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The visual system is widely used as a model in which to study neurotrauma of the central nervous system and to assess the effects of experimental therapies. Adult mammalian retinal ganglion cell axons do not normally regenerate their axons for long distances following injury. Trauma to the visual system, particularly damage to the optic nerve or central visual tracts, causes loss of electrical communication between the retina and visual processing areas in the brain. After optic nerve crush or transection, axons degenerate and retinal ganglion cells (RGCs) are lost over a period of days. To promote and maintain axonal growth and connectivity, strategies must be developed to limit RGC death and provide regenerating axons with permissive substrates and a sustainable growth milieu that will ultimately provide long term visual function. This review explores the role olfactory glia can play in this repair. We describe the isolation of these cells from the olfactory system, transplantation to the brain, gene therapy and the possible benefits that these cells may have over other cellular therapies to initiate repair, in particular the stimulation of axonal regeneration in visual pathways. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Purpose: To assess the accuracy of human blood oximetry measurements in a model eye with a hyperspectral fundus camera., Methods: Seven human whole blood samples (two arterial, five venous) were obtained, the oxygen saturations measured with a CO oximeter, and the samples inserted into quartz tubes with internal diameters of 100 and 150 μm. The tubes (n = 20; ten 100 μm and ten 150 μm) were placed within a model eye in front of a background reflectance surface with reflectivities of 20%, 60%, and 99%. Spectral images at wavelengths between 500 and 650 nm were acquired with a hyperspectral fundus camera and analyzed with an oximetric model to calculate the oxygen saturation of blood within the tubes. The calculated oxygen saturations were compared with the measured oxygen saturations. The effects of the background reflectivity and tube size on the accuracy of the calculated oxygen saturations were evaluated., Results: Background reflectivity and tube size had no significant effect on the mean oxygen saturation difference (P = 0.18 and P = 0.99, respectively; repeated-measures, two-way ANOVA). The mean differences (SD) between the measured and calculated oxygen saturations in segments of the 100 and 150 μm tubes overlying the 20%, 60%, and 99% background reflectivities were (100 μm) -4.0% (13.4%), -6.4% (9.9%), and -5.5% (10.2%) and (150 μm) -5.3% (10.8%), -5.2% (10.7%), and -5.2% (10.9%), respectively., Conclusions: There was reasonable agreement between the measured oxygen saturation values and those calculated by the oximetry model. The oximetry model could be used to determine the functional health of the retina.

We describe the achromatization of Wollaston prisms to reduce the angular dispersion in the splitting angle. Analytical theory and ray-tracing modeling is presented. In an example application, a sixfold reduction in dispersion is achieved for wavelengths in the region 400 nm to 1.7 μm. Experimental proof of concept is demonstrated, and in an example application, the spectral dispersion of extended images recorded through cascaded Wollaston prisms is shown to be reduced by an order of magnitude.

Recent clinical trials have shown that the use of replication deficient viral vectors to genetically modify cells in the retina can be of therapeutic benefit in the treatment of certain inherited degenerative conditions that compromise photoreceptor, and hence visual, function. This review is focussed primarily on the use of recombinant adeno-associated viral (rAAV) vectors to target neurons in inner retina, specifically retinal ganglion cells (RGCs). Genetic modification of RGCs may be of value in various ophthalmic conditions in which there is documented loss of RGCs or damage to their centrally projecting axons. Such conditions include glaucoma, optic neuritis, vascular disruption or trauma, and neurological degenerative conditions such as Alzheimer's disease. Furthermore, because the retina and optic nerve (ON) form part of the CNS, the visual system is a useful experimental model in which to study the molecular and cellular mechanisms that underlie degenerative as well as regenerative responses of adult CNS neurons after injury. Gene therapy studies from a number of laboratories are first reviewed, involving not only rAAV-based treatments but also application of lentiviral and adenoviral vectors. Recent work from our own laboratory is then summarized, in which intravitreal injection of rAAV2 serotype vectors is used to introduce growth promoting genes into injured RGCs. rAAV encoding a secretable form of ciliary neurotrophic factor (CNTF) has proved to be particularly effective in promoting RGC survival and axon regeneration after optic nerve crush or after transection followed by a peripheral nerve autograft. In the latter situation we have found that RGCs and their regenerated axons are maintained for at least 15 months after the initial injury. We have also combined rAAV gene therapy with pharmacotherapy to determine if cAMP elevation and additional intravitreal injections of growth factors can act synergistically with vector-based delivery of growth-promoting genes.

We experimentally demonstrate a miniaturized zoom lens with a single moving element based on the concepts and analysis described in Opt. Express 17, 6118 (2009). We show that the implementation of either a cubic or a generalized cubic phase-modulation function makes miniaturization possible in addition to providing extended-depth-of-field imaging. We present recovered images for zoom lenses employing both phase-modulation functions and conclude that the generalized-cubic-phase function yields higher image quality without the artifacts present for the pure-cubic-phase function.

