Retinal ganglion cell death and optic nerve degeneration by genetic ablation in adult mice

Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER™ transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.     

Notch-1调控视网膜前体细胞向视网膜神经节细胞分化

目的研究Notch-1对视网膜前体细胞(RPC)向视网膜神经节细胞(RGC)分化的调控作用。方法分离培养胚胎14 d龄Sprague-Dawley大鼠的RPC，实验组和对照组分别用含有Notch-1反义寡核苷酸链和无关序列寡核苷酸链的培养液进行诱导分化14 d，倒置相差显微镜每天观察细胞的生长和分化情况，Thy1.1标记RGC并进行计数。结果实验组和对照组的RPC都能分化为多种视网膜细胞类型，包括Thy1.1阳性的RGC，但两组RPC向RGC分化的百分比不同。实验组和对照组RGC的百分比分别为(16.57±4.31)%和(31.19±6.90)%，两组比较差异有统计学意义（t=9.84,P＜0.001）。结论Notch-1对RPC的分化具有负向调控作用，阻断Notch-1能促进RPC向RGC分化。(中华眼底病杂志, 2007, 23: 101-103)     

Modeling the effects of aging on retinal ganglion cell density and nerve fiber layer thickness

Purpose The effects of aging on retinal nerve fiber layer (RNFL) thickness should reflect the age-related losses in retinal ganglion cells (RGCs), but published data suggest that the relative rate of thinning of RNFL thickness with age is less than predicted by age-related losses of RGCs. Therefore, the present study was undertaken to reconcile the differences in age-dependency on measures of RGCs and axons that are incorporated in normative clinical data. Methods Normative data for RNFL thickness and visual field sensitivities were obtained from the printouts of standard optical coherence tomography (OCT) and standard automated perimetry (SAP) for patients aged between 25 and 95 years, in decade steps. These data were used in models to estimate the number of RGCs underlying each measure. Results The age-related losses of RGCs derived from normative perimetry data agreed closely with published histologic data, without an age-dependent variable in the model. In contrast, the age-related losses of RGCs derived from normative total RNFL thickness data required an age-dependent decrease of 0.007 axons/μm²/year in axon density in the RNFL to account for the relatively slower rate of RNFL thinning than RGC loss. Conclusions The analysis of normative data suggests a model of age-related thinning of RNFL in which the relationship between RNFL thickness and the density of RGC axons varies with the number of neurons that are lost through normal aging. This model posits that the OCT measurement of total RNFL thickness of a normal retina represents two components: 1) an age-dependent population of RNFL axons, and 2) a non-neural component that partially compensates for the age-related decrease in axons in the nerve fiber layer.     

糖尿病大鼠早期视网膜小胶质细胞活化与神经节细胞损害的关系

目的　观察糖尿病早期视网膜小胶质细胞的活化特征与视网膜神经节细胞（RGC）损害的关系。方法　20只成年雄性Sprague-Dawley（SD）大鼠,采用链脲佐菌霉素腹腔注射方法制作糖尿病动物模型,分为糖尿病1、3个月组及相应正常对照组,每组5只大鼠。对所有大鼠行上丘定位注射逆行标记RGC,分别用免疫组织化学法标记视网膜铺片、冰冻切片小胶质细胞和RGC,共聚焦显微镜下观察小胶质细胞细胞形态及分布特征。结果　糖尿病组视网膜铺片小胶质细胞胞体增粗,形态不规则。与对照组相比,糖尿病3个月组RGC层发生吞噬的小胶质细胞密度显著增加（t=3.83,P＜0.01）。与对照组相比,糖尿病大鼠1、3月个组RGC层小胶质细胞平均密度均显著增加（t=2.71,4.22;P＜0.05）;糖尿病大鼠3个月组RGC层小胶质细胞平均密度较糖尿病1个月组显著增加（t=7.45,P＜0.0001）。糖尿病早期小胶质细胞与RGC数量之间存在相关关系（r=0.9,P＜0.05）。结论　糖尿病早期小胶质细胞活化与RGC损伤关系密切。     

