Search

PurposeWe employed in vivo, 1.0-µm axial resolution visible-light optical coherence tomography (OCT) and ex vivo electron microscopy (EM) to investigate three subcellular features in the mouse outer retina: reflectivity oscillations inner to band 1 (study 1); hyperreflective band 2, attributed to the ellipsoid zone or inner segment/outer segment (IS/OS) junction (study 2); and the hyperreflective retinal pigment epithelium (RPE) within band 4 (study 3).MethodsPigmented (C57BL/6J, n = 10) and albino (BALB/cJ, n = 3) mice were imaged in vivo. Enucleated eyes were processed for light and electron microscopy. Using well-accepted reference surfaces, we compared micrometer-scale axial reflectivity of visible-light OCT with subcellular organization, as revealed by 9449 annotated EM organelles and features across four pigmented eyes.ResultsIn study 1, outer nuclear layer reflectivity peaks coincided with valleys in heterochromatin clump density (-0.34 ± 2.27 µm limits of agreement [LoA]). In study 2, band 2 depth on OCT and IS/OS junction depth on EM agreed (-0.57 ± 0.76 µm LoA), with both having similar distributions. In study 3, RPE electron dense organelle distribution did not agree with reflectivity in C57BL/6J mice, with OCT measures of RPE thickness exceeding those of EM (2.09 ± 0.89 µm LoA). Finally, RPE thickness increased with age in pigmented mice (slope = 0.056 µm/mo; P = 6.8 × 10-7).ConclusionsVisible-light OCT bands arise from subcellular organization, enabling new measurements in mice. Quantitative OCT-EM comparisons may be confounded by hydration level, particularly in the OS and RPE. Caution is warranted in generalizing results to other species.

PurposeAge-related cataracts affect the majority of older adults and are a leading cause of blindness worldwide. Treatments that delay cataract onset or severity have the potential to delay cataract surgery, but require relevant animal models that recapitulate the major types of cataracts for their development. Unfortunately, few such models are available. Here, we report the lens phenotypes of aged mice lacking the critical antioxidant transcription factor Nfe2l2 (designated as Nrf2 -/-).MethodsThree independent cohorts of Nrf2 -/- and wild-type C57BL/6J mice were evaluated for cataracts using combinations of slit lamp imaging, photography of freshly dissected lenses, and histology. Mice were fed high glycemic diets, low glycemic diets, regular chow ad libitum, or regular chow with 30% caloric restriction.ResultsNrf2 -/- mice developed significant opacities between 11 and 15 months and developed advanced cortical, posterior subcapsular, anterior subcapsular, and nuclear cataracts. Cataracts occurred similarly in male mice fed high or low glycemic diets, and were also observed in 21-month male and female Nrf2 -/- mice fed ad libitum or 30% caloric restriction. Histological observation of 18-month cataractous lenses revealed significant disruption to fiber cell architecture and the retention of nuclei throughout the cortical region of the lens. However, fiber cell denucleation and initiation of lens differentiation was normal at birth, with the first abnormalities observed at 3 months.ConclusionsNrf2 -/- mice offer a tool to understand how defective antioxidant signaling causes multiple forms of cataract and may be useful for screening drugs to prevent or delay cataractogenesis in susceptible adults.