We show that phase aberrations in an imaging system can be mitigated using binary-amplitude masks that reduce destructive interference in the image spatial frequency domain. Appropriately designed masks increase the magnitude of the optical transfer function and prevent nulls. This offers a low-cost, transmission-mode alternative to phase correction as used in active and adaptive optics, without a restriction on the waveband of operation.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Traumatic injury to the central nervous system (CNS) is accompanied by the spreading damage of secondary degeneration, resulting in further loss of neurons and function. Partial transection of the optic nerve (ON) has been used as a model of secondary degeneration, in which axons of retinal ganglion cells in the ventral ON are spared from initial dorsal injury, but are vulnerable to secondary degeneration. We have recently demonstrated that early after partial ON injury, oxidative stress spreads through the ventral ON vulnerable to secondary degeneration via astrocytes, and persists in the nerve in aggregates of cellular debris. In this study, we show that diffuse transcranial irradiation of the injury site with far red to near infrared (NIR) light (WARP 10 LED array, center wavelength 670 nm, irradiance 252 W/m(-2), 30 min exposure), as opposed to perception of light at this wavelength, reduced oxidative stress in areas of the ON vulnerable to secondary degeneration following partial injury. The WARP 10 NIR light treatment also prevented increases in NG-2-immunopositive oligodendrocyte precursor cells (OPCs) that occurred in ventral ON as a result of partial ON transection. Importantly, normal visual function was restored by NIR light treatment with the WARP 10 LED array, as assessed using optokinetic nystagmus and the Y-maze pattern discrimination task. To our knowledge, this is the first demonstration that 670-nm NIR light can reduce oxidative stress and improve function in the CNS following traumatic injury in vivo.

The extensive period of retinal ganglion cell (RGC) neurogenesis in the rat is associated with a protracted sequence of arrival of their axons into central targets such as the superior colliculus (SC) (Dallimore et al., 2002). Using in utero 5-bromo-2'-deoxyrudine (BrdU) injections to label early (embryonic day (E) 15) or late (E18 or E19) born RGCs, we now show that E15 RGCs with axons that enter the SC prenatally undergo programmed cell death earlier than late-born RGCs whose axons only reach the SC late in the first postnatal week. These late-born RGCs do not begin to die until postnatal day (P) 5/6. Removal of retrograde trophic support by P1 SC ablation initially only affects E15 RGCs; however by P5 death of late-born RGCs is increased, confirming that a switch to target dependency is delayed in this cohort. In a further experiment it was found that, following complete rostral SC transection at P2, the proportion of post-lesion axons originating from E19 RGCs was significantly greater than the proportion that normally makes up the retinotectal projection. Thus, even in neonatal brain, uninjured late-arriving axons are more likely to grow across a lesion site than injured axons undergoing regeneration. To study if birth date also affects regenerative potential in adulthood, autologous peripheral nerve (PN) was grafted onto the cut optic nerve in mature BrdU labelled rats. We found that, compared to E15 RGCs, a significantly greater proportion of late-born RGCs survived axotomy, but comparatively fewer of these surviving E19 RGCs regrew an axon into a graft. In summary, this research shows that the birthdate of RGCs significantly impacts on their subsequent life history and response to injury. Understanding how developing central nervous system (CNS) neurons acquire dependency on target-derived trophic support may lead to new strategies for enhancing survival and regeneration in adult CNS., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Hybrid imaging systems employing cubic phase modulation in the pupil-plane enable significantly increased depth of field, but artifacts in the recovered images are a major problem. We present a parametric blind-deconvolution algorithm, based on minimization of the high-frequency content of the restored image that enables recovery of artifact-free images for a wide range of defocus. We show that the algorithm enables robust matching of the image recovery kernel with the optical point-spread function to enable, for the first time, optimally low noise levels in recovered images.

The oxidative folding of proteins in the secretory pathway involves the formation and isomerisation of disulphide bonds and is catalysed by foldases in the lumen of the endoplasmic reticulum (ER). The transfer of reducing equivalents, from disulphide bond formation, to oxygen involves the participation of thiol oxidases. Here, we describe the identification and functional characterisation of the eroA and ervA genes from Aspergillus niger, encoding functional orthologues of S. cerevisiae ERO1 and ERV2, respectively. The eroA gene encodes a product of 600 amino acids, EroA, and the ervA gene encodes a product of 215 amino acids, ErvA, both of which share common motifs and features with their S. cerevisiae orthologues. In contrast to Ero1p in S. cerevisiae, A. niger EroA appears to be retained in the ER lumen by a C-terminal retention motif. Real-time PCR analysis indicated that eroA is transcriptionally up-regulated in response to ER stress, whereas ervA is slightly down-regulated in response to DTT stress yet up-regulated in response to expression of a heterologous protein. Gene disruption studies indicated that, unlike ervA, eroA is essential for viability. When expressed in the thermosensitive S. cerevisiae ero1-1 strain, both eroA and ervA were able to complement the temperature and DTT sensitive phenotype, although a truncated eroA, missing the putative HEEL ER-retention signal was unable to complement as well as the full-length eroA gene., ((c) 2010 Elsevier B.V. All rights reserved.)

We demonstrate an experimental method to obtain a continuously variable hybrid imaging system that uses two generalized cubic phase masks, to enable real-time optimization of the trade between extended depth-of-field and noise gain. We obtain point-spread functions as a function of the rotation angle and show an example of optimization based on recovered image quality.

Interferometric synthetic aperture arrays (SAA) can be refocused at any range via digital processing of the raw data, called the visibility function. Such systems are sensitive, however, to ranging errors in digital refocusing. They, therefore, exhibit a limited depth of focus, because these errors are equivalent to introducing defocus in the system. We derive an analytical expression for the Strehl ratio of synthetic aperture arrays, which accounts for both the antenna voltage pattern and the focal shift factor encountered in imaging systems with a low Fresnel number. Assessment of the depth of focus of short-range imaging arrays is allowed and is illustrated for common array designs.

