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1.  Wnt regulates proliferation and neurogenic potential of Müller glial cells through a Lin28/let-7 miRNA-dependent pathway in adult mammalian retina 
Cell reports  2016;17(1):165-178.
In cold-blooded vertebrates such as zebrafish, Müller glial cells (MGs) readily proliferate to replenish lost retinal neurons. In mammals, however, MGs lack regenerative capability as they do not spontaneously re-enter the cell cycle unless the retina is injured. Here, we show that gene transfer of β-catenin in adult mouse retina activates Wnt signaling and MG proliferation without retinal injury. Upstream of Wnt, deletion of GSK3β stabilizes β-catenin and activates MG proliferation. Downstream of Wnt, β-catenin binds to the Lin28 promoter and activates transcription. Deletion of Lin28 abolishes β-catenin-mediated effects on MG proliferation, and Lin28 gene transfer stimulates MG proliferation. We further demonstrate that let-7 miRNAs are critically involved in Wnt/Lin28-regulated MG proliferation. Intriguingly, a subset of cell cycle reactivated MGs express markers for amacrine cells. Together, these results reveal a key role of Wnt-Lin28-let7 miRNA signaling in regulating proliferation and neurogenic potential of MGs in adult mammalian retina.
In Brief
Müller glial cells (MGs) are a source of retinal stem cells. To overcome proliferation quiescence of MGs in adult mammalian retina, Yao et al. report that modulation of Wnt/Lin28/let-7 miRNA signaling stimulates MG proliferation without retinal injury. A subset of cell cycle reactivated MGs express markers for retinal interneurons.
PMCID: PMC5076887  PMID: 27681429
2.  Optogenetic Vision Restoration Using Rhodopsin for Enhanced Sensitivity 
Molecular Therapy  2015;23(10):1562-1571.
Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination.
PMCID: PMC4817926  PMID: 26137852
3.  Visual stimulation switches the polarity of excitatory input to starburst amacrine cells 
Neuron  2014;83(5):1172-1184.
Direction-selective ganglion cells (DSGCs) are tuned to motion in one direction. Starburst amacrine cells (SACs) are thought to mediate this direction selectivity through precise anatomical wiring to DSGCs. Nevertheless, we previously found that visual adaptation can reverse DSGCs’ directional tuning, overcoming the circuit anatomy. Here we explore the role of SACs in the generation and adaptation of direction selectivity. First, using pharmaco-genetics and two-photon calcium imaging, we validate that SACs are necessary for direction selectivity. Next, we demonstrate that exposure to an adaptive stimulus dramatically alters SACs’ synaptic inputs. Specifically, after visual adaptation, On-SACs lose their excitatory input during light onset but gain an excitatory input during light offset. Our data suggest that visual stimulation alters the interactions between rod and cone-mediated inputs that converge on the terminals of On cone BCs. These results demonstrate how the sensory environment can modify computations performed by anatomically-defined neuronal circuits.
PMCID: PMC4161675  PMID: 25155960
4.  Neuron-glia signaling in developing retina mediated by neurotransmitter spillover 
eLife  null;4:e09590.
Neuron-glia interactions play a critical role in the maturation of neural circuits; however, little is known about the pathways that mediate their communication in the developing CNS. We investigated neuron-glia signaling in the developing retina, where we demonstrate that retinal waves reliably induce calcium transients in Müller glial cells (MCs). During cholinergic waves, MC calcium transients were blocked by muscarinic acetylcholine receptor antagonists, whereas during glutamatergic waves, MC calcium transients were inhibited by ionotropic glutamate receptor antagonists, indicating that the responsiveness of MCs changes to match the neurotransmitter used to support retinal waves. Using an optical glutamate sensor we show that the decline in MC calcium transients is caused by a reduction in the amount of glutamate reaching MCs. Together, these studies indicate that neurons and MCs exhibit correlated activity during a critical period of retinal maturation that is enabled by neurotransmitter spillover from retinal synapses.
eLife digest
A structure at the back of the eye known as the retina is essential for vision. When light hits the retina, cells called neurons produce electrical signals that lead to the release of chemicals known as neurotransmitters. When these chemicals reach a neighboring neuron, a corresponding electrical signal is produced in this cell. In this way, information about what we can see is ultimately transmitted to the brain.
