenvironment undergoes significant changes that are increasingly recognized to play a prominent role in determining the efficacy of therapeutic interventions. Chondroitin Sulphate Proteoglycans (CSPGs) are a major component of the extracellular matrix that have been shown to inhibit neuronal regrowth and regeneration in the brain and spinal cord, but comparatively little is known about their expression in retinal degeneration. Here we provide a comprehensive atlas of the expression patterns of four individual CSPGs in three models of inherited retinal degeneration and wildtype mice. In wildtype mice, Aggrecan presented a biphasic expression, while Neurocan and Phosphacan expression declined dramatically with time and Versican expression remained broadly constant. In degeneration, Aggrecan expression increased markedly in Aipl1-/- and Pde6brd1/rd1, while Versican showed regional increases in the periphery of Rho-/- mice. Conversely, Neurocan and Phosphacan broadly decrease with time in all models. Our data reveal significant heterogeneity in the expression of individual CSPGs. Moreover, there are striking differences in the expression patterns of specific CSPGs in the diseased retina, compared with those reported following injury elsewhere in the CNS. Better understanding of the distinct distributions of individual CSPGs will contribute to creating more permissive microenvironments for neuro-regeneration and repair.

Measurement of the bone endocortical region using clinical CT.

The extent of the endocortical region and cortical bone mineral density (cBMD) throughout the proximal femur are of interest as both have been linked to fracture risk and osteoporosis treatment response. Non-invasive in-vivo clinical CT-based techniques capable of measuring the cortical bone attributes of thickness, density and mass over a bone surface have already been proposed. Several studies have robustly shown these methods to be capable of producing cortical thickness measurements to a sub-millimetre accuracy. Unfortunately, these methods are unable to provide high quality cBMD estimates, and are not designed to measure any attributes over the endocortical region of cortical bone. In this paper, we develop a cortical bone mapping based technique capable of providing an improved cBMD estimate and a measure of the endocortical width, while maintaining similar quality cortical thickness and trabecular bone mineral density (tBMD) estimates. The performance of the technique was assessed using a paired dataset of ex-vivo QCT and HR-pQCT scans across 72 proximal femurs. The HR-pQCT scans were analysed using a new method developed for this study: high resolution tissue classification (HRTC). In HRTC the cortical, endocortical and sub-surface trabecular bone features are extracted from the partially resolvable microarchitectural details in the HR-pQCT scan. We demonstrate that measurement of the endocortical extent from QCT is possible with an accuracy of -0.15±0.71mm, and that local cBMD can be measured down to densities of 300 mg/cm3.

Donor and host photoreceptors engage in material transfer following transplantation of post-mitotic photoreceptor precursors.

Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor-host nuclear or cell-cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders.

Outside-the-(cavity-prep)-box thinking.

Direct placement restorative materials must interface with tooth structures that are often compromised by caries or trauma. The material must seal the interface while providing sufficient strength and wear resistance to assure function of the tooth for, ideally, the lifetime of the patient. Needed are direct restorative materials that are less technique-sensitive than current resin-based composite systems while having improved properties. The ideal material could be successfully used in areas of the world with limited infrastructure. Advances in our understanding of the interface between the restoration adhesive system and the stages of carious dentin can be used to promote remineralization. Application of fracture mechanics to adhesion at the tooth-restoration interface can provide insights for improvement. Research in polymer systems suggests alternatives to current composite resin matrix systems to overcome technique sensitivity, while advances in nano- and mesoparticle reinforcement and alignment in composite systems can increase material strength, toughness, and wear resistance, foreshadowing dental application.

Restoration of vision after transplantation of photoreceptors.

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.

Manipulation of the recipient retinal environment by ectopic expression of neurotrophic growth factors can improve transplanted photoreceptor integration and survival.

