Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing.

After damage to the adult mammalian central nervous system (CNS), surviving neurons have limited capacity to regenerate and restore functional connectivity. Conditional genetic deletion of PTEN results in robust CNS axon regrowth, while PTEN repression with short hairpin RNA (shRNA) improves regeneration but to a lesser extent, likely due to suboptimal PTEN mRNA knockdown using this approach. Here we employed the CRISPR/dCas9 system to repress PTEN transcription in neural cells. We targeted the PTEN proximal promoter and 5' untranslated region with dCas9 fused to the repressor protein Krüppel-associated box (KRAB). dCas9-KRAB delivered in a lentiviral vector with one CRISPR guide RNA (gRNA) achieved potent and specific PTEN repression in human cell line models and neural cells derived from human iPSCs, and induced histone (H)3 methylation and deacetylation at the PTEN promoter. The dCas9-KRAB system outperformed a combination of four shRNAs targeting the PTEN transcript, a construct previously used in CNS injury models. The CRISPR system also worked more effectively than shRNAs for Pten repression in rat neural crest-derived PC-12 cells, and enhanced neurite outgrowth after nerve growth factor stimulation. PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.

Neurotrophic Factors Used to Treat Spinal Cord Injury.

The application of neurotrophic factors as a therapy to improve morphological and behavioral outcomes after experimental spinal cord injury (SCI) has been the focus of many studies. These studies vary markedly in the type of neurotrophic factor that is delivered, the mode of administration, and the location, timing, and duration of the treatment. Generally, the majority of studies have had significant success if neurotrophic factors are applied in or close to the lesion site during the acute or the subacute phase after SCI. Comparatively fewer studies have administered neurotrophic factors in order to directly target the somata of injured neurons. The mode of delivery varies between acute injection of recombinant proteins, subacute or chronic delivery using a variety of strategies including osmotic minipumps, cell-mediated delivery, delivery using polymer release vehicles or supporting bridges of some sort, or the use of gene therapy to modify neurons, glial cells, or precursor/stem cells. In this brief review, we summarize the state of play of many of the therapies using these factors, most of which have been undertaken in rodent models of SCI.

In vivo oximetry of human bulbar conjunctival and episcleral microvasculature using snapshot multispectral imaging.

Multispectral imaging (MSI) is a well-established technique for non-invasive oximetry of retinal blood vessels, which has contributed to the understanding of a variety of retinal conditions, including glaucoma, diabetes, vessel occlusion, and retinal auto-regulation. We report the first study to use snapshot multi-spectral imaging (SMSI) for oximetry of the bulbar conjunctival and episcleral microvasculature in the anterior segment of the eye. We report the oxygen dynamics of the bulbar conjunctival and episcleral microvasculature at normoxia and at acute mild hypoxia conditions. A retinal-fundus camera fitted with a custom Image-Replicating Imaging Spectrometer was used to image the bulbar conjunctival and episcleral microvasculature in ten healthy human subjects at normoxia (21% Fraction of Inspired Oxygen [FiO2]) and acute mild hypoxia (15% FiO2) conditions. Eyelid closure was used to control oxygen diffusion between ambient air and the sclera surface. Four subjects were imaged for 30 seconds immediately following eyelid opening. Vessel diameter and Optical Density Ratio (ODR: a direct proxy for oxygen saturation) of vessels was computed automatically. Oximetry capability was validated using a simple phantom that mimicked the scleral vasculature. Acute mild hypoxia resulted in a decrease in blood oxygen saturation (SO2) (i.e. an increase in ODR) when compared with normoxia in both bulbar conjunctival (p < 0.001) and episcleral vessels (p = 0.03). Average episcleral diameter increased from 78.9 ± 8.7 µm (mean ± standard deviation) at normoxia to 97.6 ± 14.3 µm at hypoxia (p = 0.02). Diameters of bulbar conjunctival vessels showed no significant change from 80.1 ± 7.6 µm at normoxia to 80.6 ± 7.0 µm at hypoxia (p = 0.89). When exposed to ambient air, hypoxic bulbar conjunctival vessels rapidly reoxygenated due to oxygen diffusion from ambient air. Reoxygenation occured in an exponential manner, and SO2 reached normoxia baseline levels. The average ½ time to full reoxygenation was 3.4 ± 1.4 s. As a consequence of oxygen diffusion, bulbar conjunctival vessels will be highly oxygenated (i.e. close to 100% SO2) when exposed to ambient air. Episcleral vessels were not observed to undergo any significant oxygen diffusion, instead behaving similarly to pulse oximetry measurements. This is the first study to the image oxygen dynamics of bulbar conjunctival and episcleral microvasculature, and consequently, the first study to directly observe the rapid reoxygenation of hypoxic bulbar conjunctival vessels when exposed to ambient air. Oximetry of bulbar conjunctival vessels could potentially provide insight into conditions where oxygen dynamics of the microvasculature are not fully understood, such as diabetes, sickle-cell diseases, and dry-eye syndrome. Oximetry in the bulbar conjunctival and episcleral microvasculature could be complimentary or alternative to retinal oximetry.