The retina also contains cells that are not neurons, such as Müller glial cells. These cells span the thickness of the retina, and appear well placed to interact with all of the different types of neuron in the retina. Other types of glial cells help the neural circuits in the brain to develop, but it is not clear whether Müller cells perform the same role in the developing retina.
Newborn mice do not open their eyes until around two weeks after they are born, during which time their retina continues to develop. The retinal neurons are already active throughout this period, and spontaneously generate periodic bursts of electrical activity that spreads across the developing retina in waves. Now, Rosa, Bos et al. show that in newborn mice, these waves also trigger brief, or ‘transient’, increases in the concentration of calcium ions inside Müller cells. Exactly how the Müller cells respond depends on which neurotransmitters are released during waves.
A few days before the mice open their eyes, the number of calcium transients in Müller cells decreases sharply. At the same time, neurons continue to spontaneously release waves of one particular neurotransmitter called glutamate. Rosa, Bos et al. used a sensor that showed where glutamate is found in the developing retina. This revealed that the decrease in calcium transients as the retina matures is due to less glutamate reaching the Müller cells.
These findings reveal that Müller cells are involved in the spontaneous electrical activity seen in the retina during a critical period of retinal development. The next challenge is to determine how this neuronal-glial cell signaling helps the retina to mature.
PMCID: PMC4566075  PMID: 26274565
iGluSnFR; GCaMP; retinal ganglion cell; mouse
5.  Advancing Therapeutic Strategies for Inherited Retinal Degeneration: Recommendations From the Monaciano Symposium 
Although rare in the general population, retinal dystrophies occupy a central position in current efforts to develop innovative therapies for blinding diseases. This status derives, in part, from the unique biology, accessibility, and function of the retina, as well as from the synergy between molecular discoveries and transformative advances in functional assessment and retinal imaging. The combination of these factors has fueled remarkable progress in the field, while at the same time creating complex challenges for organizing collective efforts aimed at advancing translational research. The present position paper outlines recent progress in gene therapy and cell therapy for this group of disorders, and presents a set of recommendations for addressing the challenges remaining for the coming decade. It is hoped that the formulation of these recommendations will stimulate discussions among researchers, funding agencies, industry, and policy makers that will accelerate the development of safe and effective treatments for retinal dystrophies and related diseases.
PMCID: PMC4554258  PMID: 25667399
retinal dystrophy; gene therapy; cell therapy; disease phenotypes; outcome measures
6.  Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina 
Biomedical Optics Express  2015;6(6):2191-2210.
Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.
PMCID: PMC4473753  PMID: 26114038
(170.4460) Ophthalmic optics and devices; (110.4500) Optical coherence tomography; (110.1080) Active or adaptive optics; (170.0110) Imaging systems; (330.7324) Visual optics, comparative animal models; (170.4470) Ophthalmology
7.  CRALBP supports the mammalian retinal visual cycle and cone vision 
Mutations in the cellular retinaldehyde–binding protein (CRALBP, encoded by RLBP1) can lead to severe cone photoreceptor–mediated vision loss in patients. It is not known how CRALBP supports cone function or how altered CRALBP leads to cone dysfunction. Here, we determined that deletion of Rlbp1 in mice impairs the retinal visual cycle. Mice lacking CRALBP exhibited M-opsin mislocalization, M-cone loss, and impaired cone-driven visual behavior and light responses. Additionally, M-cone dark adaptation was largely suppressed in CRALBP-deficient animals. While rearing CRALBP-deficient mice in the dark prevented the deterioration of cone function, it did not rescue cone dark adaptation. Adeno-associated virus–mediated restoration of CRALBP expression specifically in Müller cells, but not retinal pigment epithelial (RPE) cells, rescued the retinal visual cycle and M-cone sensitivity in knockout mice. Our results identify Müller cell CRALBP as a key component of the retinal visual cycle and demonstrate that this pathway is important for maintaining normal cone–driven vision and accelerating cone dark adaptation.
PMCID: PMC4319437  PMID: 25607845
8.  Antibody Neutralization Poses a Barrier to Intravitreal Adeno-Associated Viral Vector Gene Delivery to Non-Human Primates 
Gene therapy  2014;22(2):116-126.
Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that pre-existing immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene, or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, neutralizing antibody levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of pre-existing neutralizing antibody titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying, or no transgene expression following intravitreal administration of AAV. Investigating anti-AAV antibody development will aid in understanding the interactions between gene therapy vectors and the immune system during ocular administration and can form a basis for future clinical studies applying intravitreal gene delivery.
