Changes in concentrations of NMDA receptor subunit GluN2B, Arc and syntaxin-1 in dorsal hippocampus Schaffer collateral synapses in a rat learned helplessness model of depression

Major depressive disorder involves changes in synaptic structure and function, but the molecular underpinnings of these changes are still not established. In an initial pilot experiment, whole-brain synaptosome screening with quantitative western blotting was performed to identify synaptic proteins that may show concentration changes in a congenital rat learned helplessness model of depression. We found that the N-methyl-D-aspartate receptor (NMDAR) subunits GluN2A/GluN2B, activityregulated cytoskeleton-associated protein (Arc) and syntaxin-1 showed significant concentration differences between congenitally learned helpless (LH) and nonlearned helpless (NLH) rats. Having identified these three proteins, we then performed more elaborate quantitative immunogold electron microscopic analyses of the proteins in a specific synapse type in the dorsal hippocampus: the Schaffer collateral synapse in the CA1 region. We expanded the setup to include also unstressed wild-type (WT) rats. The concentrations of the proteins in the LH and NLH groups were compared to WT animals. In this specific synapse, we found that the concentration of NMDARs was increased in postsynaptic spines in both LH and NLH rats. The concentration of Arc was significantly increased in postsynaptic densities in LH animals as well as in presynaptic cytoplasm of NLH rats. The concentration of syntaxin-1 was significantly increased in both presynaptic terminals and postsynaptic spines in LH animals, while pre- and postsynaptic syntaxin-1 concentrations were significantly decreased in NLH animals. These protein changes suggest pathways by which synaptic plasticity may be increased in dorsal hippocampal Schaffer collateral synapses during depression, corresponding to decreased synaptic stability.publishedVersio

Eccentric lamellar keratolimbal grafts harvested with a manually guided microkeratome

Background: To perform lamellar keratolimbal allograft transplantation in a one- step procedure with a single graft, we investigated the feasibility of harvesting eccentric lamellar keratolimbal grafts from conventionally processed corneoscleral buttons using a manually guided microkeratome in conjunction with an artificial anterior chamber system. Methods: We used the Moria LSK- One microkeratome and the automated lamellar therapeutic keratoplasty ( ALTK) system ( Antony, France). Ten human donor eyes were used to obtain single- piece lamellar keratolimbal grafts. Specimens were processed for light and electron microscopy. Results: Eccentric keratolimbal grafts could be obtained from all human donor buttons. Grafts include a crescent- shaped limbal and a large corneal portion. No visible damage to the limbal region was discernible. Conclusion: Our data show that the LSK- One microkeratome in conjunction with the ALTK system allows harvesting eccentric keratolimbal grafts from donor corneoscleral buttons. Copyright (c) 2007 S. Karger AG, Basel

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background: Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker. Methodology/Principal Findings: K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker. Conclusions/Significance: K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo

The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P</=0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins

Completing Baseline Mapping of Trachoma in Uganda: Results of 14 Population-Based Prevalence Surveys Conducted in 2014 and 2018.

PURPOSE: We aimed to estimate the prevalence of trachomatous inflammation-follicular (TF) in children aged 1-9 years, trichiasis in adults aged ≥15 years, and water and sanitation (WASH) indicators in 12 suspected-endemic districts in Uganda. METHODS: Surveys were undertaken in 14 evaluation units (EUs) covering 12 districts. Districts were selected based on a desk review in 2014 (four districts) and trachoma rapid assessments in 2018 (eight districts). We calculated that 1,019 children aged 1-9 years were needed in each EU to estimate TF prevalence with acceptable precision and used three-stage cluster sampling to select 30 households in each of 28 (2014 surveys) or 24 (2018 surveys) villages. Participants living in selected households aged ≥1 year were examined for trachoma; thus enabling estimation of prevalences of TF in 1-9 year-olds and trichiasis in ≥15 year-olds. Household-level WASH access data were also collected. RESULTS: A total of 11,796 households were surveyed; 22,465 children aged 1-9 years and 24,652 people aged ≥15 years were examined. EU-level prevalence of TF ranged from 0.3% (95% confidence interval [CI] 0.1-0.7) to 3.9% (95% CI 2.1-5.8). EU-level trichiasis prevalence ranged from 0.01% (95% CI 0-0.11) to 0.81% (95% CI 0.35-1.50). Overall proportions of households with improved drinking water source, water source in yard or within 1km, and improved sanitation facilities were 88.1%, 23.0% and 23.9%, respectively. CONCLUSION: TF was not a public health problem in any of the 14 EUs surveyed: antibiotic mass drug administration is not required in these districts. However, in four EUs, trichiasis prevalence was ≥ 0.2%, so public health-level trichiasis surgery interventions are warranted. These findings will facilitate planning for elimination of trachoma in Uganda

Changes in Corneal Basal Epithelial Phenotypes in an Altered Basement Membrane

To examine the corneal epithelial phenotype in an altered basement membrane.Corneas from 9 patients with symptoms of continuous unstable corneal curvature (CUCC) were harvested by penetrating keratoplasty and subjected to histology examination and immunohistochemical staining with transactivating and N-terminally truncated pP63 transcript (ΔNp63), cytokeratin 3 (Krt3), ATP-binding cassette sub-family G member 2 (ABCG2), connexin 43 (CX43), p38 mitogen-activated protein kinases (p38MAPK), activating protein 2 (TFAP2), and extracellular signal-regulated kinase (Erk1/2) monoclonal antibodies. Positive immunostaining with ABCG2, p38MAPK, and TFAP2 monoclonal antibodies was observed in the basal epithelial cells of CUCC patients, and CX43 and ΔNp63 were detected in the full-thickness epithelial cells of CUCC patients.Our results indicate that alteration of the corneal basement membrane induces a de-differentiation-like phenotype in corneal basal epithelial cells

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing

Abstract Background The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/− hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. Results Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/− hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/− hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. Conclusions Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing