Interrelationships between Cellular Density,Mosaic Patterning,and Dendritic Coverage of VGluT3 Amacrine Cells

Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax-mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax-KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population.SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina.     

A novel PAX6 mutation causes congenital aniridia with or without retinal detachment

Background: Aniridia is a rare developmental eye disorder characterized by complete or partial iris hypoplasia often accompanied with other ocular changes that affect the cornea, anterior chamber, lens, retina, and optic nerve. Most cases of aniridia are inherited with an autosomal dominant mode of inheritance caused by PAX6 mutations or deletions. To reveal the underlying genetic defect in a four-generation Iranian family with aniridia, we carried out a genetic screening of PAX6.

Methods: Complete ophthalmic examinations were performed for available affected family members. All PAX6 exons and their flanking regions were sequenced for affected individuals. Candidate variation was screened for segregation in the pedigree by Sanger sequencing. Bioinformatics prediction was done to evaluate the deleterious effects of the mutation on protein product. Real-time PCR was used to investigate the impact of the variant on PAX6 mRNA expression.

Results: All patients were diagnosed with isolated aniridia associated with variable phenotypic features including retinal detachment. A novel heterozygous deletion c.320_348delTGTCCGAGGGGGTCTGTACCAACGATAAC (p.Leu107HisfsX16) on PAX6 gene was detected. Decreased mRNA level of PAX6 in the affected individuals indicated that the mutation caused nonsense-mediated mRNA decay (NMD).

Conclusions: To the best of our knowledge, it is the first report on the genetics of aniridia in Iran. Segregation analysis, bioinformatics prediction and confirmation of NMD, all support the proposition that the novel observed PAX6 mutation is the cause of aniridia in the pedigree. Retinal detachment in some of the affected members, which is a rare reported phenotypic feature of aniridia patients, may be associated with this mutation.     

OCTA对青少年近视人群视网膜微血管密度的观察

目的：基于光学相干断层扫描血管成像（OCTA）技术探讨青少年儿童近视与视网膜表层微血管密度 及视网膜厚度的相关性。方法：横断面研究。共纳入2018年5─11月于四川大学华西医院眼科门 诊就诊的7～14岁青少年近视患者105例（193眼）。对所有受检者进行光学相干断层扫描（OCT）和 OCTA检查，量化分析黄斑中心凹视网膜厚度和各部位视网膜表层微血管密度。单因素方差分析比 较低、中、高度近视组各部位视网膜微血管密度及视网膜厚度的差异。采用Pearson相关系数探讨视 网膜厚度与各部位视网膜表层微血管密度的相关性。Spearman相关系数用于探讨等效球镜与中心凹、 旁中心凹视网膜表层微血管密度以及视网膜厚度的关系；分段多项式函数分析等效球镜度与外环及 直径6 mm完整视网膜表层微血管密度的关系。结果：旁中心凹、外环、直径6 mm完整区域视网膜 表层微血管密度在低、中、高度近视组间比较差异均具有统计学意义（F=11.651、14.499、14.232， 均P<0.001）。年龄与中心凹视网膜厚度之间有较弱正相关关系（r=0.187，P=0.011），与各部位微血管 密度均无相关性。等效球镜度与旁中心凹视网膜微血管密度有相关性（r=-0.301，P<0.001），与外环、 直径6 mm完整区域视网膜表层微血管密度呈曲线相关（r=-0.319，P<0.001；r=-0.307，P<0.001）。 但与中心凹视网膜表层微血管密度及视网膜厚度无显著相关性。此外，中心凹处视网膜厚度与微血 管密度呈正相关（r=0.691，P<0.001），与其余部位微血管密度无相关性。结论：青少年近视程度数 与旁中心凹、外环及直径6 mm完整区域视网膜表层微血管密度呈负相关；中心凹处视网膜厚度与年 龄、微血管密度呈正相关。     