PMCID: PMC4393652  PMID: 25503696
9.  Viral-mediated RdCVF and RdCVFL expression protects cone and rod photoreceptors in retinal degeneration 
Alternative splicing of nucleoredoxin-like 1 (Nxnl1) results in 2 isoforms of the rod-derived cone viability factor. The truncated form (RdCVF) is a thioredoxin-like protein secreted by rods that promotes cone survival, while the full-length isoform (RdCVFL), which contains a thioredoxin fold, is involved in oxidative signaling and protection against hyperoxia. Here, we evaluated the effects of these different isoforms in 2 murine models of rod-cone dystrophy. We used adeno-associated virus (AAV) to express these isoforms in mice and found that both systemic and intravitreal injection of engineered AAV vectors resulted in RdCVF and RdCVFL expression in the eye. Systemic delivery of AAV92YF vectors in neonates resulted in earlier onset of RdCVF and RdCVFL expression compared with that observed with intraocular injection using the same vectors at P14. We also evaluated the efficacy of intravitreal injection using a recently developed photoreceptor-transducing AAV variant (7m8) at P14. Systemic administration of AAV92YF-RdCVF improved cone function and delayed cone loss, while AAV92YF-RdCVFL increased rhodopsin mRNA and reduced oxidative stress by-products. Intravitreal 7m8-RdCVF slowed the rate of cone cell death and increased the amplitude of the photopic electroretinogram. Together, these results indicate different functions for Nxnl1 isoforms in the retina and suggest that RdCVF gene therapy has potential for treating retinal degenerative disease.
PMCID: PMC4382269  PMID: 25415434
10.  Advances in AAV Vector Development for Gene Therapy in the Retina 
Adeno-associated virus (AAV) is a small, non-pathogenic dependovirus that has shown great potential for safe and long-term expression of a genetic pay-load in the retina. AAV has been used to treat a growing number of animal models of inherited retinal degeneration, though drawbacks—including a limited carrying capacity, slow onset of expression, and a limited ability to transduce some retinal cell types from the vitreous—restrict the utility of AAV for treating some forms of inherited eye disease. Next generation AAV vectors are being created to address these needs, through rational design efforts such as the creation of self-complementary AAV vectors for faster onset of expression and specific mutations of surface-exposed residues to increase transduction of viral particles. Furthermore, directed evolution has been used to create, through an iterative process of selection, novel variants of AAV with newly acquired, advantageous characteristics. These novel AAV variants have been shown to improve the therapeutic potential of AAV vectors, and further improvements may be achieved through rational design, directed evolution, or a combination of these approaches, leading to broader applicability of AAV and improved treatments for inherited retinal degeneration.
PMCID: PMC4267879  PMID: 24664759
Adeno-associated virus; Gene therapy; Mutagenesis; Directed evolution; Retinal degeneration
11.  Retinoschisin gene therapy in photoreceptors, Müller glia, or all retinal cells in the Rs1h−/− mouse 
Gene therapy  2014;21(6):585-592.
X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors, or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h−/− mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, though photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.
PMCID: PMC4047144  PMID: 24694538
Gene therapy; X-linked retinoschisis; AAV vectors; photoreceptors; Müller glia; cell targeting
12.  Imaging Light Responses of Foveal Ganglion Cells in the Living Macaque Eye 
The Journal of Neuroscience  2014;34(19):6596-6605.
The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses.
PMCID: PMC4012315  PMID: 24806684
calcium imaging; in vivo adaptive optics imaging; intrinsic signal imaging; primate fovea; retinal ganglion cells
13.  Targeting Müller Cell–Derived VEGF164 to Reduce Intravitreal Neovascularization in the Rat Model of Retinopathy of Prematurity 
To determine whether knockdown of Müller cell–derived VEGFA-splice variant, VEGF164, which is upregulated in the rat retinopathy of prematurity (ROP) model, safely inhibits intravitreal neovascularization (IVNV).