This study examined inter-rater reliability of the Functional Mobility Scale (FMS) for children with cerebral palsy (CP) and the presence of rater bias. A consecutive sample of 118 children with CP, 2-18 years old (mean 10.3 years, SD 3.6), was recruited from a hospital setting. Children were classified using the gross motor function classification system (GMFCS) with 13 in Level I, 49 in Level II, 44 in Level III, and 12 in Level IV. Each child was independently scored on the FMS by two raters. Raters were randomly assigned from a sample of 44 orthopaedic surgeons, hospital-based physiotherapists, and community-based physiotherapists. Quadratic weighted kappa coefficients for mobility ratings varied from 0.86 to 0.92 for the three distances, indicating substantial chance corrected agreement. Levels of agreement were similar when administering the scale in person and by telephone, suggesting that the FMS can be administered by either method. There was a tendency for surgeons to rate mobility higher than physiotherapists, however, only one of the comparisons was statistically significant. The FMS is a reliable tool that can be used by clinicians to assess mobility in children with CP.

Several phase-modulation functions have been reported to decrease the aberration variance of the modulation-transfer-function (MTF) in aberration-tolerant hybrid imaging systems. The choice of this phase-modulation function is crucial for optimization of the overall system performance. To prevent a significant loss in signal-to-noise ratio, it is common to enforce restorability constraints on the MTF, requiring trade of aberration-tolerance and noise-gain. Instead of optimizing specific MTF characteristics, we directly minimize the expected imaging-error of the joint design. This method is used to compare commonly used phase-modulation functions: the antisymmetric generalized cubic polynomial and fourth-degree rotational symmetric phase-modulation. The analysis shows how optimal imaging performance is obtained using moderate phase-modulation, and more importantly, the relative merits of the above functions.

We present the first analytical analysis of image artifacts in defocused hybrid imaging systems that employ a cubic phase-modulation function. We show that defocus artifacts have the form of image replications and are caused by a net phase modulation of the optical transfer function. Both numerical simulations and experimental images are presented that exhibit replication artifacts that are compatible with the analytical expressions.

Secondary degeneration is a process encompassing damage adjacent to a primary injury, usually involving increased Ca(2+) influx into neurons and glia. Lomerizine dihydrochloride is a calcium channel blocker with relatively selective CNS effects, currently in clinical trials for glaucoma. We have recently demonstrated that, following partial transection of the optic nerve (ON), 1 month of lomerizine treatment protects retinal ganglion cells (RGCs), incompletely preserves visual function and also limits elements of secondary degeneration, including macrophage infiltration. However, under some circumstances macrophages have been shown to have different supportive effects on RGC protection and regeneration, casting doubt on the benefit of longer term therapies that reduce macrophage numbers. Here, we determined whether shorter treatment times (1 day or 1 week) result in improved effects on RGC survival and visual function, and whether benefits are maintained after cessation of treatment. We demonstrate that 1 month of lomerizine is the minimum period required to restore the fast reset phase of the optokinetic nystagmus and maintain it for a further 2 months after cessation of treatment (p>0.05, not different from normal). While 1 week of lomerizine treatment results in temporary recovery of numbers of fast reset phases, the recovery is not maintained after treatment cessation. Similarly, protection of RGC densities requires 1 month of lomerizine treatment, but protection is not maintained after treatment cessation. Importantly, none of the lomerizine treatment protocols resulted in full restoration of visual function, confirming the necessity of combining lomerizine with other treatment modalities., (Copyright 2009 Elsevier Inc. All rights reserved.)

A snapshot multi-spectral imaging technique is described which employs multiple cascaded birefringent interferometers to simultaneously spectrally filter and demultiplex multiple spectral images onto a single detector array. Spectral images are recorded directly without the need for inversion and without rejection of light and so the technique offers the potential for high signal-to-noise ratio. An example of an eight-band multi-spectral movie sequence is presented; we believe this is the first such demonstration of a technique able to record multi-spectral movie sequences without the need for computer reconstruction.

Secondary degeneration in the central nervous system involves indirect damage to neurons and glia away from the initial injury. Partial transection of the dorsal optic nerve (ON) results in precise spatial separation of the primary trauma from delayed degenerative events in ventrally placed axons and parent somata. Here we conduct an immunohistochemical survey of secondary cellular changes in and around axons and their parent retinal ganglion cell (RGC) somata during the first 3 days after a restricted, dorsal ON transection. This is before the secondary loss of RGCs and axons projecting through the uninjured, ventral portion of the ON. Within 5 min, manganese superoxide dismutase (MnSOD; a marker of oxidative stress) co-localizes within the astrocytic network across the entire profile of the ON. Secondary astrocyte hypertrophy of immunofluorescent labeling was evident from 3 h, with sustained increases in myelin basic protein immunoreactivity across the nerve by 24 h. Increases in NG-2-positive oligodendrocyte precursor cells, ED-1-positive activated microglia/macrophages, and Iba1-positive reactive resident microglia/macrophage numbers were only seen in ON vulnerable to secondary degeneration by 3 days. Changes within RGC somata exclusively vulnerable to secondary degeneration were detected at 24 h, as evidenced by increases in MnSOD immunoreactivity, followed by increases in c-jun immunoreactivity at 3 days. Treatment with the voltage-gated calcium channel blocker lomerizine did not alter any measured outcome. We conclude that oxidative stress spreading via the astrocytic network and from injured axons to parent RGC somata is an early event during secondary degeneration, and containment is likely to be required in order to prevent further damage to the nerve.

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.