Degeneration of the neural retina is the leading cause of untreatable blindness in the developed world. Stem cell replacement therapy offers a novel strategy for retinal repair. Postmitotic photoreceptor precursors derived from the early postnatal (P) retina are able to migrate and integrate into the adult mouse retina following transplantation into the subretinal space, but it is likely that a large number of these cells would be required to restore vision. The adult recipient retina presents a very different environment to that from which photoreceptor precursor donor cells isolated from the developing postnatal retina are derived. Here we considered the possibility that modulation of the recipient environment by ectopic expression of developmentally regulated growth factors, normally present during photoreceptor development, might enhance the migration and integration of transplanted cells into the adult neural retina. Adeno-associated viral (AAV) vectors were used to introduce three growth factors previously reported to play a role in photoreceptor development, IGF1, FGF2, and CNTF, into the adult retina, prior to transplantation of P4 cells derived from the Nrl.GFP(+ve) neural retina. At 3 weeks posttransplantation the number of integrated, differentiated photoreceptor cells present in AAV-mediated neurotrophic factor-treated eyes was assessed and compared to control treated contralateral eyes. We show, firstly, that it is possible to manipulate the recipient retinal microenvironment via rAAV-mediated gene transfer with respect to these developmentally relevant growth factors. Moreover, when combined with cell transplantation, AAV-mediated expression of IGF1 led to significantly increased levels of cell integration, while overexpression of FGF2 had no significant effect on integrated cell number. Conversely, expression of CNTF led to a significant decrease in cell integration and an exacerbated glial response that led to glial scarring. Together, these findings demonstrate the importance of the extrinsic environment of the recipient retina for photoreceptor cell transplantation and show for the first time that it is possible to manipulate this environment using viral vectors to influence photoreceptor transplantation efficiency.

Effective transplantation of photoreceptor precursor cells selected via cell surface antigen expression.

Retinal degenerative diseases are a major cause of untreatable blindness. Stem cell therapy to replace lost photoreceptors represents a feasible future treatment. We previously demonstrated that postmitotic photoreceptor precursors expressing an NrlGFP transgene integrate into the diseased retina and restore some light sensitivity. As genetic modification of precursor cells derived from stem cell cultures is not desirable for therapy, we have tested cell selection strategies using fluorochrome-conjugated antibodies recognizing cell surface antigens to sort photoreceptor precursors. Microarray analysis of postnatal NrlGFP-expressing precursors identified four candidate genes encoding cell surface antigens (Nt5e, Prom1, Podxl, and Cd24a). To test the feasibility of using donor cells isolated using cell surface markers for retinal therapy, cells selected from developing retinae by fluorescence-activated cell sorting based on Cd24a expression (using CD24 antibody) and/or Nt5e expression (using CD73 antibody) were transplanted into the wild-type or Crb1(rd8/rd8) or Prph2(rd2/rd2) mouse eye. The CD73/CD24-sorted cells migrated into the outer nuclear layer, acquired the morphology of mature photoreceptors and expressed outer segment markers. They showed an 18-fold higher integration efficiency than that of unsorted cells and 2.3-fold higher than cells sorted based on a single genetic marker, NrlGFP, expression. These proof-of-principle studies show that transplantation competent photoreceptor precursor cells can be efficiently isolated from a heterogeneous mix of cells using cell surface antigens without loss of viability for the purpose of retinal stem cell therapy. Refinement of the selection of donorphotoreceptor precursor cells can increase the number of integrated photoreceptor cells,which is a prerequisite for the restoration of sight.

Cone and rod photoreceptor transplantation in models of the childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive donor cells.