Lens-specific beaded filament (BF) proteins CP49 and filensin interact with the C-terminus of the water channel protein Aquaporin 0 (AQP0). Previously we have reported that a C-terminally end-deleted AQP0-expressing transgenic mouse model AQP0ΔC/ΔC developed abnormal optical aberrations in the lens. This investigation was undertaken to find out whether the total loss of the BF structural proteins alter the optical properties of the lens and cause optical aberrations similar to those in AQP0ΔC/ΔC lenses; also, to map the changes in the optical quality as a function of age in the single or double BF protein knockouts as well as to assess whether there is any significant change in the water channel function of AQP0 in these knockouts. A double knockout mouse (2xKO) model for CP49 and filensin was developed by crossing CP49-KO and filensin-KO mice. Wild type, CP49-KO, filensin-KO, and 2xKO lenses at different ages, and AQP0ΔC/ΔC lenses at postnatal day-17 were imaged through the optical axis and compared for optical quality and focusing property. All three knockout models showed loss of transparency, and development of abnormal optical distortion aberration similar to that in AQP0ΔC/ΔC. Copper grid focusing by the lenses at 6, 9 and 12 months of age showed an increase in aberrations as age advanced. With progression in age, the grid images produced by the lenses of all KO models showed a transition from a positive barrel distortion aberration to a pincushion distortion aberration with the formation of three distinct aberration zones similar to those produced by AQP0ΔC/ΔC lenses. Water permeability of fiber cell membrane vesicles prepared from CP49-KO, filensin-KO and 2xKO models, measured using the osmotic shrinking method, remained similar to that of the wild type without any statistically significant alteration (P > 0.05). Western blotting and quantification revealed the expression of comparable quantities of AQP0 in all three BF protein KOs. Our study reveals that loss of single or both beaded filament proteins significantly affect lens refractive index gradient, transparency and focusing ability in an age-dependent manner and the interaction of BF proteins with AQP0 is critical for the proper functioning of the lens. The presence of BF proteins is necessary to prevent abnormal optical aberrations and maintain homeostasis in the aging lens.

PurposeTo investigate diurnal variation in the length of mouse rod outer segments in vivo.MethodsThe lengths of rod inner and outer segments (RIS, ROS) of dark-adapted albino mice maintained on a 12-hour dark:12-hour light cycle with light onset 7 AM were measured at prescribed times (6:30 AM, 11 AM, 3:30 PM) during the diurnal cycle with optical coherence tomography (OCT), taking advantage of increased visibility, after a brief bleaching exposure, of the bands corresponding to RIS/ROS boundaries and ROS tips (ROST).ResultsDeconvolution of OCT depth profiles resolved two backscatter bands located 7.4 ± 0.1 and 10.8 ± 0.2 µm (mean ± SEM) proximal to Bruch's membrane (BrM). These bands were identified with histology as arising from the apical surface of RPE and ROST, respectively. The average length of dark-adapted ROS at 6:30 AM was 17.7 ± 0.8 µm. By 11 AM, the average ROS length had decreased by 10% to 15.9 ± 0.7 µm. After 11 AM, the ROS length increased steadily at an average rate of 0.12 µm/h, returning to baseline length by 23.5 hours in the cycle.ConclusionsThe diurnal variation in ROS length measured in these experiments is consistent with prior histological investigations showing that rodent rod discs are phagocytosed by the RPE maximally over several hours around the time of normal light onset. The rate of recovery of ROS to baseline length before normal light onset is consistent with the hypothesis that disc membrane synthesis is fairly constant over the diurnal cycle.

PurposeTo investigate the molecular and cellular mechanisms of cataract induced by cold temperatures in young lenses of wild-type C57BL/6J (B6), wild-type 129SvJae (129), and filensin knockout (KO) mice. To determine how lens intermediate filament proteins, filensin (BFSP1) and CP49 (BFSP2), are involved in the formation of cold cataract.MethodsThe formation of cold cataract was examined in enucleated lenses at different temperatures and was imaged under a dissecting microscope. Lens vibratome sections were prepared, immunostained with different antibodies and fluorescent probes, and then imaged with a laser confocal microscope to evaluate the protein distribution and the membrane and cytoskeleton structures in the lens fibers.ResultsPostnatal day 14 (P14) wild-type B6 lenses showed cataracts dependent on cold temperatures in interior fibers about 420-875 µm (zone III) and 245-875 µm (zone II and zone III) from the lens surface, under 25 °C and 4 °C, respectively. In contrast, wild-type 129 (with CP49 gene deletion) and filensin KO (on the B6 background) lenses did not have cold cataracts at 25 °C but displayed a reduced cold cataract, especially in zone III, at 4 °C. Immunofluorescent staining data revealed that CP49 and filensin proteins were uniformly distributed in fiber cell cytosols without cold cataracts but accumulated or aggregated in the cell boundaries of the fibers where cold cataracts appeared.ConclusionsCP49 and filensin are important components for the formation of cold cataract in young B6 mouse lenses. Accumulated or aggregated CP49 and filensin beaded intermediate filaments in fiber cell boundaries might directly or indirectly contribute to the light scattering of cold cataract. Cold cataract in zone II is independent of beaded intermediate filaments. CP49 and filensin intermediate filaments and other lens proteins probably form distinct high molecular organizations to regulate lens transparency in interior fibers.