N-乙酰-5-羟色胺对视网膜缺血-再灌注损伤大鼠视网膜 Fas、FasL 蛋白表达的影响

目的　探讨N-乙酰-5-羟色胺（N-acetylserotonin，NAS）对视网膜缺血-再灌注损伤（retina ischemia-reperfusion injury，RIRI）大鼠视网膜Fas、FasL蛋白表达的影响。方法　取健康成年Sprague Dawley大鼠54只，将大鼠随机分为正常组（6只）、RIRI组（24只）与NAS组（24只）；采用高眼压法建立大鼠RIRI模型，依据造模后不同时间点将RIRI组与NAS组大鼠又分为6 h、12 h、24 h及72 h四个亚组。NAS组于造模前30 min腹腔注射NAS（5 mg·kg^-1），RIRI组腹腔注射等剂量的生理盐水。通过HE染色在光学显微镜下观察各组大鼠视网膜形态学变化，并记录各组大鼠视网膜厚度及视网膜神经节细胞数，采用免疫组织化学染色法检测NAS对RIRI大鼠视网膜Fas、FasL蛋白表达的影响。结果　HE染色显示，正常组大鼠视网膜各层细胞分界清晰，形态正常，神经细胞排列整齐；RIRI组大鼠再灌注后6 h视网膜各层出现水肿，以神经节细胞层及内核层较显著，神经节细胞数较正常组减少；随后视网膜水肿进一步加重，神经节细胞继续减少；NAS组大鼠在再灌注后6 h、12 h、24 h 视网膜水肿程度较 RIRI组轻，NAS组在再灌注后72 h视网膜厚度较 RIRI组厚，NAS组各时间点神经节细胞数均较 RIRI组多，差异均有统计学意义（均为P＜0.05）。免疫组织化学染色显示，正常组几乎未见 Fas⁺细胞。再灌注后6 h，RIRI组视网膜神经节细胞及内核层开始出现少量 Fas⁺细胞；再灌注后12 h，RIRI组视网膜 Fas⁺细胞表达逐渐增多；再灌注后24 h视网膜Fas⁺细胞数达到高峰，棕色阳性染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层；再灌注后 72 h 视网膜 Fas⁺细胞较再灌注后 24 h 减少。NAS组在再灌注后6 h、12 h、24 h、72 h 视网膜 Fas⁺细胞数均较 RIRI组各时间点减少，再灌注后24 h，Fas⁺细胞数达较高水平，随后下降，差异均有统计学意义（均为P＜0.05）。正常组视网膜可见 FasL 全层低表达。RIRI组再灌注后 6 h，视网膜神经节细胞层和神经纤维层存在少量 FasL⁺细胞；再灌注后12 h FasL蛋白表达逐渐增多；再灌注后24 h FasL⁺细胞数达高峰，可见深棕色的细胞膜及细胞质染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层；再灌注后72 h FasL蛋白的阳性表达逐渐减少。NAS组再灌注后6 h、12 h、24 h、72 h 视网膜FasL⁺细胞数均少于 RIRI组各时间点阳性细胞数，差异均有统计学意义（均为P＜0.05）。结论　NAS可通过抑制RIRI大鼠视网膜细胞Fas、FasL蛋白的表达，减轻缺血再灌注对大鼠视网膜细胞造成的损伤。     

大鼠皮质神经元的体外培养和纯化

目的：探讨大鼠皮质神经元分离纯化的有效方法。方法：取胎鼠大脑皮质细胞，分别在不同的培养液中进行原代培养，利用相差显微镜对培养的细胞进行动态观察；并以免疫细胞化学技术对神经元进行染色。结果：在相差显微镜下可见加用N2添加剂和胎牛血清培养的神经元生长良好，胞体形态多样，突起数目多，长度长，与邻近神经元的胞体或突起形成接触，而胶质细胞则大量死亡，神经元纯化率达94％以上，未加用N2添加剂培养的神经元胶质细胞大量存在，神经元纯化率达50％左右。结论：N2添加剂和胎牛血清联合应用可使大鼠皮质神经元得到良好的生长和有效纯化。     

补阳还五汤对大鼠脑皮层神经元生长的影响

目的观察含补阳还五汤的药物血清对体外培养大鼠脑皮层神经元生长的影响,为补阳还五汤治疗缺血性脑损伤提供实验依据。方法以体外培养新生SD乳鼠大脑皮层神经元为观察对象,采用四唑盐(MTT)比色法测定细胞生长情况。观察含药血清对常规培养及在缺氧状态下神经元生长的影响。结果补阳还五汤对常规培养及在缺氧条件下培养的神经元有显著促进生长作用,与空白血清组比较有显著差(P<0.05)。结论大鼠以补阳还五汤灌胃给药后,其血清中的药物成份有促进体外培养神经元细胞生长的作用。     

pp60c-src encoded by the proto-oncogene c-src is a product of sensory neurons

The advent of recombinant DNA technology has led to the identification in the DNA of normal animal cells of over 30 proto-oncogenes that are homologous to retroviral transforming genes. One of these encodes a protein kinase (pp60c-src) of unknown function, that is preferentially synthesized in brain and neural retina. Here the expression of pp60c-src in the peripheral nervous system was examined in sensory neurons from chick dorsal root ganglia with antisera raised against the transforming protein of Rous sarcoma virus (pp60v-src) expressed in Escherichia coli carrying the cloned v-src gene. This antiserum recognizes pp60c-src specifically in normal chicken cells. Western immunoblotting showed that dorsal root ganglia of stage 30 (day 6.5) chick embryos contained elevated levels of pp60c-src. Immunoperoxidase staining of neuron-enriched cultures prepared from chick dorsal root ganglia showed pp60c-src immunoreactivity in cells with neuronal morphology; flat, fibroblastic cells contained no detectable immunoreactivity. Indirect double immunofluorescence with pp60src antibodies and monoclonal antibodies against the 200-kD subunit of neurofilament protein confirmed that the cells expressing pp60c-src were neurons. Ninety-six percent of the neurofilament-positive cells were immunoreactive with pp60src antibodies, and conversely, all pp60c-src-positive cells were immunoreactive with neurofilament antibodies. pp60c-src immunofluorescence appeared to be distributed over the cell body, processes, and growth cones. These results clearly demonstrate that pp60c-src is a product of neurons and is expressed in sensory neurons in culture.