The retina sends spatially ordered visual information to the superior colliculus (SC) directly and indirectly via the thalamus and primary visual cortex (V1). Gradients of Ephs and ephrins are present in all of these regions, and have been shown to be involved in establishing topography of at least some of these interconnected visual pathways. Studies in ephrin-A knockout mice show that abnormal retinotectal termination zones (TZs) are present in a majority of mice lacking (-/-) ephrin-A2 (57%), and ephrin-A2 and -A5 (89%). A similar but seemingly less disordered pattern is detected in the retina-to-dorsal lateral geniculate nucleus (dLGN) and dLGN-to-V1 projections. Here we analyse the dLGN-to-V1 and V1-to-SC projections in ephrin-A(-/-) mice to determine the extent to which topographic errors are transmitted across synaptic relays. Fluorescent tracers were injected into V1 of wild-type (WT), ephrin-A2(-/-) or ephrin-A2A5(-/-) mice. We examined the number, location and size of anterograde TZs in SC, and mapped the distribution of retrogradely labelled neurons in dLGN. Compared with WT and ephrin-A2(-/-) mice, the volume of individual TZs in the SC was smaller in ephrin-A2A5(-/-) mice (P = 0.002). Single V1 injections labelled two foci of dLGN neurons in 70%, and two SC TZs in 80% of ephrin-A2A5(-/-) mice. Abnormalities in one or other of the projections were detected in 10% of ephrin-A2(-/-) mice. Importantly, there was no consistent correspondence between the organization of geniculocortical and corticotectal projections in either genotype, suggesting a role for ephrin-As in maintaining topographic organization in register across multiple interconnected central visual pathways.

We describe the use of wavefront coding for the mitigation of optical aberrations in a thermal imaging system. Diffraction-limited imaging is demonstrated with a simple singlet which enables an approximate halving in length and mass of the optical system compared to an equivalent two-element lens.

Purpose: After partial optic nerve (ON) injury, intact retinal ganglion cells (RGCs) undergo secondary death, but the topographic distribution of this death is unknown, and it is unclear which cell death pathways are involved. Although the calcium channel blocker lomerizine reduces RGC death after partial ON injury, it is unknown whether this drug alleviates necrotic or apoptotic death., Methods: The dorsal ON was transected in adult Piebald-Virol-Glaxo (PVG) rats, and the site of secondary RGC death was determined using anterograde and retrograde DiI tracing. RGC death was assessed at 2 and 3 weeks. Retrograde tracing with fluorogold injected into the superior colliculus 3 days before euthanatization was used to identify RGCs undergoing secondary death. Overall cell loss was quantified using betaIII-tubulin immunohistochemistry. Lomerizine (30 mg/kg, oral) or vehicle was given twice daily, and retinal wholemounts were analyzed for necrotic morphology (nucleic acid stain) or anticleaved caspase-3 expression at 2 and 3 weeks., Results: Ventral retina was identified as the site of secondary RGC death, and central and dorsal retinae were defined as sites of both primary and secondary death. Overall RGC loss occurred by 2 weeks in central and ventral retina (P < 0.05) and by 3 weeks in dorsal retina (P < 0.05). Secondary RGC death was characterized mainly by necrotic morphology, with caspase-3 expression in some RGCs. Lomerizine reduced secondary necrosis at 2 weeks and secondary caspase-3 expression at 3 weeks., Conclusions: Lomerizine had differential effects on necrotic and apoptotic death with time, but its inability to completely prevent secondary death suggests that full neuroprotection will require combinatorial treatments.

The adult olfactory epithelium has maintained the ability to reconstitute its olfactory sensory neurons (OSNs) from a basal progenitor cell compartment. This allows for life-long turnover and replacement of receptor components as well as repair of the primary olfactory pathway in response to injury and environmental insults. The present study investigated whether fibulin-3, a glycoprotein in the extracellular matrix and binding partner of tissue inhibitor of metalloproteinases-3 (TIMP-3), plays a role in ongoing plasticity and regenerative events in the adult primary olfactory pathway. In wild-type control mice, fibulin-3 protein was detected on IB4(+)CD31(+) blood vessels, nerve fascicles and the basement membrane underneath the olfactory epithelium. After target ablation (olfactory bulbectomy), fibulin-3 was also abundantly present in the central nervous system (CNS) scar tissue that occupied the bulbar cavity. Using two different lesion models, i.e. intranasal Triton X-100 lesion and olfactory bulbectomy, we show that fibulin-3 deficient (Efemp1(-/-)) mice have impaired recovery of the olfactory epithelium after injury. Ten days post-injury, Efemp1(-/-) mice showed altered basal stem/progenitor cell proliferation and increased overall numbers of mature (olfactory marker protein (OMP) -positive) versus immature OSNs. However, compromised regenerative capacity of the primary olfactory pathway in Efemp1(-/-) mice was evidenced by reduced numbers of mature OSNs at the later time point of 42 days post-injury. In addition to these neural differences there were consistent changes in blood vessel structure in the olfactory lamina propria of Efemp1(-/-) mice. Overall, these data suggest a role for fibulin-3 in tissue maintenance and regeneration in the adult olfactory pathway.