Clinically Unsuspected Prion Disease Among Patients With Dementia Diagnoses in an Alzheimer's Disease Database.

BACKGROUND: Brain tissue analysis is necessary to confirm prion diseases. Clinically unsuspected cases may be identified through neuropathologic testing. METHODS: National Alzheimer's Coordinating Center (NACC) Minimum and Neuropathologic Data Set for 1984 to 2005 were reviewed. Eligible patients had dementia, underwent autopsy, had available neuropathologic data, belonged to a currently funded Alzheimer's Disease Center (ADC), and were coded as having an Alzheimer's disease clinical diagnosis or a nonprion disease etiology. For the eligible patients with neuropathology indicating prion disease, further clinical information, collected from the reporting ADC, determined whether prion disease was considered before autopsy. RESULTS: Of 6000 eligible patients in the NACC database, 7 (0.12%) were clinically unsuspected but autopsy-confirmed prion disease cases. CONCLUSION: The proportion of patients with dementia with clinically unrecognized but autopsy-confirmed prion disease was small. Besides confirming clinically suspected cases, neuropathology is useful to identify unsuspected clinically atypical cases of prion disease.

Development of a cell-based treatment for long-term neurotrophin expression and spiral ganglion neuron survival.

Spiral ganglion neurons (SGNs), the target cells of the cochlear implant, undergo gradual degeneration following loss of the sensory epithelium in deafness. The preservation of a viable population of SGNs in deafness can be achieved in animal models with exogenous application of neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3. For translation into clinical application, a suitable delivery strategy that provides ongoing neurotrophic support and promotes long-term SGN survival is required. Cell-based neurotrophin treatment has the potential to meet the specific requirements for clinical application, and we have previously reported that Schwann cells genetically modified to express BDNF can support SGN survival in deafness for 4 weeks. This study aimed to investigate various parameters important for the development of a long-term cell-based neurotrophin treatment to support SGN survival. Specifically, we investigated different (i) cell types, (ii) gene transfer methods and (iii) neurotrophins, in order to determine which variables may provide long-term neurotrophin expression and which, therefore, may be the most effective for supporting long-term SGN survival in vivo. We found that fibroblasts that were nucleofected to express BDNF provided the most sustained neurotrophin expression, with ongoing BDNF expression for at least 30 weeks. In addition, the secreted neurotrophin was biologically active and elicited survival effects on SGNs in vitro. Nucleofected fibroblasts may therefore represent a method for safe, long-term delivery of neurotrophins to the deafened cochlea to support SGN survival in deafness.

Oxygen saturation measurements of the retinal vasculature in treated asymmetrical primary open-angle glaucoma using hyperspectral imaging.

