Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are limited by the opacity of brain tissue.In this study,we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury.Specifically,we performed light-sheet imaging of whole brain tissue after a clearing process.We found that pseudorabies virus 724(PRV724)mostly infected retinal ganglion cells,and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains.Unexpectedly,PRV724-traced neurons were more widely distributed compared with data from previous studies.We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus,intergeniculate leaflet,ventral lateral geniculate nucleus,central amygdala,basolateral amygdala,Edinger-Westphal nucleus,and oculomotor nucleus,but not the superior vestibular nucleus,red nucleus,locus coeruleus,gigantocellular reticular nucleus,or facial nerve nucleus.Our findings demonstrate that the tissue clearing technique,combined with retrograde trans-synaptic viral tracing,can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury.Thus,our approach may be useful for future estimations of optic nerve injury and regeneration.

Evaluation of regional bulbar redness using an imagebased objective method

AIM: To develop an image-based objective method to precisely evaluate regional ocular bulbar injection. · METHODS: Six healthy adult volunteers were photographed in four orientations(superior,inferior,nasal and temporal sides) with and without stimulating eye drops. Six line segments(covering 30°) were drawn 4mm away from the limbus on each image using ImageJ software. The graph peaks,which were derived from the areas under the line segments and corresponded to the cross-sectional grey-level of the vessels,were analyzed to obtain peak area,peak height/width(PH/PW),and peak numbers. Different-sized areas were selected to calculate the pixels based on the edge-detection algorithm. Also,conjunctival and superficial scleral vessels were analyzed separately. ·RESULTS: This method had a smaller coefficient of variation,especially for PH/PW,in all four orientations. Hyperaemia parameters changed the least after challenging in the superior region. Moreover,95% of the PH/PW ratios were greater than 0.87 in conjunctival vessels and less than 1.00 in superficial scleral vessels. PH/PW significantly increased in conjunctival vessels and changed less in superficial scleral vessels. ·CONCLUSION: A new method of objectively assessing bulbar injection based on ocular surface images was developed. This method can be used to quantify ocular regional injection and to distinguish the superficial scleral and conjunctival vessels.

Anti-viral activity of Staphylococcus aureus lysates against herpes simplex virus type-Ⅰinfection:an in vitro and in vivo study

AIM:To investigate the effect of Staphylococcus aureus(S.aures)lysates(SALs)on herpes simplex virus type-Ⅰ(HSV1)infection in human corneal epithelial(HCE)cells and in a mouse model of HSV1 keratitis.METHODS:HCE,Vero,HeLa,and BV2 cells were infected with HSV1[HSV1f strain,HSV1f;HSV-1-H129 with green fluorescent protein(GFP)knock-in,HSV1g].Pre-or post-infection,SAL at various concentrations was added to the culture medium for 24 h.GFP fluorescence in HSV1g or plaque formation by HSV1f were examined.The effects of heat-treated SAL,precooled acetone-precipitated SAL,and SAL subjected to ultrafiltration(100 kDa)were evaluated.The effects of other bacterial components and lysates on HSV1 infection were also tested,including lipoteichoic acid(LTA),peptidoglycan(PGN),staphylococcal protein A(SPA),andα-hemolysin from S.aureus(α-toxin)as well as lysates from a wild-type S.aureus strain,S.epidermidis,and Escherichia coli(W-SAL,SEL,and ECL,respectively).In addition,SAL eye drops were applied topically to BALB/c mice with HSV1 keratitis,followed by in vivo observations.RESULTS:The cytopathic effect,plaque formation(HSV1f),and GFP expression(HSV1g)in infected cells were inhibited by SAL in a dose-dependent manner.The active component of SAL(≥100 kDa)was heat-sensitive and retained activity after acetone precipitation.In HSV1ginfected cells,treatment with LTA-sa,α-toxin,PGN-sa,or SPA did not inhibit GFP expression.SAL,W-SAL,and SEL(but not ECL)decreased GFP expression.In mice with HSV1 keratitis,SAL reduced corneal lesions by 71%.CONCLUSION:The results of this study demonstrate that SAL can be used to inhibit HSV1 infection,particularly keratitis.Further studies are needed to determine the active components and mechanism underlying the effects of SAL.

Bacterial spectrum and resistance patterns in corneal infections at a Tertiary Eye Care Center in South China

AIM： To investigate the spectrum and antibiotic susceptibility of bacteria isolated from patients with suspected corneal infections in Zhongshan Ophthalmic Center in South China over the past four years retrospectively. METHODS： Totally 1943 corneal scrapes from patients with corneal infections from 2010 to 2013 were cultured and processed using standard microbiological procedures to identify bacterial isolates. Simultaneously, the bacterial isolates were tested for antibiotic susceptibility to 8 antibiotics （ceftazidime, cefuroxim, cefazolin, levofloxacin, ofloxacin, neomycin, tobramycin, chloramphenicol） using the Kirby-Bauer disc diffusion technique. RESULTS： Of the total 1943 scrapes, 397 （20.43%） were culture-positive, of which 294 （74.06%） were gram- positive （GP） and 103 （25.94%） were gram-negative （GN） bacteria. Of the GP organisms, the most prevalent genera were Staphylococcus spp. （56.17%, n=223）, Kocuria spp. （5.29%, n=21） and Micrococcus spp. （1.26%, n=5）. On the other hand, the most prevalent genera were Pseudomonas spp. （12.85%, n=51）, Burkholderia spp. （2.02%, n=8） and Acinetobacter spp. （1.51%, n =6） for the GN organisms. Among five antibiotics that have eye drop products, the resistant to neomycin of GP （7.82%, 95% Ch 4.72%-10.92%） and GN isolates （9.71%, 95% Ch 4.01%-15.41% ） was lowest, while the resistant to chloramphenicol was highest （GP： 34.35%, 95% Ch 28.92%-39.78%; GN： 60.19%, 95% Ch 50.74%-69.64%）. CONCLUSION： Staphylococcus spp. was the most common bacterial pathogens isolated from patients with corneal infections in this setting. High percentages of GP and GN bacteria were mostly susceptible to neomycin and highly resistant to chloramphenicol.

Overexpression of carbonic anhydrase 1 in pterygium

Carbonic anhydrase （CA） is a kind of zinc enzyme that reversibly catalyzes hydration of CO2. Sixteen CAisoenzymes have been described in mammals . These isozymes play physiological roles in erythrocytes, including CO2 transport, ion secretion, pH regulation and so forth. CA1 and 2 are both cytosolic enzymes that are found in large quantities in erythrocytes. Excluding hemoglobin, CA I is the most abundant protein in erythrocytes