Electroconvulsive therapy (ECT) is a highly effective neuromodulatory intervention for treatment-resistant major depressive disorder (MDD). Presently, however, understanding of its neurophysiological effects remains incomplete. In the present study, we utilised resting-state electroencephalography (RS-EEG) to explore changes in functional connectivity, network topology, and spectral power elicited by an acute open-label course of ECT in a cohort of 23 patients with treatment-resistant MDD. RS-EEG was recorded prior to commencement of ECT and again within 48 h following each patient's final treatment session. Our results show that ECT was able to enhance connectivity within lower (delta and theta) frequency bands across subnetworks largely confined to fronto-central channels, while, conversely, more widespread subnetworks of reduced connectivity emerged within faster (alpha and beta) bands following treatment. Graph-based topological analyses revealed changes in measures of functional segregation (clustering coefficient), integration (characteristic path length), and small-world architecture following ECT. Finally, post-treatment enhancement of delta and theta spectral power was observed, which showed a positive association with the number of ECT sessions received. Overall, our findings indicate that RS-EEG can provide a sensitive measure of dynamic neural activity following ECT and highlight network-based analyses as a promising avenue for furthering mechanistic understanding of the effects of convulsive therapies.

Aberrant connectivity between the dorsolateral prefrontal cortex (DLPFC) and the subgenual cingulate cortex (SGC) has been linked to the pathophysiology of depression. Indirect evidence also links hippocampal activation to the cognitive side effects of seizure treatments. Magnetic seizure therapy (MST) is a novel treatment for patients with treatment resistant depression (TRD). Here we combine transcranial magnetic stimulation with electroencephalography (TMS-EEG) to evaluate the effects of MST on connectivity and activation between the DLPFC, the SGC and hippocampus (Hipp) in patients with TRD. The TMS-EEG was collected from 31 TRD patients prior to and after an MST treatment trial. Through TMS-EEG methodology we evaluated significant current scattering (SCS) as an index of effective connectivity between the SGC and left DLPFC. Significant current density (SCD) was used to assess activity at the level of the Hipp. The SCS between the SGC and DLPFC was reduced after the course of MST (p

Electron paramagnetic resonance (EPR) spectroscopy of full-length vimentin and X-ray crystallography of vimentin peptides has provided concordant structural data for nearly the entire central rod domain of the protein. In this report, we use a combination of EPR spectroscopy and molecular modeling to determine the structure and dynamics of the missing region and unite the separate elements into a single structure. Validation of the linker 1-2 (L1-2) modeling approach is demonstrated by the close correlation between EPR and X-ray data in the previously solved regions. Importantly, molecular dynamic (MD) simulation of the constructed model agrees with spin label motion as determined by EPR. Furthermore, MD simulation shows L1-2 heterogeneity, with a concerted switching of states among the dimer chains. These data provide the first ever experimentally driven model of a complete intermediate filament rod domain, providing research tools for further modeling and assembly studies.

Myo/Nog cells are identified by their expression of the skeletal muscle specific transcription factor MyoD and the bone morphogenetic protein inhibitor noggin, and binding of the G8 monoclonal antibody. Their release of noggin is critical for morphogenesis and skeletal myogenesis. In the adult, Myo/Nog cells are present in normal tissues, wounds and skin tumors. Myo/Nog cells in the lens give rise to myofibroblasts that synthesize skeletal muscle proteins. The purpose of this study was to screen human lens tissue, rhabdomyosarcoma cell lines, and tissue sections from rhabdomyosarcoma, Wilms and tumors lacking features of skeletal muscle for co-localization of antibodies to Myo/Nog cell markers and the lens beaded filament proteins filensin and CP49. Immunofluorescence localization experiments revealed that Myo/Nog cells of the lens bind antibodies to beaded filament proteins. Co-localization of antibodies to G8, noggin, filensin and CP49 was observed in most RC13 and a subpopulation of RD human rhabdomyosarcoma cell lines. Western blotting with beaded filament antibodies revealed bands of similar molecular weights in RC13 and murine lens cells. Human alveolar, embryonal, pleomorphic and spindle cell rhabdomyosarcomas and Wilms tumors contained a subpopulation of cells immunoreactive for G8, noggin, MyoD and beaded filaments. G8 was also co-localized with filensin mRNA. Staining for beaded filament proteins was not detected in G8 positive cells in leiomyosarcomas, squamous and basal cell carcinomas, syringocarciomas and malignant melanomas. Lens beaded filament proteins were thought to be present only in the lens. Myo/Nog-like cells immunoreactive for beaded filaments may be diagnostic of tumors related to the skeletal muscle lineage.

Background: Caloric vestibular stimulation (CVS) is a well-established neurological diagnostic technique that also induces many phenomenological modulations, including reductions in phantom limb pain (PLP), spinal cord injury pain (SCIP), and central post-stroke pain. Objective: We aimed to assess in a variety of persistent pain (PP) conditions (i) short-term pain modulation by CVS relative to a forehead ice pack cold-arousal control procedure and (ii) the duration and repeatability of CVS modulations. The tolerability of CVS was also assessed and has been reported separately. Methods: We conducted a convenience-based non-randomised single-blinded placebo-controlled study. Thirty-eight PP patients were assessed (PLP, n = 8; SCIP, n = 12; complex regional pain syndrome, CRPS, n = 14; non-specific PP, n = 4). Patients underwent 1–3 separate-day sessions of iced-water right-ear CVS. All but four also underwent the ice pack procedure. Analyses used patient-reported numerical rating scale pain intensity (NRS-PI) scores for pain and allodynia. Results: Across all groups, NRS-PI for pain was significantly lower within 30 min post-CVS than post-ice pack (p < 0.01). Average reductions were 24.8% (CVS) and 6.4% (ice pack). CRPS appeared most responsive to CVS, while PLP and SCIP responses were less than expected from previous reports. The strongest CVS pain reductions lasted hours to over three weeks. CVS also induced substantial reductions in allodynia in three of nine allodynic CRPS patients, lasting 24 h to 1 month. As reported elsewhere, only one patient experienced emesis and CVS was widely rated by patients as a tolerable PP management intervention. Conclusions: Although these results require interpretative caution, CVS was found to modulate pain relative to an ice pack control. CVS also modulated allodynia in some cases. CVS should be examined for pain management efficacy using randomised controlled trials. [ABSTRACT FROM AUTHOR]

Automated EEG pre‐processing pipelines provide several key advantages over traditional manual data cleaning approaches; primarily, they are less time‐intensive and remove potential experimenter error/bias. Automated pipelines also require fewer technical expertise as they remove the need for manual artefact identification. We recently developed the fully automated Reduction of Electroencephalographic Artefacts (RELAX) pipeline and demonstrated its performance in cleaning EEG data recorded from adult populations. Here, we introduce the RELAX‐Jr pipeline, which was adapted from RELAX and designed specifically for pre‐processing of data collected from children. RELAX‐Jr implements multi‐channel Wiener filtering (MWF) and/or wavelet‐enhanced independent component analysis (wICA) combined with the adjusted‐ADJUST automated independent component classification algorithm to identify and reduce all artefacts using algorithms adapted to optimally identify artefacts in EEG recordings taken from children. Using a dataset of resting‐state EEG recordings (N = 136) from children spanning early‐to‐middle childhood (4–12 years), we assessed the cleaning performance of RELAX‐Jr using a range of metrics including signal‐to‐error ratio, artefact‐to‐residue ratio, ability to reduce blink and muscle contamination, and differences in estimates of alpha power between eyes‐open and eyes‐closed recordings. We also compared the performance of RELAX‐Jr against four publicly available automated cleaning pipelines. We demonstrate that RELAX‐Jr provides strong cleaning performance across a range of metrics, supporting its use as an effective and fully automated cleaning pipeline for neurodevelopmental EEG data. [ABSTRACT FROM AUTHOR]

