Quantitative distribution of six amino acids in rat retinal layers

Concentrations of glutamate, aspartate, glutamine, glycine, GABA, and taurine were determined in samples microdissected from rat retinal layers and assayed by HPLC. Glutamate and glutamine were relatively high in the inner nuclear (INL) and ganglion cell (GCL) layers; aspartate was relatively high in the outer nuclear layer (ONL), outer plexiform layer, and INL. Distributions of glutamate and aspartate did not correlate well with those of enzymes involved in their metabolism. Glycine and GABA were highest in the inner plexiform layer, with increasing concentrations through the INL, and were relatively high in the GCL. Taurine was highest in the ONL.     

大麻素受体在大鼠视网膜中的表达和分布

目的 研究大麻素CB1和CB2受体在幼年、成年和老龄大鼠视网膜组织的表达和分布.方法 研究采用免疫组织化学、Western印迹法和视网膜神经节细胞的上丘逆行追踪标记技术.结果 CB1和CB2受体在成年大鼠视网膜上均有较为丰富的表达,其中CB1受体的主要表达部位是外核层（ONL）、内核层（INL）和视神经节细胞层（GCL）,以及外网状层（OPL）和内网状层（IPL）;CB2的主要表达部位是GCL、INL、ONL的细胞膜和OPL,以及Müller胶质细胞的终足和突起上.免疫荧光双标结果显示,CB1和CB2受体在几乎所有的大鼠视网膜神经节细胞均有表达.CB1和CB2受体在幼年和老龄大鼠视网膜的表达分布与成年大鼠相似.结论 大麻素CB1和CB2受体在大鼠视网膜广泛分布和表达,从幼年到老龄,其表达和分布相似,提示在出生后早期这些受体的发育已经基本完成.大麻素受体系统在视网膜信息处理的调控和视网膜神经保护中可能发挥重要作用.     

No age-related cell loss in three retinal nuclear layers of the Long-Evans rat

The retina mainly contains ganglion, bipolar and photoreceptor cells which are distributed in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL), respectively. Whether there is an age-related loss of these retinal cells remains not well understood. Cell density and the total number of cells were two commonly used measures to evaluate such age-related changes in most previous studies and provided controversial conclusions. The use of density measures as decisive data is problematic because the total area of the retina was expanded in aging, whereas the application of the total number of cells was limited for assessing ganglion cells. In this study, thus, we wanted to test whether there is an age-related cell loss in the GCL, INL and ONL and if so, whether such a loss is correlated to the convergence ratio of these cells. We used stereological procedures to quantify the total number of cells in the three retinal nuclear layers in six young and six aged Long-Evans rats. We found that during aging, the total volume of the retina remained unchanged, but the retina became thinner. There was no cell loss in each individual nuclear layer, and the ratio of the ONL to INL to GCL was preserved.     

Effects of nerve growth factor for retinal cell survival in experimental retinal detachment

PURPOSE: To investigate the neuron protective effect of recombined nerve growth factor (rNGF) on retinal cell damage induced by experimental retinal detachment. METHODS: Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate. Intravitreal injection of rNGF (5 microg/eye) or phosphate-buffered saline (PBS) was separately applied every 4 days after retinal detachment. The rat eyes were then observed and sacrificed at various time points. Morphologic changes were observed by light microscopy, electron microscopy, and cell counts. Apoptosis of retinal cells was detected by TUNEL assay. RESULTS: After retinal detachment, most eyes from NGF-treated groups showed better organized structure of retinal cells than those from the PBS-treated control groups. Cell counts indicated that the nuclei numbers in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of NGF-treated groups were significantly more than those from PBS-treated control group (p < 0.05) after retinal reattachment. TUNEL-positive cells were identified in ONL, INL, and GCL. They peaked at the fourth day after retinal detachment in both the NGF-treated groups and the control groups. But the cell counts of apoptosis revealed that the NGF-treated retina had less TUNEL-positive cells than the control groups (p < 0.05). CONCLUSIONS: The results showed that intravitreal injection of exogenous NGF can protect retinal cells from degeneration and apoptosis in experimental retinal detachment. It may exert its neuroprotection effect by preventing the apoptosis of retinal cells after retinal detachment.     