PURPOSE: To determine whether there are differences in retinal vascular oxygen saturation measurements, estimated using a hyperspectral fundus camera, between normal eyes and treated eyes of subjects with asymmetrical primary open-angle glaucoma (POAG). METHODS: A noninvasive hyperspectral fundus camera was used to acquire spectral images of the retina at wavelengths between 556 and 650 nm in 2-nm increments. In total, 14 normal eyes and both eyes of 11 treated POAG subjects were imaged and analyzed using algorithms that use the spectral variation of the optical densities of blood vessels to estimate the oxygen saturation of blood within the retinal vasculature. In the treated POAG group, each of the eyes were categorized, based on the mean deviation of the Humphrey visual-field analyzer result, as either more-advanced or less-advanced, glaucomatous eyes. Unpaired t-tests (two-tailed) with Welch's correction were used to compare the mean oxygen saturation between the normal subjects and the treated POAG subgroups. RESULTS: In less-advanced and more-advanced-treated POAG eyes, mean retinal venular oxygen saturations (48.2±21.6% and 42.6±18.8%, respectively) were significantly higher than in normal eyes (27.9±9.9%; P=0.03 and 0.01, respectively). Arteriolar oxygen saturation was not significantly different between normal eyes and treated POAG eyes. CONCLUSIONS: The increased oxygen saturation of the retinal venules in advanced-treated POAG eyes may indicate reduced metabolic consumption of oxygen in the inner retinal tissues.

An outpatient low-intensity locomotor training programme for paediatric chronic incomplete spinal cord injury.

STUDY DESIGN: Single case study. OBJECTIVES: Intensive locomotor training programmes have recently been implemented in paediatric settings for patients with chronic incomplete spinal cord injury. This case study examines whether a lower-intensity locomotor training programme can improve functional ambulation. SETTING: Tertiary care setting in Melbourne, Australia. METHODS: A pretest-post-test design was used for a 17-year-old boy, 16 months after incomplete spinal injury at T6, who was classified as American Spinal Injury Association (ASIA) level C. He participated in two weekly sessions of locomotor training for a period of 6 weeks. Lower Extremity Motor Score (LEMS), Walking Index for Spinal Cord Injury (WISCI II), 6-min walk test (6MWT), 10-m walk test (10MWT), Timed Up and Go (TUG), and the PedsQL were measured before training, immediately after training and 6 weeks after training had ceased. RESULTS: The WISCI II score improved from 6 at baseline to 9 immediately post treatment and this was maintained at follow-up. The PedsQL score was also significantly improved immediately post treatment and at 6 weeks follow-up. The LEMS and 6MWT improved after the intervention also. CONCLUSION: This case study provides evidence of improvements following a less-intensive locomotor training programme in an outpatient setting. Studies with larger samples are required to fully examine the benefits of this programme.

Scaffolds to promote spinal cord regeneration.

Substantial research effort in the spinal cord injury (SCI) field is directed towards reduction of secondary injury changes and enhancement of tissue sparing. However, pathway repair after complete transections, large lesions, or after chronic injury may require the implantation of some form of oriented bridging structure to restore tissue continuity across a trauma zone. These matrices or scaffolds should be biocompatible and create an environment that facilitates tissue growth and vascularization, and allow axons to regenerate through and beyond the implant in order to reconnect with "normal" tissue distal to the injury. The myelination of regrown axons is another important requirement. In this chapter, we describe recent advances in biomaterial technology designed to provide a terrain for regenerating axons to grow across the site of injury and/or create an environment for endogenous repair. Many different types of scaffold are under investigation; they can be biodegradable or nondegradable, natural or synthetic. Scaffolds can be designed to incorporate immobilized signaling molecules and/or used as devices for controlled release of therapeutic agents, including growth factors. These bridging structures can also be infiltrated with specific cell types deemed suitable for spinal cord repair.

The development of encapsulated cell technologies as therapies for neurological and sensory diseases.

Cell encapsulation therapies involve the implantation of cells that secrete a therapeutic factor to provide clinical benefits. The transplanted cells are protected from immunorejection via encapsulation in a semipermeable membrane. This treatment strategy was originally investigated as a method for protecting pancreatic islets from immunorejection, thus allowing them to secrete insulin as a chronic treatment for diabetes. Since then a significant body of work has been conducted in developing cell encapsulation therapies to treat a variety of different diseases. Many of these conditions involve neurodegeneration, such as Alzheimer's and Parkinson's disease, as cell encapsulation therapies have proven to be particularly suitable for delivering therapeutics to the central nervous system. This is mainly because they offer chronic delivery of a therapeutic and can be implanted proximal to the affected tissue, bypassing the blood brain barrier, which is impermeable to many agents. Whilst these therapies are not yet widely available in the clinic, promising results have been obtained in several advanced clinical trials and further developmental work is currently underway. This review specifically examines the development of encapsulated cell therapies as treatments for neurological and sensory diseases and evaluates the challenges that are yet to be overcome before they can be made available for clinical use.

Spectral imaging of the retina.

INTRODUCTION: The work described here involved the use of a modified fundus camera to obtain sequential hyperspectral images of the retina in 14 normal volunteers and in 1 illustrative patient with a retinal vascular occlusion. METHODS: The paper describes analysis techniques, which allow oximetry within retinal vessels; these results are presented as retinal oximetry maps. RESULTS: Using spectral images, with wavelengths between 556 and 650 nm, the mean oxygen saturation (OS) value in temporal retinal arterioles in normal volunteers was 104.3 (± 16.7), and in normal temporal retinal venules was 34.8 (± 17.8). These values are comparable to those quoted in the literature, although, the venular saturations are slightly lower than those values found by other authors; explanations are offered for these differences. DISCUSSION: The described imaging and analysis techniques produce a clinically useful map of retinal oximetric values. The results from normal volunteers and from one illustrative patient are presented. Further developments, including the recent development of a 'snapshot' spectral camera, promises enhanced non-invasive retinal vessel oximetry mapping.

Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons.

Intravitreal injections of recombinant ciliary neurotrophic factor (rCNTF) protect adult rat retinal ganglion cells (RGCs) after injury and stimulate regeneration, an effect enhanced by co-injection with a cAMP analogue (CPT-cAMP). This effect is partly mediated by PKA and associated signaling pathways, but CPT-cAMP also moderates upregulation of suppressor of cytokine signaling (SOCS) pathways after rCNTF injection, which will also enhance the responsiveness of RGCs to this and perhaps other cytokines. We now report that intravitreal injections of CPT-cAMP do not potentiate RGC axonal regeneration when CNTF is expressed via an adeno-associated viral vector (rAAV2), and concomitantly we show that increases in retinal SOCS mRNA expression are less when CNTF is delivered using the vector. We also directly tested the impact of elevated SOCS3 expression on the survival and regeneration of injured adult RGCs by injecting a bicistronic rAAV2-SOCS3-GFP vector into the vitreous of eyes in rats with a peripheral nerve graft sutured onto the cut optic nerve. Overexpression of SOCS3 resulted in an overall reduction in axonal regrowth and almost complete regeneration failure of RGCs transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal rCNTF injections. In summary, CNTF delivery to the retina using viral vectors may be more effective than bolus rCNTF injections because the gene therapy approach has a less pronounced effect on neuron-intrinsic SOCS repressor pathways. Our new gain of function data using rAAV2-SOCS3-GFP demonstrate the negative impact of enhanced SOCS3 expression on the regenerative potential of mature CNS neurons.

Novel phage peptides attenuate beta amyloid-42 catalysed hydrogen peroxide production and associated neurotoxicity.