Purpose To compare the CT imaging performance of a carboxybetaine zwitterionic-coated tantalum oxide (TaCZ) nanoparticle CT contrast agent with that of a conventional iodinated contrast agent in a swine model meant to simulate overweight and obese patients. Materials and Methods Four swine were evaluated inside three different-sized adipose-equivalent encasements emulating abdominal girths of 102, 119, and 137 cm. Imaging was performed with a 64-detector row CT scanner at six scan delays after intravenous injection of 240 mg element (Ta or I) per kilogram of body weight of TaCZ or iopromide. For each time point, contrast enhancement of the aorta and liver were measured by using regions of interest. Two readers independently recorded the clarity of vasculature using a five-point Likert scale. Findings were compared by using paired t tests and Wilcoxon signed-rank tests. Results Mean peak enhancement was higher for TaCZ than for iopromide in the aorta (270 HU [σ = 24.5] vs 199 HU [σ = 10.2], P < .001) and liver (61.3 HU [σ = 11.7] vs 45.2 HU [σ = 8], P < .001). Vascular clarity was higher for TaCZ than for iopromide in 63% (132 of 208), 82% (170 of 208), and 86% (178 of 208) of the individual vessels at the 102-, 119-, and 137-cm girths, respectively (P < .01). Arterial clarity scores were higher for TaCZ than for iopromide in 62% (208 of 336) of vessels. Venous clarity scores were higher for TaCZ than for iopromide in 89% (128 of 144) of the veins in the venous phase and in 100% (144 of 144) of veins in the delayed phase (P < .01). No vessel showed higher clarity score with iopromide than with TaCZ. Conclusion An experimental tantalum nanoparticle-based contrast agent showed greater contrast enhancement compared with iopromide in swine models meant to simulate overweight and obese patients. © RSNA, 2018.

The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1loxP/loxP mice to generate Slc2a1loxP/loxP;GFAP-Cre+/0 (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.

Myo/Nog cells, named for their expression of MyoD and noggin, enter the eye during early stages of embryonic development. Their release of noggin is critical for normal morphogenesis of the lens and retina. Myo/Nog cells are also present in adult eyes. Single nucleated skeletal muscle cells designated as myofibroblasts arise from Myo/Nog cells in cultures of lens tissue. In this report we document the presence of Myo/Nog cells in the lens, ciliary body and on the zonule of Zinn in mice, rabbits and humans. Myo/Nog cells were rare in all three structures. Their prevalence increased in the lens and ciliary body of rabbits 24 h following cataract surgery. Rabbits developed posterior capsule opacification (PCO) within one month of surgery. The number of Myo/Nog cells continued to be elevated in the lens and ciliary body. Myo/Nog cells containing alpha smooth muscle actin and striated muscle myosin were present on the posterior capsule and overlaid deformations in the capsule. Myo/Nog cells also were present on the zonule fibers and external surface of the posterior capsule. These findings suggest that Myo/Nog contribute to PCO and may use the zonule fibers to migrate between the ciliary processes and lens.

The introduction of spectral CT imaging in the form of fast clinical dual-energy CT enabled contrast material to be differentiated from other radiodense materials, improved lesion detection in contrast-enhanced scans, and changed the way that existing iodine and barium contrast materials are used in clinical practice. More profoundly, spectral CT can differentiate between individual contrast materials that have different reporter elements such that high-resolution CT imaging of multiple contrast agents can be obtained in a single pass of the CT scanner. These spectral CT capabilities would be even more impactful with the development of contrast materials designed to complement the existing clinical iodine- and barium-based agents. New biocompatible high-atomic number contrast materials with different biodistribution and X-ray attenuation properties than existing agents will expand the diagnostic power of spectral CT imaging without penalties in radiation dose or scan time.