Short hairpin RNAs for VEGF164 (VEGF164.shRNAs) or luciferase.shRNA control were cloned into lentivectors with CD44 promoters that specifically target Müller cells. Knockdown efficiency, off-target effects, and specificity were tested in HEK reporter cell lines that expressed green fluorescent protein (GFP)-tagged VEGF164 or VEGF120 with flow cytometry or in rat Müller cells (rMC-1) by real-time PCR. In the rat oxygen-induced retinopathy (OIR) ROP model, pups received 1 μL subretinal lentivector-driven luciferase.shRNA, VEGFA.shRNA, or VEGF164.shRNA at postnatal day 8 (P8). Analyses at P18 and P25 included: IVNV and avascular retina (AVA); retinal and serum VEGF (ELISA); density of phosphorylated VEGFR2 (p-VEGFR2) in lectin-labeled retinal endothelial cells (ECs; immunohistochemistry); TUNEL staining and thickness of inner nuclear (INL) and outer nuclear layers (ONL) in retinal cryosections; and pup weight gain.
In HEK reporter and in rMC-1 cells and in comparison to lucifferase.shRNA, VEGFA.shRNA reduced both VEGF120 and VEGF164, but VEGF164.shRNA only reduced VEGF164 and not VEGF120. Compared with luciferase.shRNA, VEGFA.shRNA and VEGF164.shRNA reduced retinal VEGF and IVNV without affecting AVA at P18 and P25. At P25, VEGF164.shRNA more effectively maintained IVNV inhibition than VEGFA.shRNA. VEGFA.shRNA and VEGF164.shRNA reduced pVEGFR2 in retinal ECs at P18, but VEGFA.shRNA increased it at P25. VEGFA.shRNA increased TUNEL+ cells at P18 and decreased ONL thickness at P18 and P25. VEGFA.shRNA and VEGF164.shRNA did not affect pup weight gain and serum VEGF.
Short hairpin RNA to Müller cell VEGF164 maintained long-term inhibition of IVNV and limited cell death compared with shRNA to VEGFA.
Using a relevant model of current ROP, the rat 50/10 oxygen-induced retinopathy model, we determined that targeting Müller cell VEGF164 by lentivector-delivered shRNA achieved better inhibition of intravitreal neovascularization without causing retinal cell death compared to shRNA to VEGFA.
PMCID: PMC3920823  PMID: 24425851
vascular endothelial growth factor; lentivector; short hairpin RNA; intravitreal neovascularization; Müller cells
14.  Quantitative Analyses of Retinal Vascular Area and Density After Different Methods to Reduce VEGF in a Rat Model of Retinopathy of Prematurity 
Targeted inhibition of Müller cell (MC)–produced VEGF or broad inhibition of VEGF with an intravitreal anti-VEGF antibody reduces intravitreal neovascularization in a rat model of retinopathy of prematurity (ROP). In this study, we compared the effects of these two approaches on retinal vascular development and capillary density in the inner and deep plexi in the rat ROP model.
In the rat model of ROP, pups received 1 μL of (1) subretinal lentivector-driven short hairpin RNA (shRNA) to knockdown MC-VEGFA (VEGFA.shRNA) or control luciferase shRNA, or (2) intravitreal anti-VEGF antibody (anti-VEGF) or control isotype goat immunoglobulin G (IgG). Analyses of lectin-stained flat mounts at postnatal day 18 (p18) included: vascular/total retinal areas (retinal vascular coverage) and pixels of fluorescence/total retinal area (capillary density) of the inner and deep plexi determined with the Syncroscan microscope, and angles between cleavage planes of mitotic vascular figures labeled with anti-phosphohistone H3 and vessel length.
Retinal vascular coverage and density increased in both plexi between p8 and p18 in room air (RA) pups. Compared with RA, p18 ROP pups had reduced vascular coverage and density of both plexi. Compared with respective controls, VEGFA.shRNA treatment significantly increased vascular density in the deep plexus, whereas anti-VEGF reduced vascular density in the inner and deep plexi. Vascular endothelial growth factor-A.shRNA caused more cleavage angles predicting vessel elongation and fewer mitotic figures, whereas anti-VEGF treatment led to patterns of pathologic angiogenesis.
Targeted treatment with lentivector-driven VEGFA.shRNA permitted physiologic vascularization of the vascular plexi and restored normal orientation of dividing vascular cells, suggesting that regulation of VEGF signaling by targeted treatment may be beneficial.