视网膜下移植睫状神经营养因子编码基因修饰的细胞保护SD大鼠视神经横断伤后视网膜节细胞变性

目的探讨移植表达睫状神经营养因子(CNTF)的细胞对SD大鼠视神经横断伤后视网膜节细胞的保护作用。方法通过脂质体将CNTF表达质粒转移至人胚肺成纤维细胞,建立稳定、高水平表达CNTF的细胞株。采用双侧背外侧膝状体及上丘核团注射3%荧光金逆行标记视网膜节细胞。将标记后的大鼠分为两组,于标记后7d手术切断眶内段视神经其中一组左眼不做手术作为正常对照组,右眼切断视神经作为手术对照组;另一组双眼均手术切断视神经,左眼注射PBS作为治疗对照组,右眼视网膜下移植表达CNTF的细胞作为实验组。术后5、14、17、21及28d取出眼球,铺片后荧光显微镜观察并计数视网膜内存活的节细胞。结果手术切断眶内段视神经后2周,视网膜内节细胞数减少6744%,视网膜下移植表达CNTF的细胞后第5、17、21d视网膜内存活的节细胞数明显多于治疗对照组(P<005)。结论视网膜下移植高水平表达CNTF的细胞对视网膜节细胞有保护作用。     

Imaging retinal ganglion cells: Enabling experimental technology for clinical application

Recent advances in clinical ophthalmic imaging have enhanced patient care. However, the ability to differentiate retinal neurons, such as retinal ganglion cells (RGCs), would advance many areas within ophthalmology, including the screening and monitoring of glaucoma and other optic neuropathies. Imaging at the single cell level would take diagnostics to the next level. Experimental methods have provided techniques and insight into imaging RGCs, however no method has yet to be translated to clinical application. This review provides an overview of the importance of non-invasive imaging of RGCs and the clinically relevant capabilities. In addition, we report on experimental data from wild-type mice that received an in vivo intravitreal injection of a neuronal tracer that labelled RGCs, which in turn were monitored for up to 100 days post-injection with confocal scanning laser ophthalmoscopy. We were able to demonstrate efficient and consistent RGC labelling with this delivery method and discuss the issue of cell specificity. This type of experimental work is important in progressing towards clinically applicable methods for monitoring loss of RGCs in glaucoma and other optic neuropathies. We discuss the challenges to translating these findings to clinical application and how this method of tracking RGCs in vivo could provide valuable structural and functional information to clinicians.     

Optic Nerve Engraftment of Neural Stem Cells

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm² vs. 199 ± 58 RGCs/mm²; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.     

Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas

Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory’s previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.     

Retinal ganglion cell population in adult albino and pigmented mice: A computerized analysis of the entire population and its spatial distribution

In adult Swiss albino and C57 pigmented mice, RGCs were identified with a retrogradely transported neuronal tracer applied to both optic nerves (ON) or superior colliculi (SCi). After histological processing, the retinas were prepared as whole-mounts, examined and photographed under a fluorescence microscope equipped with a motorized stage controlled by a commercial computer image analysis system: Image-Pro Plus^® (IPP). Retinas were imaged as a stack of 24-bit color images (140 frames per retina) using IPP with the Scope-Pro plug-in 5.0 and the images montaged to create a high-resolution composite of the retinal whole-mount when required. Single images were also processed by specific macros written in IPP that apply a sequence of filters and transformations in order to separate individual cells for automatic counting. Cell counts were later transferred to a spreadsheet for statistical analysis and used to generate a RGC density map for each retina. Results: The mean total numbers of RGCs labeled from the ON, in Swiss (49,493 ± 3936; n = 18) or C57 mice (42,658 ± 1540; n = 10) were slightly higher than the mean numbers of RGCs labeled from the SCi, in Swiss (48,733 ± 3954; n = 43) or C57 mice (41,192 ± 2821; n = 42), respectively. RGCs were distributed throughout the retina and density maps revealed a horizontal region in the superior retina near the optic disk with highest RGC densities. In conclusion, the population of mice RGCs may be counted automatically with a level of confidence comparable to manual counts. The distribution of RGCs adopts a form of regional specialization that resembles a horizontal visual streak.     

压力对大鼠纯化视网膜神经节细胞诱导型一氧化氮合酶mRNA及其蛋白表达的影响

目的 研究不同压力下纯化培养的大鼠视网膜神经节细胞（retinal ganglion cells,RGCs)中诱导一氧化氮合酶（inducible nitric oxide synthase,iNOS)mRNA及其蛋白质的表达,探讨青光眼患者RGCs损伤的机制。方法 将纯化培养的Sprague-Dawley大鼠RGCs随机分成对照组A,B,C,及D组,分别在0,20,40,60及80mmHg(1mmHg=0.133KPa)的压力下培养48h后,用原位杂交,RTPCR及Western blot法检测RGCs中iNOSmRNA及其蛋白质的表达,并用全自动图像分析系统测定其吸光度值。结果 纯化培养的RGCs纯度为98％。原位杂交,RT－PCR及Western blot法检测RGCs中iNOS mRNA及其蛋白表达结果变化均呈平行关系;对照组无表达,A组有较弱的表达信号,B,C及D组表达逐渐增强;3项检测结果用全自动图像分析显示;A组iNOSmRNA均弱表达,与对照组比较差异有显著意义（P＜0．05）;B,C及D组均为强表达,各组与对照组比较差异有非常显著意义（P＜0．01）。结论 压力可激活RGCs中iNOSmRNA及其蛋白质的表达,由此产生过量的NO损伤RGCs成为青光眼的发病因素之一。     