Injured adult retinal ganglion cells (RGCs) regrow axons into peripheral nerve (PN) grafted onto cut optic nerve. Survival and regeneration of RGCs is increased by intraocular injections of ciliary neurotrophic factor (CNTF) and axonal regeneration is further enhanced by co-injection of a cyclic AMP analogue (CPT-cAMP). Based on these data, and because cytokine signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins, we set out to determine whether CNTF injections increase retinal SOCS expression and whether any changes are attenuated by co-injection with CPT-cAMP. Using quantitative PCR we found increased SOCS1, SOCS2 and SOCS3 mRNA levels at various times after a single CNTF injection. Expression remained high for many days. SOCS protein levels were also increased. In situ hybridization revealed that RGCs express SOCS3 mRNA, and SOCS expression in cultured RGCs was increased by CNTF. Co-injection of CPT-cAMP reduced CNTF induced expression of SOCS1 and SOCS3 mRNA and decreased SOCS3 protein expression. CNTF injection also transiently increased retinal leukemia inhibitory factor (LIF) expression, an effect that was also moderated by CPT-cAMP. We propose that, along with known reparative effects of elevated cAMP on neurons, reducing SOCS upregulation may be an additional way in which cyclic nucleotides augment cytokine-induced regenerative responses in the injured CNS.

To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages., (Copyright 2009 Wiley-Liss, Inc.)

For over a decade, a number of social work scholars have advocated for an Africentric paradigm shift in social work practice with African Americans; yet the paradigm shift has been slow in coming with respect to infusing Africentric theory and interventions into social work practice, education, and research. Interventions that infuse Africentric values (such as interdependence, collectivism, transformation, and spirituality) have been shown to create significant change across a number of areas important to social work practice with African Americans. However, a barrier to the full integration of Africentric models into social work practice is that Africentric programs lack cohesive documentation and replication and, thus, have limited potential to be established as evidence-based practices. The authors present an overview of various Africentric interventions, including their program components and methods of evaluation, with the aim of establishing guideposts or next steps in developing a discourse on Africentric interventions that are promising best practices or are emerging as evidence-based practices. The authors conclude with implications for social work practice, education, and research and a call for Africentric scholars to engage in increased discussion, dissemination of manualized treatments, and collaborative research to build the evidence-based Africentric knowledge base and foster replication of studies.

Purpose of Review: To discuss the relationship between androgens, cognition and Alzheimer's disease., Recent Findings: It has been found that low circulating levels of androgens are a risk factor for Alzheimer's disease. Decreased circulating androgens are also associated with declining cognitive performance, particularly in memory-related tasks. Conversely, androgen supplementation to hypogonadal men results in improved memory performance. It has therefore been hypothesized that androgen supplementation may be beneficial in Alzheimer's disease. In recent studies, animal models have been used to elucidate the molecular mechanism behind this relationship between androgens and Alzheimer's disease. These studies have shown that androgen depletion results in increased levels of beta amyloid and hyperphosphorylated tau, changes which are thought to be associated with subsequent neuronal death., Summary: Androgen depletion results in molecular changes associated with Alzheimer's disease. Further human trials are needed to determine whether androgen modulating therapy for Alzheimer's disease has clinical significance.

We present an analysis of single-moving-element zoom lenses in the thin-lens limit and show how the length of these zoom lenses is determined by the zoom-factor, sensor-dimension and the depth-of-focus. By decreasing the sensor size and extending the depth-of-focus, the lengths of these zoom lenses can be reduced significantly. As an example we present a ray-traced design of a miniaturized single-moving-element zoom lens with a 2.3 x zoom-factor and show how the exploitation of modern miniaturized detector array combined with wavefront coding enables a reduction in length of almost three orders-of-magnitude to 10mm.

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.

Secondary degeneration is a form of 'bystander' damage that can affect neural tissue both nearby and remote from an initial injury. Partial optic nerve transection is an excellent model in which to unequivocally differentiate events occurring during secondary degeneration from those resulting from primary CNS injury. We analysed the primary injury site within the optic nerve (ON) and intact areas vulnerable to secondary degeneration. Areas affected by the primary injury showed morphological disruption, loss of beta-III tubulin axonal staining, reduced myelinated axon density, greater proteoglycan expression (phosphacan), increased microglia and macrophage numbers and increased oxidative stress. Similar, but less extreme, changes were seen in areas of the optic nerve undergoing secondary degeneration. The CNS-specific L- and T-type calcium channel blocker lomerizine alleviated some of the changes in areas vulnerable to secondary degeneration. Lomerizine reduced morphological disruption, oxidative stress and phosphacan expression, and limited early increases in macrophage numbers. However, lomerizine failed to prevent progressive de-myelination of ON axons. Within the retina, secondary retinal ganglion cell (RGC) death was significant in areas vulnerable to secondary degeneration. Lomerizine protected RGCs from secondary death at 4 weeks but did not fully restore behavioural function (optokinetic nystagmus). We conclude that blockade of calcium channels is neuroprotective and limits secondary degenerative changes following CNS injury. However such an approach may need to be combined with other treatments to ensure long-term maintenance of full visual function.

The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. A specialized glial cell within the olfactory nerve, called olfactory ensheathing cell (OEC), and their associated extracellular matrix are thought to play an important role during regenerative events in this system. To gain insight into novel molecules that could mediate the OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments were conducted which revealed high expression of the glycoprotein fibulin-3 in OECs. This observation was confirmed with quantitative PCR. In vivo, the distribution of all members of the fibulin family, fibulin-3 included, was localized to the lamina propria underneath the olfactory epithelium, in close association within olfactory nerve bundles. To manipulate fibulin-3 gene expression in cultured OECs, lentiviral vector constructs were designed to either transgenically express or knock-down fibulin-3. Experimental data showed that increased levels of fibulin-3 induced profound morphological changes in cultured OECs, impeded with their migratory abilities and also suppressed OEC-mediated neurite outgrowth. Knock-down of fibulin-3 levels resulted in reduced OEC proliferation. In conclusion, the data provide novel insights into a putative role for fibulin-3 in the regulation of cell migration and neurite outgrowth within the primary olfactory pathway.