Targeted inhibition of Müller cell (MC)-produced VEGF or broad inhibition of VEGF with anti-VEGF antibody reduces intravitreal neovascularization (IVNV) in the rat retinopathy of prematurity (ROP) model. We compared the effects of these two approaches on retinal vascular area and capillary density of the inner and deep plexi.
PMCID: PMC3915769  PMID: 24425858
VEGF; vascular extent; vascular density; intrvitreal neovscularization; rat model of retinopathy of prematurity
15.  Specific tools for targeting and expression in Müller glial cells 
Despite their physiological roles, Müller glial cells are involved directly or indirectly in retinal disease pathogenesis and are an interesting target for therapeutic approaches for retinal diseases and regeneration such as CRB1 inherited retinal dystrophies. In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells. ShH10Y and AAV9 were the most powerful capsids to infect mouse Müller glial cells. Retinaldehyde-binding protein 1 (RLBP1) promoter was the most powerful promoter to transduce Müller glial cells. ShH10Y capsids and RLBP1 promoter targeted human Müller glial cells in vitro. We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors. Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller glial cells. In summary, ShH10Y and AAV9 capsids, and RLBP1 or minimal CMV promoters are of interest as specific tools to target and express in mouse or human Müller glial cells.
PMCID: PMC4362388  PMID: 26015954
16.  Evaluation of patient-centered rehabilitation model targeting older persons with a hip fracture, including those with cognitive impairment 
BMC Geriatrics  2013;13:136.
The purpose of this study was to evaluate outcomes for older persons post-hip fracture repair, including those with cognitive impairment (CI), following implementation of a novel model of care – the Patient-Centered Rehabilitation Model including persons with CI (PCRM-CI). The PCRM-CI is an interdisciplinary rehabilitation program that incorporates education for healthcare professionals (HCPs), including nurses, which is focused on geriatric care including management of dementia and delirium, support for HCPs from an Advanced Practice Nurse, and family support and education. Primary outcome measures were mobility gain from admission to discharge and whether or not patients returned home post-discharge.
The PCRM-CI intervention was evaluated using a quasi-experimental design, following implementation in two community hospital inpatient rehabilitation units. One hundred forty-nine patients aged 65 and older participated as patients in the usual care (76) or PCRM-CI intervention (73) groups. Patient mobility was assessed at admission and discharge by the Functional Independence Measure Motor Subscale (FIMM); the difference in mobility scores was defined as mobility gain. Patient discharge location was also captured to determine whether or not patients returned home from inpatient rehabilitation.
No difference in mobility gain was found between the usual care and PCRM-CI groups as measured by the FIMM. Patients in the intervention group were more likely to return home post-discharge than those in the usual care group (p = 0.02).
Results of the PCRM-CI evaluation suggest that older adults with CI can successfully be rehabilitated post-hip fracture repair using this novel, interdisciplinary rehabilitation program.
Trial registration
This trial has been registered with the US National Institutes of Health (ID: NCT01566136)
PMCID: PMC4028934  PMID: 24330470
Person-centered care; Cognitive impairment; Rehabilitation; Healthcare restructuring; Hip fracture
17.  Norovirus Genotypes Present in Oysters and in Effluent from a Wastewater Treatment Plant during the Seasonal Peak of Infections in Ireland in 2010 
We determined norovirus (NoV) concentrations in effluent from a wastewater treatment plant and in oysters during the peak period of laboratory-confirmed cases of NoV infection in Ireland in 2010 (January to March). Weekly samples of influent, secondary treated effluent, and oysters were analyzed using real-time quantitative reverse transcription-PCR for NoV genogroup I (GI) and genogroup II (GII). The mean concentration of NoV GII (5.87 × 104 genome copies 100 ml−1) in influent wastewater was significantly higher than the mean concentration of NoV GI (1.40 × 104 genome copies 100 ml−1). The highest concentration of NoV GII (2.20 × 105 genome copies 100 ml−1) was detected in influent wastewater during week 6. Over the study period, a total of 931 laboratory-confirmed cases of NoV GII infection were recorded, with the peak (n = 171) occurring in week 7. In comparison, 16 cases of NoV GI-associated illness were reported during the study period. In addition, the NoV capsid N/S domain was molecularly characterized for selected samples. Multiple genotypes of NoV GI (GI.1, GI.4, GI.5, GI.6, and GI.7) and GII (GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, and GII.17), as well as 4 putative recombinant strains, were detected in the environmental samples. The NoV GII.4 variant 2010 was detected in wastewater and oyster samples and was the dominant strain detected in NoV outbreaks at that time. This study demonstrates the diversity of NoV genotypes present in wastewater during a period of high rates of NoV infection in the community and highlights the potential for the environmental spread of multiple NoV genotypes.