Amyloid-beta (Abeta) peptides play a central role in the pathogenesis of Alzheimer's disease. There is accumulating evidence that supports the notion that the toxicity associated with human Abeta (both 40 and 42) is dependent on its superoxide dismutase (SOD)-like activity. We developed a novel screening method involving phage display technology to identify novel peptides capable of inhibiting Abeta's neurotoxicity. Two random peptide libraries containing 6-mer and 15-mer peptide inserts were used and resulted in the identification of 25 peptides that bound human Abeta (40 or 42). Here, we show that two of the three most enriched peptides obtained significantly reduced Abeta42's SOD-like activity. A 15-mer peptide reduced Abeta42 neurotoxicity in a dose-dependent manner as evidenced by a reduction in LDH release. These findings were confirmed in the independent MTT assay. Furthermore, comparative analysis of the 15-mer peptide with Clioquinol, a known inhibitor of Abeta's metal-mediated redox activity, showed the 15-mer peptide to be equipotent to this metal chelator, under the same experimental conditions. These agents represent novel peptides that selectively target and neutralise Abeta-induced neurotoxicity and thus provide promising leads for rational drug development.

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection.

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP(+) cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.

EVALUATION OF INTERFACIAL FLUID DYNAMICAL STRESSES USING THE IMMERSED BOUNDARY METHOD.

The goal of this paper is to examine the evaluation of interfacial stresses using a standard, finite difference based, immersed boundary method (IMBM). This calculation is not trivial for two fundamental reasons. First, the immersed boundary is represented by a localized boundary force which is distributed to the underlying fluid grid by a discretized delta function. Second, this discretized delta function is used to impose a spatially averaged no-slip condition at the immersed boundary. These approximations can cause errors in interpolating stresses near the immersed boundary.To identify suitable methods for evaluating stresses, we investigate three model flow problems at very low Reynolds numbers. We compare the results of the immersed boundary calculations to those achieved by the boundary element method (BEM). The stress on an immersed boundary may be calculated either by direct evaluation of the fluid stress (FS) tensor or, for the stress jump, by direct evaluation of the locally distributed boundary force (wall stress or WS). Our first model problem is Poiseuille channel flow. Using an analytical solution of the immersed boundary formulation in this simple case, we demonstrate that FS calculations should be evaluated at a distance of approximately one grid spacing inward from the immersed boundary. For a curved immersed boundary we present a procedure for selecting representative interfacial fluid stresses using the concepts from the Poiseuille flow test problem. For the final two model problems, steady state flow over a bump in a channel and unsteady peristaltic pumping, we present an 'exclusion filtering' technique for accurately measuring stresses. Using this technique, these studies show that the immersed boundary method can provide reliable approximations to interfacial stresses.

3-D retinal surface inference: stereo or monocular fundus camera?

Three-dimensional (3-D) imaging of the eye fundus, and in particular of the optic disc, is widely used to assess glaucoma progression over time. In the literature, 3-D images of the optic disc have been obtained from stereo and monocular fundus cameras. While stereo systems are the gold standard for optic disc examination, monocular systems are less expensive, and therefore of more practical use. This stimulated a thorough investigation of the limits and advantages of these two imaging modalities. Our conclusion is that monocular imaging is generally not suitable for 3-D estimation. This is attributed to the fact that monocular systems do not allow a change in the vantage point from which the retinal surface is observed, despite variations in the relative pose between the eye and the fundus camera. To validate this analysis we carry out several experiments on both stereo and monocular fundus cameras with standard 3-D reconstruction algorithms. Furthermore, we devise a calibration procedure to quantify experimentally the highest accuracy achievable with a stereo system.

Strain-specific differences in the effects of cyclosporin A and FK506 on the survival and regeneration of axotomized retinal ganglion cells in adult rats.