We here describe various approaches using GFP that are being used in the morphological and functional analysis of specific cell types in the normal and injured central nervous system. Incorporation of GFP into viral vectors allows phenotypic characterization of transduced cells and can be used to label their axons and terminal projections. Characterization of transduced cell morphology can be enhanced by intracellular injection of living GFP-labeled cells with appropriate fluorescent dyes. Ex vivo labeling of precursor or glial cells using viral vectors that encode GFP permits long-term identification of these cells after transplantation into the brain or spinal cord. In utero electroporation methods result in expression of gene products in developing animals, allowing both functional and morphological studies to be carried out. GFPCre has been developed as a marker gene for viral vector-mediated expression of the bacterial recombinase Cre in the brain of adult mice with "floxed" transgenes. GFPCre-mediated induction of transgene expression can be monitored by GFP expression in defined populations of neurons in the adult brain. Finally, GFP can be used to tag proteins, permitting dynamic visualization of the protein of interest in living cells.

The mature CNS has limited intrinsic capacity for repair after injury; therefore, strategies are needed to enhance the viability and regrowth of damaged neurons. Here we review gene therapy studies in the eye, aimed at improving the survival and regeneration of injured retinal ganglion cells (RGCs). To target RGCs most current methods use recombinant adeno-associated viral vectors (AAV), usually serotype-2 (AAV2), that are injected into the vitreal chamber of the eye. This vector provides long-term transduction of adult RGCs. Strong, constitutive promoters such as CMV and/or beta-actin are commonly used but cell-specific promoters have also been tested. Transgenes encoded by AAV have been selected to limit cell death, enhance growth factor expression, or promote growth cone responsiveness. We have assessed the effects of AAV vectors in adult rodent models (i) after optic nerve (ON) crush and (ii) after transplantation of peripheral nerve (PN) onto the cut ON, a procedure that induces injured RGCs to regenerate axons over longer distances. AAV-CNTF-GFP promotes RGC survival and axonal regrowth in mice after ON crush, and in rats after ON crush or PN transplantation. In rats, intravitreal injection of AAV-BDNF-GFP also increases RGC viability but does not promote regeneration. RGC viability and axonal regrowth is further enhanced when AAV-CNTF-GFP is injected into transgenic mice that over-express bcl-2. Reconstituted PN grafts containing Schwann cells that were transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of CNTF support RGC axonal regrowth, however grafts containing Schwann cells transduced with LV-BDNF or LV-GDNF are less successful. We have also quantified the transduction efficiency and tropism of different AAV vectors injected intravitreally. AAV 2/2 and AAV 2/6 showed highest levels of transduction, AAV 2/8 the lowest, and each serotype displayed different transduction profiles for retinal cells. We are also studying the long-term impact of AAV2-mediated CNTF or BDNF expression on the dendritic morphology of RGCs in normal and PN grafted retinas. Analysis of regenerating RGCs intracellularly injected with lucifer yellow indicates gene-specific changes in dendritic structure that likely impact upon visual function.

In order to promote neurite outgrowth, we constructed a plasmid producing RhoA-specific small hairpin RNA (shRNA) to block RhoA expression and tested its actions in PC12 cells. Our results show that the shRNA-mediated RNA interference (RNAi) successfully suppressed RhoA expression. As a consequence of RhoA knockdown, F-actin levels were significantly reduced and processes were markedly induced. These processes express two neurite markers, neurofilament and betaIII tubulin. This study demonstrates that plasmid-producing shRNA specific for RhoA can act as an effective tool to induce neurite outgrowth and further confirms the neurite growth-inhibitory role of RhoA. This shRNA may thus be a useful tool in promoting neurite outgrowth and could be applicable in neural repair after CNS injury.

The immune response after neural injury influences the survival and regenerative capacity of neurons. In the primary visual pathway, previous studies have described beneficial effects of macrophages and T-cells in promoting neural survival and axonal regeneration in some rat strains. However, the contributions of specific cell populations to these responses have been unclear. In adult Fischer (F344) rats, we confirm prior reports that intravitreal macrophage activation promotes the survival of retinal ganglion cells (RGCs) and greatly enhances axonal regeneration through a peripheral nerve graft. Neonatal thymectomy that results in elimination of T-cell production enhanced RGC survival after axotomy, but diminished the effect of intravitreal macrophage activation on axon regeneration. Thus, in F344 rats, lymphocytes appear to suppress RGC survival but augment the pro-regenerative effects of macrophages. The cytotoxic effect of lymphocytes on RGCs was confirmed in in vitro studies; coculture of retinal explants with lymphocytes led to a 60% reduction in viable RGCs. Similar in vivo results were obtained in Sprague Dawley rats. By comparison, in adult Lewis rats, neither RGC survival nor axonal regeneration was increased after intravitreal macrophage activation. Neonatal thymectomy had only a small beneficial effect on RGC survival, and although Lewis lymphocytes reduced RGC viability in culture, they did so to a lesser extent. Thus, in addition to a complex role of lymphocytes, particularly T-cells, after central nervous system injury, the present results demonstrate that the impact of macrophages is also influenced by genetic background.