PMCID: PMC3623187  PMID: 23396337
18.  AAV-Mediated, Optogenetic Ablation of Müller Glia Leads to Structural and Functional Changes in the Mouse Retina 
PLoS ONE  2013;8(9):e76075.
Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.
PMCID: PMC3785414  PMID: 24086689
19.  Activation of Specific Interneurons Improves V1 Feature Selectivity and Visual Perception 
Nature  2012;488(7411):379-383.
Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABAergic interneurons have been identified based on their morphology, molecular markers, biophysical properties, and innervation pattern1,2,3. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV+) interneurons in mouse V1 sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes4,5 and channelrhodopsin 2 (ChR2)-mediated optical activation6, we found that elevated spiking of PV+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than decreased spiking of excitatory neurons, since archaerhodopsin-3 (Arch)-mediated optical silencing7 of calcium/calmodulin-dependent protein kinase IIα-positive (CaMKIIα+) excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV+ neuron activation, since activating somatostatin (SOM+) or vasointestinal peptide (VIP+) interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by elevated spiking of a specific subtype of cortical inhibitory interneurons.
PMCID: PMC3422431  PMID: 22878719
20.  Concentration of Norovirus during Wastewater Treatment and Its Impact on Oyster Contamination 
The concentrations of Escherichia coli, F-specific RNA bacteriophage (FRNA bacteriophage), and norovirus genogroup I (NoV GI) and norovirus genogroup II (NoV GII) in wastewater were monitored weekly over a 1-year period at a wastewater treatment plant (WWTP) providing secondary wastewater treatment. A total of 49 samples of influent wastewater and wastewater that had been treated by primary and secondary wastewater treatment processes (primary and secondary treated wastewater) were analyzed. Using a real-time reverse transcription-quantitative PCR (RT-qPCR), the mean NoV GI and NoV GII concentrations detected in effluent wastewater were 2.53 and 2.63 log10 virus genome copies 100 ml−1, respectively. The mean NoV concentrations in wastewater during the winter period (January to March) (n = 12) were 0.82 (NoV GI) and 1.41 (NoV GII) log units greater than the mean concentrations for the rest of the year (n = 37). The mean reductions of NoV GI and GII during treatment were 0.80 and 0.92 log units, respectively, with no significant difference detected in the extent of NoV reductions due to season. No seasonal trend was detected in the concentrations of E. coli or FRNA bacteriophage in wastewater influent and showed mean reductions of 1.49 and 2.13 log units, respectively. Mean concentrations of 3.56 and 3.72 log10 virus genome copies 100 ml−1 for NoV GI and GII, respectively, were detected in oysters sampled adjacent to the WWTP discharge. A strong seasonal trend was observed, and the concentrations of NoV GI and GII detected in oysters were correlated with concentrations detected in the wastewater effluent. No seasonal difference was detected in concentrations of E. coli or FRNA bacteriophage detected in oysters.
PMCID: PMC3346491  PMID: 22367079
21.  An inpatient rehabilitation model of care targeting patients with cognitive impairment 
BMC Geriatrics  2012;12:21.
The course and outcomes of hip fracture patients are often complicated by the presence of dementia and delirium, referred to as cognitive impairment (CI), which limits access to in-patient rehabilitation. In response to this concern, members of our team developed and piloted an in-patient rehabilitation model of care (Patient-Centred Rehabilitation Model; PCRM) targeting patients with hip fracture and CI (PCRM-CI). We are now conducting a 3-year study comparing an inpatient rehabilitation model of care for community dwelling individuals with hip fracture and CI (PCRM-CI) to usual care to determine whether it results in improved mobility at the time of discharge from inpatient rehabilitation.