Potential mechanisms of retinal ganglion cell type-specific vulnerability in glaucoma

Glaucoma is a neurodegenerative disease characterised by progressive damage to the retinal ganglion cells (RGCs), the output neurons of the retina. RGCs are a heterogenous class of retinal neurons which can be classified into multiple types based on morphological, functional and genetic characteristics. This review examines the body of evidence supporting type-specific vulnerability of RGCs in glaucoma and explores potential mechanisms by which this might come about. Studies of donor tissue from glaucoma patients have generally noted greater vulnerability of larger RGC types. Models of glaucoma induced in primates, cats and mice also show selective effects on RGC types – particularly OFF RGCs. Several mechanisms may contribute to type-specific vulnerability, including differences in the expression of calcium-permeable receptors (for example pannexin-1, P2X7, AMPA and transient receptor potential vanilloid receptors), the relative proximity of RGCs and their dendrites to blood supply in the inner plexiform layer, as well as differing metabolic requirements of RGC types. Such differences may make certain RGCs more sensitive to intraocular pressure elevation and its associated biomechanical and vascular stress. A greater understanding of selective RGC vulnerability and its underlying causes will likely reveal a rich area of investigation for potential treatment targets.     

Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG⁺ or DTMR⁺RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR⁺RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR⁺RGCs and FG⁺RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.     

Dominant optic atrophy: Culprit mitochondria in the optic nerve

Dominant optic atrophy (DOA) is an inherited mitochondrial disease leading to specific degeneration of retinal ganglion cells (RGCs), thus compromising transmission of visual information from the retina to the brain. Usually, DOA starts during childhood and evolves to poor vision or legal blindness, affecting the central vision, whilst sparing the peripheral visual field. In 20% of cases, DOA presents as syndromic disorder, with secondary symptoms affecting neuronal and muscular functions. Twenty years ago, we demonstrated that heterozygous mutations in OPA1 are the most frequent molecular cause of DOA. Since then, variants in additional genes, whose functions in many instances converge with those of OPA1, have been identified by next generation sequencing. OPA1 encodes a dynamin-related GTPase imported into mitochondria and located to the inner membrane and intermembrane space. The many OPA1 isoforms, resulting from alternative splicing of three exons, form complex homopolymers that structure mitochondrial cristae, and contribute to fusion of the outer membrane, thus shaping the whole mitochondrial network. Moreover, OPA1 is required for oxidative phosphorylation, maintenance of mitochondrial genome, calcium homeostasis and regulation of apoptosis, thus making OPA1 the Swiss army-knife of mitochondria. Understanding DOA pathophysiology requires the understanding of RGC peculiarities with respect to OPA1 functions. Besides the tremendous energy requirements of RGCs to relay visual information from the eye to the brain, these neurons present unique features related to their differential environments in the retina, and to the anatomical transition occurring at the lamina cribrosa, which parallel major adaptations of mitochondrial physiology and shape, in the pre- and post-laminar segments of the optic nerve. Three DOA mouse models, with different Opa1 mutations, have been generated to study intrinsic mechanisms responsible for RGC degeneration, and these have further revealed secondary symptoms related to mitochondrial dysfunctions, mirroring the more severe syndromic phenotypes seen in a subgroup of patients. Metabolomics analyses of cells, mouse organs and patient plasma mutated for OPA1 revealed new unexpected pathophysiological mechanisms related to mitochondrial dysfunction, and biomarkers correlated quantitatively to the severity of the disease. Here, we review and synthesize these data, and propose different approaches for embracing possible therapies to fulfil the unmet clinical needs of this disease, and provide hope to affected DOA patients.     

Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats

Previous studies evaluating neural stem cells transplanted into the mature retina have demonstrated limited levels of graft-host integration and photoreceptor differentiation. The purpose of this investigation is to enhance photoreceptor cell differentiation and integration of retinal progenitor cells (RPC) following subretinal transplantation into retinal degenerate rats by optimization of isolation, expansion, and transplantation procedures. RPCs were isolated from human placental alkaline phosphatase (hPAP)-positive embryonic day 17 (E17) rat retina and expanded in serum-free defined media. RPCs at passage 2 underwent in vitro induction with all trans retinoic acid or were transplanted into the subretinal space of post-natal day (P) 17 S334ter-3 and S334ter-5 transgenic rats. Animals were examined post-operatively by ophthalmoscopy and optical coherence tomography (OCT) at weeks 1 and 4. Differentiation profiles of RPCs, both in vitro and in vivo were analysed microscopically by immunohistochemistry for various retinal cell specific markers. Our results demonstrated that the majority of passage 2 RPCs differentiated into retina-specific neurons expressing rhodopsin after in vitro induction. Following subretinal transplantation, grafted cells formed a multi-layer cellular sheet in the subretinal space in both S334ter-3 and S334ter-5 rats. Prominent retina-specific neuronal differentiation was observed in both rat lines as evidenced by recoverin or rhodopsin staining in 80% of grafted cells. Less than 5% of the grafted cells expressed glial fibrillary acidic protein. Synapsin-1 (label for nerve terminals) positive neural processes were present at the graft-host interface. Expression profiles of the grafted RPCs were similar to those of RPCs induced to differentiate in vitro using all-trans retinoic acid. In contrast to our previous study, grafted RPCs can demonstrate extensive rhodopsin expression, organize into layers, and show some features of apparent integration with the host retina following subretinal transplantation in slow and fast retinal degenerate rats. The similarity of the in vitro and in vivo RPC differentiation profiles suggests that intrinsic signals may have a significant contribution to RPC cell fate determination.     

大鼠视网膜脱离及复位状态下存活的视网膜神经节细胞计数

目的 探讨神经节细胞（RGC）在实验性视网膜脱离及复位状态下的反应，检测白介素1β（IL-1β）、白介素-1受体拮抗物(IL-1Ra)在RGC丢失过程中的作用。 方法 73只SD大鼠用1％荧光金(FG)经上丘、外侧膝状体逆行标记，healon GV（1.4％透明质酸钠）鼻侧视网膜下注射。10 ng IL-1Ra和500 ngIL-1β抗体随healon GV一同注入视网膜下。对逆行标记后10 d，视网膜脱离后2h、1 、3、5、 7、10、20、50、90 d,视网膜脱离10 d复位30 d,视网膜脱离90 d复位20 d，IL-1Ra 和IL-1β抗体视网膜下注入后1 d和10 d取材的视网膜进行铺片，在荧光显微镜下拍片，进行RGC计数。对照组则在球内注入相应计量healon GV。 结果 视网膜脱离后2h开始有RGC的丢失，1 d时达到高峰，然后缓慢下降。在未脱离区，亦有RGC的丢失。视网膜脱离10 d后复位（早期复位）可停止RGC的丢失，视网膜脱离90 d后复位（晚期复位）可使RGC进一步丢失，最后停留在某一水平。IL-1β抗体、IL-1Ra可以减少这种丢失。 结论 视网膜脱离状态下，脱离区与未脱离区都有RGC的丢失。早期复位可使RGC的丢失停止，晚期复位可进一步损伤RGC。拮抗IL-1β能减少RGC的丢失，IL-1Ra对RGC有保护作用。 （中华眼底病杂志，2004，20：233-236）     