Retinal degenerative disease causing loss of photoreceptor cells is the leading cause of untreatable blindness in the developed world, with inherited degeneration affecting 1 in 3000 people. Visual acuity deteriorates rapidly once the cone photoreceptors die, as these cells provide daylight and colour vision. Here, in proof-of-principle experiments, we demonstrate the feasibility of cone photoreceptor transplantation into the wild-type and degenerating retina of two genetic models of Leber congenital amaurosis, the Crb1(rd8/rd8) and Gucy2e(-/-) mouse. Crx-expressing cells were flow-sorted from the developing retina of CrxGFP transgenic mice and transplanted into adult recipient retinae; CrxGFP is a marker of cone and rod photoreceptor commitment. Only the embryonic-stage Crx-positive donor cells integrated within the outer nuclear layer of the recipient and differentiated into new cones, whereas postnatal cells generated a 10-fold higher number of rods compared with embryonic-stage donors. New cone photoreceptors displayed unambiguous morphological cone features and expressed mature cone markers. Importantly, we found that the adult environment influences the number of integrating cones and favours rod integration. New cones and rods were observed in ratios similar to that of the host retina (1:35) even when the transplanted population consisted primarily of cone precursors. Cone integration efficiency was highest in the cone-deficient Gucy2e(-/-) retina suggesting that cone depletion creates a more optimal environment for cone transplantation. This is the first comprehensive study demonstrating the feasibility of cone transplantation into the adult retina. We conclude that flow-sorted embryonic-stage Crx-positive donor cells have the potential to replace lost cones, as well as rods, an important requirement for retinal disease therapy.

Targeted disruption of outer limiting membrane junctional proteins (Crb1 and ZO-1) increases integration of transplanted photoreceptor precursors into the adult wild-type and degenerating retina.

Diseases culminating in photoreceptor loss are a major cause of untreatable blindness. Transplantation of rod photoreceptors is feasible, provided donor cells are at an appropriate stage of development when transplanted. Nevertheless, the proportion of cells that integrate into the recipient outer nuclear layer (ONL) is low. The outer limiting membrane (OLM), formed by adherens junctions between Müller glia and photoreceptors, may impede transplanted cells from migrating into the recipient ONL. Adaptor proteins such as Crumbs homologue 1 (Crb1) and zona occludins (ZO-1) are essential for localization of the OLM adherens junctions. We investigated whether targeted disruption of these proteins enhances donor cell integration. Transplantation of rod precursors in wild-type mice achieved 949 +/- 141 integrated cells. By contrast, integration is significantly higher when rod precursors are transplanted into Crb1(rd8/rd8) mice, a model of retinitis pigmentosa and Lebers congenital amaurosis that lacks functional CRB1 protein and displays disruption of the OLM (7,819 +/- 1,297; maximum 15,721 cells). We next used small interfering (si)RNA to transiently reduce the expression of ZO-1 and generate a reversible disruption of the OLM. ZO-1 knockdown resulted in similar, significantly improved, integration of transplanted cells in wild-type mice (7,037 +/- 1,293; maximum 11,965 cells). Finally, as the OLM remains largely intact in many retinal disorders, we tested whether transient ZO-1 knockdown increased integration in a model of retinitis pigmentosa, the rho(-/-) mouse; donor cell integration was significantly increased from 313 +/- 58 cells without treatment to 919 +/- 198 cells after ZO-1 knockdown. This study shows that targeted disruption of OLM junctional proteins enhances integration in the wild-type and degenerating retina and may be a useful approach for developing photoreceptor transplantation strategies.

Cell transplantation strategies for retinal repair.

Cell transplantation is a novel therapeutic strategy to restore visual responses to the degenerate adult neural retina and represents an exciting area of regenerative neurotherapy. So far, it has been shown that transplanted postmitotic photoreceptor precursors are able to functionally integrate into the adult mouse neural retina. In this review, we discuss the differentiation of photoreceptor cells from both adult and embryonic-derived stem cells and their potential for retinal cell transplantation. We also discuss the strategies used to overcome barriers present in the degenerate neural retina and improve retinal cell integration. Finally, we consider the future translation of retinal cell therapy as a therapeutic strategy to treat retinal degeneration.

Neuron network activity scales exponentially with synapse density.

