Longitudinal In Vivo Imaging of Retinal Ganglion Cells and Retinal Thickness Changes Following Optic Nerve Injury in Mice

Retinal ganglion cells (RGCs) die in sight-threatening eye diseases. Imaging RGCs in humans is not currently possible and proof of principle in experimental models is fundamental for future development. Our objective was to quantify RGC density and retinal thickness following optic nerve transection in transgenic mice expressing cyan fluorescent protein (CFP) under control of the Thy1 promoter, expressed by RGCs and other neurons.A modified confocal scanning laser ophthalmoscopy (CSLO)/spectral-domain optical coherence tomography (SD-OCT) camera was used to image and quantify CFP+ cells in mice from the B6.Cg-Tg(Thy1-CFP)23Jrs/J line. SD-OCT circle (1 B-scan), raster (37 B-scans) and radial (24 B-scans) scans of the retina were also obtained. CSLO was performed at baseline (n = 11) and 3 (n = 11), 5 (n = 4), 7 (n = 10), 10 (n = 6), 14 (n = 7) and 21 (n = 5) days post-transection, while SD-OCT was performed at baseline and 7, 14 and 35 days (n = 9) post-transection. Longitudinal change in CFP+ cell density and retinal thickness were computed. Compared to baseline, the mean (SD) percentage CFP+ cells remaining at 3, 5, 7, 10, 14 and 21 days post-transection was 86 (9)%, 63 (11)%, 45 (11)%, 31 (9)%, 20 (9)% and 8 (4)%, respectively. Compared to baseline, the mean (SD) retinal thickness at 7 days post-transection was 97 (3)%, 98 (2)% and 97 (4)% for the circle, raster and radial scans, respectively. The corresponding figures at 14 and 35 days post-transection were 96 (3)%, 97 (2)% and 95 (3)%; and 93 (3)%, 94 (3)% and 92 (3)%.Longitudinal imaging showed an exponential decline in CFP+ cell density and a small (≤8%) reduction in SD-OCT measured retinal thickness post-transection. SD-OCT is a promising tool for detecting structural changes in experimental optic neuropathy. These results represent an important step towards translation for clinical use

In Situ Dividing and Phagocytosing Retinal Microglia Express Nestin, Vimentin, and NG2 In Vivo

BACKGROUND: Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin(+) cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo. METHODOLOGY/PRINCIPAL FINDINGS: For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin(+) microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin(+) microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU(+) and nestin(+), although no further local cell proliferation occurred. In addition, nestin(+) microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin(+)NG2(+) microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons. CONCLUSIONS/SIGNIFICANCE: The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed

Resistance trends in gram-negative bacteria: surveillance results from two Mexican hospitals, 2005–2010

<p>Abstract</p> <p>Background</p> <p>Hospital-acquired infections caused by multiresistant gram-negative bacteria are difficult to treat and cause high rates of morbidity and mortality. The analysis of antimicrobial resistance trends of gram-negative pathogens isolated from hospital-acquired infections is important for the development of antimicrobial stewardship programs. The information obtained from antimicrobial resistant programs from two hospitals from Mexico will be helpful in the selection of empiric therapy for hospital-acquired gram-negative infections.</p> <p>Findings</p> <p>Two thousand one hundred thirty two gram-negative bacteria collected between January 2005 and December 2010 from hospital-acquired infections occurring in two teaching hospitals in Mexico were evaluated. <it>Escherichia coli</it> was the most frequently isolated gram-negative bacteria, with >50% of strains resistant to ciprofloxacin and levofloxacin. <it>Klebsiella</it> spp. showed resistance rates similar to <it>Escherichia coli</it> for ceftazidime (33.1% vs 33.2%), but exhibited lower rates for levofloxacin (18.2% vs 56%). Of the samples collected for the third most common gram-negative bacteria, <it>Pseudomonas aeruginosa</it>, >12.8% were resistant to the carbapenems, imipenem and meropenem. The highest overall resistance was found in <it>Acinetobacter</it> spp. <it>Enterobacter</it> spp. showed high susceptibility to carbapenems.</p> <p>Conclusions</p> <p><it>E. coli</it> was the most common nosocomial gram-negative bacilli isolated in this study and was found to have the second-highest resistance to fluoroquinolones (>57.9%, after <it>Acinetobacter</it> spp. 81.2%). This finding represents a disturbing development in a common nosocomial and community pathogen.</p

Ecogeographic survey and gap analysis of Lathyrus L. species

The genetic diversity of the genus Lathyrus is of significant importance, particularly for its role in sustaining the livelihoods of local communities living under very harsh conditions and its potential to adapt to climate change. Grasspea (L. sativus) is the most widely used species and to a lesser extent L. cicera and L. ochrus, each is used for animal feed in many parts of the world and food in poorer regions, but human over-consumption of the seeds can lead to lathyrism, a disease caused by neurotoxins. This study has added substantial information and accuracy to the existing global Lathyrus database by combining diverse datasets and by adding information of major herbaria from Europe. This global Lathyrus database, available at ICARDA, was used to conduct gap analysis to guide future collecting missions and in situ conservation efforts for highest priority species originating from the Mediterranean Basin, and Caucasus, Central and West Asia region. The results showed the highest concentration of Lathyrus priority species are found in the Fertile Crescent countries, France, Italy and Greece. The area either side of the Lebanese/Syrian border near Tel Kalakh, Syria was identified as the hotspot and the overall priority location for establishing the first in situ genetic reserve. The gap analysis for ex situ conservation shows that only six species (representing 16.6 %) of the 36 priority species are adequately sampled. Only L. cicera has already been well sampled among the closely related species to cultivated species L. sativus, indicating further collecting of L. amphicarpos, L. belinensis, L. chrysanthus, L. hirticarpus, L. hirsutus and L. marmoratus is required. In addition, six secondary priority Lathyrus species have no ex situ collections (L. lentiformis, L. lycicus, L. phaselitanus, L. trachycarpus, L. tremolsianus and L. undulatus) and also require targeted collecting.</p