When grafted onto the cut optic nerve, chimeric peripheral nerve (PN) sheaths reconstituted with adult Schwann cells (SCs) support the regeneration of adult rat retinal ganglion cell (RGC) axons. Regrowth can be further enhanced by using PN containing SCs transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of ciliary neurotrophic factor (CNTF). To determine whether other neurotrophic factors or different cell types also enhance RGC regrowth in this bridging model, we tested the effectiveness of (1) adult SCs transduced with brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF), and (2) fibroblasts (FBs) genetically modified to express CNTF. SCs transduced with LV-BDNF and LV-GDNF secreted measurable and bioactive amounts of each of these proteins, but reconstituted grafts containing LV-BDNF or LV-GDNF transduced SCs did not enhance RGC survival or axonal regrowth. LV-BDNF modified grafts did, however, contain many pan-neurofilament immunolabeled axons, many of which were also immunoreactive for calcitonin gene-related peptide (CGRP) and were presumably of peripheral sensory origin. Nor-adrenergic and cholinergic axons were also seen in these grafts. There were far fewer axons in LV-GDNF engineered grafts. Reconstituted PN sheaths containing FBs that had been modified to express CNTF did not promote RGC viability or regeneration, and PN reconstituted with a mixed population of SCs and CNTF expressing FBs were less effective than SCs alone. These data show that both the type of neurotrophic factor and the cell types that express these factors are crucial elements when designing bridging substrates to promote long-distance regeneration in the injured CNS.

Alzheimer's disease is a progressive neurodegenerative disorder characterised by abnormal extracellular deposition of a 4 kDa peptide termed beta-amyloid, neuronal loss, oxidative stress and chronic astrocytosis and microgliosis, but how the latter two features contribute to the progression of the disease is poorly understood. We have previously demonstrated in a novel in vivo transplantation model that chronic astro- and microgliosis resulted in molecular pathology similar to that observed in the Alzheimer's disease brain. We now report that these heterotopic, gliotic transplants exhibit prolonged oxidative stress, characterised by lipid peroxidation and protein carbonyl formation. Furthermore, we demonstrate that dietary additives can elevate endogenous anti-oxidant defences and reduce oxidative stress without attenuating astro- and microgliosis. We also show that administration of ibuprofen through the drinking water results in a similar reduction in oxidative stress but with no observable effect on glial reactivity. The present study lends support to the notion that dietary anti-oxidants and non-steroidal anti-inflammatory drugs may be potential preventative agents against some of the pathological processes associated with neurodegenerative disease.

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.

We used an in vivo transplant approach to examine how adult Schwann cells and olfactory ensheathing glia OEG influence the specificity of axon-target cell interactions when they are introduced into the CNS. Populations of either Schwann cells or OEG were mixed with dissociated fetal tectal cells presumptive superior colliculus and, after reaggregation, pieces were grafted onto newborn rat superior colliculus. Both glial types were prelabeled with lentiviral vectors encoding green fluorescent protein. Grafts rapidly established fiber connections with the host and retinal projections into co-grafts were assessed 656 days posttransplantation by injecting cholera toxin B into host eyes. In control rats that received pure dissociated-reaggregated tectal grafts, retinal ganglion cell RGC axons selectively innervated defined target areas, corresponding to the retinorecipient layer in normal superior colliculus. The pattern of RGC axon ingrowth into OEG containing co-grafts was similar to that in control grafts. However, in Schwann cell co-grafts there was reduced host retinal input into presumptive target areas and many RGC axons were scattered throughout the neuropil. Given that OEG in co-grafts had minimal impact on axon-target cell recognition, OEG might be an appropriate cell type for direct transplantation into injured neuropil when attempting to stimulate specific pathway reconstruction.

Extracellular recordings were obtained from single cochlear nucleus neurons in guinea pigs anesthetized with Nembutal and Hypnorm. Neurons were classified by their spontaneous firing rates and responses to acoustic stimuli. In addition, electrical shocks were applied to the midline at the level of the IVth ventricle and spike responses were recorded. Spikes were evoked by shocks only in neurons that were classified as onset choppers (O(c)). The shock-evoked spikes could be extinguished by acoustically evoked action potentials in the same neurons. In roughly 30% of the sample of O(c) neurons, quantitative aspects of the timing of this extinction were not compatible with the shock-evoked spike being antidromically conducted from O(c) output axons. Together with the presence of temporal jitter at high shock rates, the data suggest the possibility that at least some of the shock-evoked spikes may be generated by excitatory synaptic input to the O(c) neurons, most likely from the collaterals of the medial olivocochlear system (MOCS), whose axons pass close to the floor of the IVth ventricle. This excitatory synaptic input may operate to modulate the activity of O(c) neurons in addition to MOCS actions in the auditory periphery.

We tested whether combined therapy involving Rho inactivation, elevation of cAMP and supply of ciliary neurotrophic factor (CNTF) (i) increased axotomized adult retinal ganglion cell (RGC) survival and (ii) promoted axonal regeneration into peripheral nerve (PN) autografted onto the cut optic nerve. PN-grafted eyes were injected with combinations of a Rho-inactivating enzyme C3 transferase (C3-11), CNTF and a cell-permeant analogue of cAMP (CPT-cAMP). Four weeks after PN transplantation, RGC survival was quantified using beta-III tubulin immunohistochemistry. Regeneration was assessed using retrograde fluorogold tracing and pan-neurofilament immunostaining of grafts. Treatment with C3-11 increased RGC survival but co-injection with CPT-cAMP, CNTF or combined CNTF/CPT-cAMP did not further enhance RGC viability. There were greater numbers of regenerating RGCs after multiple C3-11 injections and regeneration was further and significantly increased after intravitreal injections of all three factors. In the combined C3-11/CNTF/CPT-cAMP treatment group about 15% of RGCs remained viable of which more than half regenerated an axon. These data emphasize the power of combinatorial pharmacotherapeutic and transplant strategies in the treatment of neurotrauma.

Purpose: We tested whether microenvironmental changes surrounding apoptotic neural degeneration, cellular pre-treatment and timing of transplant can influence the survival and differentiation of transplanted cells. This was done by transplanting adult hippocampal precursor cells (AHPCs) into normal and retinal ganglion cell (RGC) depleted rat retinae., Methods: Apoptotic RGC death was induced in neonates by removal of the contralateral superior colliculus (SC) and in adults by unilateral optic nerve transection, with or without a peripheral nerve (PN) graft. AHPCs were transplanted 24 h after SC ablation, or 5, 7 or 14 days following optic nerve (ON) transection. Hosts received untreated grafts, or grafts treated by co-culture with embryonic retinal explants or the neuropeptide somatostatin., Results: AHPCs integrated within all neonatal and 65% of adult retinae. Greater numbers of AHPCs were observed within the ganglion cell layer (GCL) in SC lesioned hosts. Explant co-culture induced proliferation of grafted AHPCs within host retinae. Somatostatin-treatment resulted in reduced overall engraftment but increased integration within the GCL. In lesioned adults, greatest GCL engraftment was observed following 7 or 14 day grafts. Some AHPCs in the inner retina expressed neuronal antigens and extended processes into the ON., Conclusions: These data indicate that various factors can influence the behaviour of grafted cells and work towards encouraging the functional restoration of retinal circuitry.

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.

Background: The mature central nervous system (CNS) has limited capacity for self-renewal and repair after injury or neurodegeneration, and therapeutic strategies are needed to promote the viability of damaged neurons and the regrowth of their axons. The retina and optic nerve (ON) are part of the CNS, and the visual system is widely used in experimental studies on injury and repair., Objective: To test various cellular and molecular approaches in attempts to replace retinal ganglion cells (RGCs) in depleted retinas or, more usually, promote the survival of endogenous injured RGCs and stimulate axonal regeneration after ON or intracranial optic tract (OT) injury., Methods and Results: Intraocular injections of brain-derived neurotrophic factor and ciliary neurotrophic factor (CNTF) temporarily increase RGC survival after ON injury. More sustained neuroprotection is obtained using adeno-associated viral vectors to transfect RGCs with brain-derived neurotrophic factor or CNTF genes. After ON crush, intravitreal adeno-associated viral CNTF injections also increase RGC axonal regrowth. Additional protective and growth effects are obtained after intraocular elevation of cAMP and by manipulation of protein kinase signalling pathways in RGCs. Regeneration is increased by transplanting a segment of peripheral nerve onto the cut ON. Schwann cells in peripheral nerve grafts can be genetically modified using lentiviral vectors to over-express CNTF, resulting in increased regrowth of RGC axons. After OT lesions, hydrogels have been used to bridge the injury, sometimes with the incorporation of signalling peptides or cells genetically modified to express neurotrophic factors., Conclusions: There is now a general consensus that combinatorial approaches are needed to elicit sustained and effective regenerative responses in injured adult CNS neurons.

We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi-cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1-1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury.

Normal visual function in humans is compromised by a range of inherited and acquired degenerative conditions, many of which affect photoreceptors and/or retinal pigment epithelium. As a consequence the majority of experimental gene- and cell-based therapies are aimed at rescuing or replacing these cells. We provide a brief overview of these studies, but the major focus of this review is on the inner retina, in particular how gene therapy and transplantation can improve the viability and regenerative capacity of retinal ganglion cells (RGCs). Such studies are relevant to the development of new treatments for ocular conditions that cause RGC loss or dysfunction, for example glaucoma, diabetes, ischaemia, and various inflammatory and neurodegenerative diseases. However, RGCs and associated central visual pathways also serve as an excellent experimental model of the adult central nervous system (CNS) in which it is possible to study the molecular and cellular mechanisms associated with neuroprotection and axonal regeneration after neurotrauma. In this review we present the current state of knowledge pertaining to RGC responses to injury, neurotrophic and gene therapy strategies aimed at promoting RGC survival, and how best to promote the regeneration of RGC axons after optic nerve or optic tract injury. We also describe transplantation methods being used in attempts to replace lost RGCs or encourage the regrowth of RGC axons back into visual centres in the brain via peripheral nerve bridges. Cooperative approaches including novel combinations of transplantation, gene therapy and pharmacotherapy are discussed. Finally, we consider a number of caveats and future directions, such as problems associated with compensatory sprouting and the reformation of visuotopic maps, the need to develop efficient, regulatable viral vectors, and the need to develop different but sequential strategies that target the cell body and/or the growth cone at appropriate times during the repair process.

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.

We describe and characterize an experimental system to perform shape measurements on deformable objects using high-speed close-range photogrammetry. The eventual application is to extract the kinematics of several marked points on an insect wing during tethered and hovering flight. We investigate the performance of the system with a small number of views and determine an empirical relation between the mean pixel error of the optimization routine and the position error. Velocity and acceleration are calculated by numerical differencing, and their relation to the position errors is verified. For a field of view of approximately 40 mm x 40 mm, a rms accuracy of 30 mum in position, 150 mm/s in velocity, and 750 m/s2 in acceleration at 5000 frames/s is achieved. This accuracy is sufficient to measure the kinematics of hoverfly flight.

A quartic phase retardation function is described that reduces the variation of the intensity of the focal point of incoherent imaging systems suffering from primary third-order aberrations limited to coma and astigmatism. Corresponding modulation transfer functions are shown to remain practically invariant for moderate amounts of coma and astigmatism.