Neuronal network output in the cortex as a function of synapse density during development has not been explicitly determined. Synaptic scaling in cortical brain networks seems to alter excitatory and inhibitory synaptic inputs to produce a representative rate of synaptic output. Here, we cultured rat hippocampal neurons over a three-week period to correlate synapse density with the increase in spontaneous spiking activity. We followed the network development as synapse formation and spike rate in two serum-free media optimized for either (a) neuron survival (Neurobasal/B27) or (b) spike rate (NbActiv4). We found that while synaptophysin synapse density increased linearly with development, spike rates increased exponentially in developing neuronal networks. Synaptic receptor components NR1, GluR1 and GABA-A also increase linearly but with more excitatory receptors than inhibitory. These results suggest that the brain's information processing capability gains more from increasing connectivity of the processing units than increasing processing units, much as Internet information flow increases much faster than the linear number of nodes and connections.

Relative economic benefits of tactical anthelmintic treatment and urea-molasses block supplementation of Boer goats raised under extensive grazing conditions at Onderstepoort, Pretoria, South Africa.

The potential economic benefits of combining tactical anthelmintic treatment for gastrointestinal nematodes and nutritional supplementation with urea-molasses blocks were examined in Boer goats raised under extensive grazing conditions in the summer rainfall area of South Africa. Eight groups of nine goats were monitored over a 12-month period from 1 October 2002 to 9 October 2003. Ad libitum nutritional supplementation with urea-molasses blocks was provided when the goats were housed at night, during the summer (wet season--December 2002 to February 2003), and/or the winter (dry season--June 2003 to August 2003). All the goats were treated symptomatically for Haemonchus contortus infection when deemed necessary by clinical examination of the conjunctiva for anaemia using the FAMACHA system. Half the groups were tactically treated for gastrointestinal nematodes in mid-summer (28 January 2003). Under the symptomatic treatment, climatic and extensive grazing conditions encountered during the trial, feed supplementation in the winter dry season had the greatest economic benefit and is therefore recommended. Tactical anthelmintic treatment afforded no additional advantage, but the nematode challenge was low.

Isolation and characterisation of neural progenitor cells from the adult Chx10(orJ/orJ) central neural retina.

Retinal stem cells have been isolated from the ciliary epithelium (CE) of the mammalian retina. However, the central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, we showed that in Chx10(orJ/orJ) mice, dividing cells persist in the adult CNR, suggesting the existence of a dormant progenitor population. Here, we show that these cells are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Müller glial cell markers. Our results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors.

Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors.

Retinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3 weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72 h, and recovers by 2 weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72 h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications.

Stem cell therapy and the retina.

Retinal degeneration culminating in photoreceptor loss is the leading cause of untreatable blindness in the developed world. In this review, we consider how photoreceptors might be replaced by transplantation and how stem cells might be optimised for use as donor cells in future clinical strategies for retinal repair. We discuss the current advances in human and animal models of retinal cell transplantation, focussing on stem cell and reproductive cloning biology, in relation to the practical issues of retinal transplantation surgery. Stem and progenitor cells can be isolated from a number of sources including embryonic tissue, adult brain and even the retina, prompting many researchers to investigate the potential for using these cells to generate photoreceptors for transplantation. Nevertheless, several obstacles need to be overcome before these techniques can be applied in a clinical setting. Embryonic or stem cells have so far shown little ability to differentiate into retinal phenotypes when transplanted into the adult retina. We have recently noted, however, that donor cells harvested much later, at the photoreceptor precursor developmental stage, can be transplanted successfully and restore visual function. The current challenge is to understand the developmental processes that guide embryonic or adult stem cells towards photoreceptor differentiation, so that large numbers of these cells might be transplanted at the optimal stage. Future advances in reproductive cloning technology could lead to the successful generation of stem cells from adult somatic cells, thereby facilitating auto-transplantation of genetically identical cells in patients requiring photoreceptor replacement.