ObjectivesThe aim of this study was to assess whether the low- to high-kVp computed tomography (CT) number ratio at dual-energy CT is affected by changes in patient diameter.MethodsSeven contrast-producing elements were housed sequentially within an abdomen phantom. Fat rings enlarged the phantom diameter from 26 to 44 cm. The phantom was scanned using single-energy CT at tube potentials of 80 and 140 kVp and rapid-kVp-switching dual-energy CT.ResultsCT numbers decreased proportionally (∼20% CT number reduction for smallest to largest phantom diameters) for low- and high-energy acquisitions but resulted in consistent dual-energy ratios for each contrast element. For 17 of 21 material pair combinations, the dual-energy ratio ranges of the two elements did not overlap, implying that discrimination should remain possible for these material pairs at all patient sizes.ConclusionsThe dual-energy ratio for different contrast materials is largely unaffected by changes in phantom diameter. This should allow for robust separation of most contrast material combinations irrespective of patient size.

PurposeTo elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein.MethodsWe investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers.ResultsF-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers.ConclusionsThese results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.

PurposeThe differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age.MethodsLocalization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6.ResultsCP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 μm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely exclude vimentin and α-tubulin. In the CP49 and filensin knockouts, this structure is absent, confirming the identity of CP49 and filensin in this structure, and suggesting a requirement for the physiologic coassembly of CP49 and filensin.ConclusionsCP49 and filensin have been considered robust markers for mouse lens fiber cell differentiation. The data reported here, however, document both proteins in the mouse lens epithelium, but only after 5 weeks of age, when lens epithelial growth and mitotic activity have slowed. Because of this, CP49 and filensin must be considered markers of differentiation for both fiber cells and the lens epithelium in the mouse. In addition, to our knowledge, no other protein has been shown to emerge so late in the development of the mouse lens epithelium, suggesting that lens epithelial differentiation may continue well into post-natal life. If this structure is related to the aggresome, it is a rare, or perhaps unique example of a large, stable aggresome in wild-type tissue.

Background: Treatment-resistant bipolar depression can be treated effectively using electroconvulsive therapy, but its use is limited because of stigma and cognitive adverse effects. Magnetic seizure therapy is a new convulsive therapy with promising early evidence of antidepressant effects and minimal cognitive adverse effects. However, there are no clinical trials of the efficacy and safety of magnetic seizure therapy for treatment-resistant bipolar depression. Methods: Participants with treatment-resistant bipolar depression were treated with magnetic seizure therapy for up to 24 sessions or until remission. Magnetic seizure therapy was applied over the prefrontal cortex at high (100 Hz; n = 8), medium (50 or 60 Hz; n = 9) or low (25 Hz; n = 3) frequency, or over the vertex at high frequency (n = 6). The primary outcome measure was the 24-item Hamilton Rating Scale for Depression. Participants completed a comprehensive battery of neurocognitive tests. Results: Twenty-six participants completed a minimally adequate trial of magnetic seizure therapy (i.e., [greater than or equal to] 8 sessions), and 20 completed full treatment per protocol. Participants showed a significant reduction in scores on the Hamilton Rating Scale for Depression. Adequate trial completers had a remission rate of 23.1% and a response rate of 38.5%. Per-protocol completers had a remission rate of 30% and a response rate of 50%. Almost all cognitive measures remained stable, except for significantly worsened recall consistency on the autobiographical memory inventory. Limitations: The open-label study design and modest sample size did not allow for comparisons between stimulation parameters. Conclusion: In treatment-resistant bipolar depression, magnetic seizure therapy produced significant improvements in depression symptoms with minimal effects on cognitive performance. These promising results warrant further investigation with larger randomized clinical trials comparing magnetic seizure therapy to electroconvulsive therapy. Clinical trial registration: NCT01596608; clinicaltrials.gov, Introduction Bipolar disorder is a recurrent neuropsychiatric disorder associated with substantial burden and impairment. (1) It is characterized by episodes of mania or hypomania and episodes of depression, with the [...]