A non-equivalent pre-post design is being used to evaluate the PCRM-CI compared to usual care. All community dwelling (private home or retirement home) patients following a hip fracture are eligible to participate. Recruitment of both cohorts is taking place at two facilities. Target accrual is 70 hip fracture patients in the PCRM-CI cohort and 70 patients in the usual care cohort. We are also recruiting 70 health care providers (HCPs), who are being trained to implement the PCRM-CI, and their unit managers. Patient data are collected at baseline, discharge, and 6 months post-discharge from an inpatient rehabilitation program. Evaluations include mobility, physical function, and living arrangement. Additional outcome variables are being collected from medical records and from the patients via their proxies. Data on the prevalence and severity of dementia and delirium are being collected. Staff data are collected at baseline and one year after implementation of the model to determine change in staff knowledge and attitudes toward patients with hip fracture and CI. Bi-monthly semi-structured interviews with unit managers have been conducted to examine factors and barriers influencing the model implementation. Data collection began in 2009 and is expected to be completed in 2012. The control cohort of 70 patients has been recruited, and 45 patients have been accrued to the intervention group to date.
Evaluation of this model of care is timely given the increasing proportion of persons with cognitive impairment and hip fractures.
Trial registration
The study is registered at, Identifier NCT01566136.
PMCID: PMC3444411  PMID: 22631877
Hip fractures; Dementia; Delirium; Cognitive impairment; Rehabilitation; Models of care; Mobility outcomes; Evaluation; Controlled investigation
22.  Role for a Novel Usher Protein Complex in Hair Cell Synaptic Maturation 
PLoS ONE  2012;7(2):e30573.
The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23), protocadherin-15 (PCDH15) and the very large G-protein coupled receptor 1 (VLGR1) have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1−/− mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzerav3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well.
PMCID: PMC3281840  PMID: 22363448
23.  Alternative splice variants of the USH3A gene Clarin 1 (CLRN1) 
Clarin 1 (CLRN1) is a four-transmembrane protein expressed in cochlear hair cells and neural retina, and when mutated it causes Usher syndrome type 3 (USH3). The main human splice variant of CLRN1 is composed of three exons that code for a 232-aa protein. In this study, we aimed to refine the structure of CLRN1 by an examination of transcript splice variants and promoter regions. Analysis of human retinal cDNA revealed 11 CLRN1 splice variants, of which 5 have not been previously reported. We studied the regulation of gene expression by several promoter domains using a luciferase assay, and identified 1000 nt upstream of the translation start site of the primary CLRN1 splice variant as the principal promoter region. Our results suggest that the CLRN1 gene is significantly more complex than previously described. The complexity of the CLRN1 gene and the identification of multiple splice variants may partially explain why mutations in CLRN1 result in substantial variation in clinical phenotype.
PMCID: PMC3039507  PMID: 20717163
Clarin 1; CLRN1; Usher syndrome; USH3; alternative splicing; promoter
24.  Intravitreal Injection of AAV2 Transduces Macaque Inner Retina 
Intravitreally injected AAV2 transduced inner retinal cells in a restricted region at the macaque fovea. Because macaque and human eyes are similar, the results suggest a need to improve transduction methods in gene therapy for the human inner retina.
Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans.
In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters.
AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-β-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters—human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)—AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis.
The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.
PMCID: PMC3088562  PMID: 21310920
25.  Educational Needs of Patients Undergoing Total Joint Arthroplasty 
Physiotherapy Canada  2010;62(3):206-214.
Purpose: To identify the educational needs of adults who undergo total hip and total knee replacement surgery.
Methods: A qualitative research design using a semi-standardized interviewing method was employed. A purposive sampling technique was used to recruit participants, who were eligible if they were scheduled to undergo total hip or total knee replacement or had undergone total hip or total knee replacement in the previous 3 to 6 months. A comparative contrast method of analysis was used.
Results: Of 22 potential participants who were approached, 15 participated. Five were booked for upcoming total hip or total knee replacement and 10 had undergone at least one total hip or total knee replacement in the previous 3 to 6 months. Several themes related to specific educational needs and factors affecting educational needs, including access, preoperative phase, surgery and medical recovery, rehabilitation process and functional recovery, fears, and expectations counterbalanced with responsibility, emerged from the interviews.
Conclusions: Educational needs of adults who undergo total hip and knee replacement surgery encompass a broad range of topics, confirming the importance of offering an all-inclusive information package regarding total hip and total knee replacement.
PMCID: PMC2909857  PMID: 21629598
educational needs; patient education; qualitative research; total hip arthroplasty; total knee arthroplasty; arthroplastie totale de la hanche; arthroplastie totale du genou; besoins éducatifs; éducation des patients; recherche qualitative

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