低氧诱导趋化因子受体表达对视网膜前体细胞迁移能力的影响

背景体外研究表明，趋化性细胞因子受体4（CXCR4）及其配体基质细胞衍生因子-1（SDF．1）在诱导视网膜前体细胞（RPCs）定向迁移的过程中可能起重要作用。RPCs表达CXCR4升高能增强干细胞的趋化活性，从而提高移植细胞的定向迁移能力。目的探讨RPCs在低氧条件下CXCR4受体的表达。方法分离孕龄17d的NIH小鼠的胚胎视网膜细胞并制备成含5×10^6～10×10^6个／L细胞的悬液，将细胞接种到25cm2培养瓶中，用全神经球贴壁培养法进行培养。RPCs在正常O2（体积分数16％O2）和低O2（体积分数10％O2）环境中培养12h和24h后，用逆转录聚合酶链反应（RT—PCR）法检测CXCR4和缺氧诱导因子-1（HIF-1）mRNA的表达；流式细胞仪（FACS）检测RPCs中CXCR4阳性细胞的百分比；Boyden小室实验观察30μg／L的SDF-1对RPCs的趋化效应。结果10％O2培养12h和24h后，RPCs中CXCR4mRNA的表达量（CXCR4mRNA／B—actinmRNA）分别为0．28±0．07和0．48±0．17，比正常氧培养组的0．16±0．02升高了1．75倍和3．00倍，10％O2培养12h和24h后RPCs中HIF-1mRNA表达量（HIF—1mRNA／B—actinmRNA）分别为0．18±0．07和0．38±0．13，比正常氧培养组的0．06±0．01升高了3．00倍和6．30倍，差异有统计学意义（P〈0．01）。Boyden小室实验表明，10％O2培养12h和24h后SDF-1对RPCs的趋化效应由正常氧的13．00％分别上升到36．00％和46．00％。FACS检测表明，10％O2诱导12h和24h后，RPCs中CXCR4阳性细胞率由正常氧浓度的9．01％分别上升到26．90％和46．10％，差异均有统计学意义（P〈0．01）。结论RPCs在低氧条件下CXCR4受体表达增加，同时对SDF-1的趋化能力增强。HIF-1的表达增加是CXCR4表达增高的可能机制。     

Identifying the Retinal Layers Linked to Human Contrast Sensitivity Via Deep Learning

PurposeLuminance contrast is the fundamental building block of human spatial vision. Therefore contrast sensitivity, the reciprocal of contrast threshold required for target detection, has been a barometer of human visual function. Although retinal ganglion cells (RGCs) are known to be involved in contrast coding, it still remains unknown whether the retinal layers containing RGCs are linked to a person''s contrast sensitivity (e.g., Pelli-Robson contrast sensitivity) and, if so, to what extent the retinal layers are related to behavioral contrast sensitivity. Thus the current study aims to identify the retinal layers and features critical for predicting a person''s contrast sensitivity via deep learning.MethodsData were collected from 225 subjects including individuals with either glaucoma, age-related macular degeneration, or normal vision. A deep convolutional neural network trained to predict a person''s Pelli-Robson contrast sensitivity from structural retinal images measured with optical coherence tomography was used. Then, activation maps that represent the critical features learned by the network for the output prediction were computed.ResultsThe thickness of both ganglion cell and inner plexiform layers, reflecting RGC counts, were found to be significantly correlated with contrast sensitivity (r = 0.26 ∼ 0.58, Ps < 0.001 for different eccentricities). Importantly, the results showed that retinal layers containing RGCs were the critical features the network uses to predict a person''s contrast sensitivity (an average R² = 0.36 ± 0.10).ConclusionsThe findings confirmed the structure and function relationship for contrast sensitivity while highlighting the role of RGC density for human contrast sensitivity.     

Neuroprotective effects of brimonidine against transient ischemia-induced retinal ganglion cell death: a dose response in vivo study

The purpose of this study was to investigate the dose-response effects of topically administered brimonidine (BMD) on retinal ganglion cell (RGC) survival, short and long periods of time after transient retinal ischemia. In adult Sprague-Dawley rats, RGCs were retrogradely labeled with the fluorescent tracer fluorogold (FG) applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 90 min. One hr prior to retinal ischemia, two 5 microl drops of saline alone or saline containing 0.0001, 0.001, 0.01 or 0.1% BMD were instilled on the left eye. Rats were processed 7, 14 or 21 days later and densities of surviving RGCs were estimated by counting FG-labeled RGCs in 12 standard regions of each retina. The following have been found. (1) Seven days after 90 min of transient ischemia there is loss of approximately 46% of the RGC population. (2) topical pre-treatment with BMD prevents ischemia-induced RGC death in a dose-dependent manner. Administration of 0.0001% BMD resulted in the loss of approximately 37% of the RGC population and had no significant neuroprotective effects. Administration of higher concentrations of BMD (0.001 or 0.01%) resulted in the survival of 76 or 90%, respectively, of the RGC population, and 0.1% BMD fully prevented RGC death in the first 7 days after ischemia. (3) Between 7 and 21 days after ischemia there was an additional slow cell loss of approximately 25% of the RGC population. Pre-treatment with 0.1% BMD also reduced significantly this slow cell death. These results indicate that the neuroprotective effects of BMD, when administered topically, are dose-dependent and that the 0.1% concentration achieves optimal neuroprotective effects against the early loss of RGCs. Furthermore, this concentration is also effective to diminish the protracted loss of RGCs that occurs with time after transient ischemia.