The immune response can influence neuronal viability and plasticity after injury, effects differing in strains of rats with different susceptibility to autoimmune disease. We assessed the effects of i.p. injections of cyclosporin A (CsA) or FK506 on adult retinal ganglion cell (RGC) survival and axonal regeneration into peripheral nerve (PN) autografted onto the cut optic nerve of rats resistant (Fischer F344) or vulnerable (Lewis) to autoimmune disease. Circulating and tissue CsA and FK506 levels were similar in both strains. Three weeks after autologous PN transplantation the number of viable beta-III tubulin-positive RGCs was significantly greater in CsA- and FK506-treated F344 rats compared with saline-injected controls. RGC survival in Lewis rats was not significantly altered. In F344 rats, retrograde labeling of RGCs revealed that CsA or FK506 treatment significantly increased the number of RGCs that regenerated an axon into a PN autograft; however these agents had no beneficial effect on axonal regeneration in Lewis rats. PN grafts in F344 rats also contained comparatively more pan-neurofilament immunoreactive axons. In both strains, 3 weeks after transplantation CsA or FK506 treatment resulted in increased retinal macrophage numbers, but only in F344 rats was this increase significant. At this time-point PN grafts in both strains contained many macrophages and some T cells. T cell numbers in Lewis rats were significantly greater than in F344 animals. The increased RGC axonal regeneration seen in CsA- or FK506-treated F344 but not Lewis rats shows that modulation of immune responses after neurotrauma has complex and not always predictable outcomes.

AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

Lengthening and transfer of hamstrings for a flexion deformity of the knee in children with bilateral cerebral palsy: technique and preliminary results.

Between July 2000 and April 2004, 19 patients with bilateral spastic cerebral palsy who required an assistive device to walk had combined lengthening-transfer of the medial hamstrings as part of multilevel surgery. A standardised physical examination, measurement of the Functional Mobility Scale score and video or instrumented gait analysis were performed pre- and post-operatively. Static parameters (popliteal angle, flexion deformity of the knee) and sagittal knee kinematic parameters (knee flexion at initial contact, minimum knee flexion during stance, mean knee flexion during stance) were recorded. The mean length of follow-up was 25 months (14 to 45). Statistically significant improvements in static and dynamic outcome parameters were found, corresponding to improvements in gait and functional mobility as determined by the Functional Mobility Scale. Mild hyperextension of the knee during gait developed in two patients and was controlled by adjustment of their ankle-foot orthosis. Residual flexion deformity > 10 degrees occurred in both knees of one patient and was treated by anterior distal femoral physeal stapling. Two children also showed an improvement of one level in the Gross Motor Function Classification System.

Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro.

In vivo, transplanted adult olfactory ensheathing glia (OEG) and adult Schwann cells (SC) can support the regrowth of at least some transected axons within adult CNS neuropil. In the present study, we developed an in vitro adult rat retinal explant model to explore the influence of primary adult SC and OEG on retinal ganglion cell (RGC) neurite regrowth in the presence of glial cells endogenous to the retina. Retinal quadrants were plated RGC-side down onto aclar hats coated with either pure collagen (type 1), collagen with OEG, collagen with SCs, or collagen coated with both OEG and SCs. Regrowing retinal neurites extended onto the pure collagen substrate, largely in association with astrocytes that migrated out from the explants (mean number of neurites: 144+/-65 SEM). The additional presence of OEG (669+/-122), but not SCs (97+/-41), supported the regrowth of significantly greater numbers of RGC neurites. Furthermore, this OEG-stimulated regeneration was over significantly greater distances; >68% of neurites extended >500 microm from the explant, compared with explants plated onto SCs or collagen alone (15% and 29%, respectively). When OEG and SCs were co-cultured the number of regenerating neurites was reduced (397+/-81) compared with the pure OEG treatment. Analysis of explants on pure collagen substrates fed with media conditioned by purified OEG or SC showed no increase in neurite outgrowth compared with control treatments, suggesting that the enhanced growth in the presence of OEG is a contact-mediated effect. The observed differences between the abilities of OEG and SC to support the growth of CNS-derived fibers in the presence of astrocytes support the suggestion that OEG may be better suited for direct transplantation into CNS neuropil following injury.