PurposeA form of retinal degeneration suspected to be hereditary was discovered in a family of Bengal cats. A breeding colony was established to characterize disease progression clinically, electrophysiologically, and morphologically, and to investigate the mode of inheritance.MethodsAffected and related cats were donated by owners for breeding trials and pedigree analysis. Kittens from test and complementation breedings underwent ophthalmic and neuro-ophthalmic examinations and ERG, and globes were evaluated using light microscopy.ResultsPedigree analysis, along with test and complementation breedings, indicated autosomal recessive inheritance and suggested that this disease is nonallelic to a retinal degeneration found in Persian cats. Mutation analysis confirmed the disease is not caused by CEP290 or CRX variants found predominantly in Abyssinian and Siamese cats. Ophthalmoscopic signs of retinal degeneration were noted at 9 weeks of age and became more noticeable over the next 4 months. Visual deficits were behaviorally evident by 1 year of age. Electroretinogram demonstrated reduced rod and cone function at 7 and 9 weeks of age, respectively. Rod responses were mostly extinguished at 14 weeks of age; cone responses were minimal by 26 weeks. Histologic degeneration was first observed at 8 weeks, evidenced by reduced photoreceptor numbers, then rapid deterioration of the photoreceptor layer and, subsequently, severe outer retinal degeneration.ConclusionsA recessively inherited primary photoreceptor degeneration was characterized in the Bengal cat. The disease is characterized by early onset, with histologic, ophthalmoscopic, and electrophysiological signs evident by 2 months of age, and rapid progression to blindness.

Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent ex vivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

PurposeSeveral properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques.MethodsA simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed.ResultsAn inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.ConclusionsA simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

Microglia dynamically prune synaptic contacts during development, and digest waste that accumulates in degeneration and aging. In many neurodegenerative diseases, microglial activation and phagocytosis gradually increase over months or years, with poorly defined initial triggering events. Here, we describe rapid retinal microglial activation in response to physiological light levels in a mouse model of photoreceptor degeneration that arises from defective rhodopsin deactivation and prolonged signaling. Activation, migration and proliferation of microglia proceeded along a well-defined time course apparent within 12 h of light onset. Retinal imaging in vivo with optical coherence tomography revealed dramatic increases in light-scattering from photoreceptors prior to the outer nuclear layer thinning classically used as a measure of retinal neurodegeneration. This model is valuable for mechanistic studies of microglial activation in a well-defined and optically accessible neural circuit, and for the development of novel methods for detecting early signs of pending neurodegeneration in vivo.

SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes, as highlighted by the pleiotropic defects observed in Mowat-Wilson syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases, where it functionally links TGFβ signaling to the loss of epithelial cell characteristics and gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers, such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation, and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes. Furthermore, 34% of the genes with increased expression in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes with decreased expression in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts.

Determining which biological traits affect taxonomic durations is critical for explaining macroevolutionary patterns. Two approaches are commonly used to investigate the associations between traits and durations and/or extinction and origination rates: analyses of taxonomic occurrence patterns in the fossil record and comparative phylogenetic analyses, predominantly of extant taxa. By capitalizing upon the empirical record of past extinctions, paleontological data avoid some of the limitations of existing methods for inferring extinction and origination rates from molecular phylogenies. However, most paleontological studies of extinction selectivity have ignored phylogenetic relationships because there is a dearth of phylogenetic hypotheses for diverse non-vertebrate higher taxa in the fossil record. This omission inflates the degrees of freedom in statistical analyses and leaves open the possibility that observed associations are indirect, reflecting shared evolutionary history rather than the direct influence of particular traits on durations. Here we investigate global patterns of extinction selectivity in Devonian terebratulide brachiopods and compare the results of taxonomic vs. phylogenetic approaches. Regression models that assume independence among taxa provide support for a positive association between geographic range size and genus duration but do not indicate an association between body size and genus duration. Brownian motion models of trait evolution identify significant similarities in body size, range size, and duration among closely related terebratulide genera. We use phylogenetic regression to account for shared evolutionary history and find support for a significant positive association between range size and duration among terebratulides that is also phylogenetically structured. The estimated range size - duration relationship is moderately weaker in the phylogenetic analysis due to the down-weighting of closely related genera that were both broadly distributed and long lived; however, this change in slope is not statistically significant. These results provide evidence for the phylogenetic conservatism of organismal and emergent traits, yet also the general phylogenetic independence of the relationship between range size and duration.