Expression of the chemokines MIP-1alpha, MCP-1, and RANTES in experimental autoimmune uveitis

PURPOSE: To determine the location of the CC chemokines macrophage inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein (MCP)-1, and regulated on activation of normal T-cell-expressed and secreted (RANTES) during disease progression in experimental autoimmune uveitis (EAU) and their relationship with the presence of the T helper cell (Th)1-type cytokine IFNgamma. METHODS: EAU was induced by immunization of Lewis rats with retinal extract. Consecutive cryostat sections were prepared from eyes at different stages of EAU, graded for severity of uveitis and stained by using antibodies to MCP-1, MIP-1alpha, and RANTES and to cell surface markers. Supernatants from superficial cervical lymph node cells were examined by ELISA for IFNgamma, IL-4, and IL-10. RESULTS: MIP-1alpha and IFNgamma were present most frequently and most extensively at peak disease but also were detectable in the choroid 8 days after immunization, before clinical disease onset. MCP-1 and RANTES were present at peak disease, but much less frequently. RANTES was occasionally found in the choroid before clinical disease. By days 19 to 21 after immunization, although infiltrating cells were present, there were only residual low levels of chemokine staining. MCP-1 and RANTES were detected on CD3-positive cells and on some ED1-positive cells, whereas MIP-1alpha was also associated with vessels and areas of exudate. Lymph node cells cultured from animals with peak disease had increased levels of IFNgamma and IL-10, but for IFNgamma this occurred only after stimulation in vitro with retinal extract. CONCLUSIONS: Although MCP-1 and RANTES were associated predominantly with cells infiltrating the retina, MIP-1alpha was also associated with resident cells. All three are likely to exacerbate EAU-MIP-1alpha, to the greatest degree.     

Cell-specific expression of tubby gene family members (tub, Tulp1,2, and 3) in the retina.

PURPOSE: The family of tubby-like proteins (TULPs), consisting of four family members, are all expressed in-the retina at varying levels. Mutations within two members, tub and TULP1, are known to lead to retinal degeneration in mouse and humans, respectively, suggesting the functional importance of this family of proteins in the retina. Despite a high degree of conservation in the carboxy-terminal region (e.g., putative functional domain of the genes) among family members, they are unable to compensate for one another. The purpose of this study was to provide a rationale for this lack of compensation by investigating the spatial distribution of tubby gene family members in the retina and to investigate the mechanism of photoreceptor cell death in tubby mice. METHODS: In situ hybridization using riboprobes specific for each tubby gene family member and immunohistochemistry for TUB and TULP1 were performed to determine their expression patterns in the retina of tubby and wild-type control mice. The terminal dUTP nick-end labeling (TUNEL) assay was performed to detect apoptotic cells in the retina of tubby and wild-type control mice. RESULTS: tub mRNA was found to be expressed throughout the retina, with highest expression in the ganglion cell layer (GCL) and photoreceptor cells. In contrast, Tulp1 expression was observed only in photoreceptor cells and Tulp3 mRNA was expressed at a moderate level only in the inner nuclear layer (INL) and GCL. The results of the immunohistochemical analysis paralleled those observed in the in situ studies. TUB immunoreactivity was most highly concentrated in the GCL, in the inner and outermost regions of the INL, in the outer plexiform layer (OPL), and in the inner segments of photoreceptor cells. Similarly, TULP1 immunoreactivity was observed in the OPL and inner segments of the photoreceptor cells. No differences in expression at the mRNA or protein level were observed for any of the molecules tested in tubby or wild-type mice. TUNEL-positive cells were detected in the ONL of tubby mice, whereas very few were seen in the same layer of age-matched control mice. CONCLUSIONS: Although all tubby gene family members are expressed in the retina, they each have different cell-specific expression patterns, which may account in part for their inability to compensate for the loss of one family member. The photoreceptor cell death in tubby mice occurs through an apoptotic mechanism, which is known to be the common final outcome of other forms of retinal degeneration.     

Upregulation of P-selectin and intercellular adhesion molecule-1 after retinal ischemia-reperfusion injury

PURPOSE: Vascular endothelial cells and leukocytes express several inflammatory adhesion receptors, such as selectins and cell adhesion molecules, after retinal ischemia. They mediate the transmigration process of leukocytes. For further understanding of the role of leukocytes after retinal ischemia-reperfusion injury, the responses of the leukocyte-endothelial cell adhesion molecules P-selectin and intercellular adhesion molecule (ICAM)-1 during retinal ischemia and reperfusion were examined. METHODS: Male pigmented rats were subjected to retinal ischemia by a 1-hour ligation of the optic nerve followed by reperfusion. Gene and protein expression of P-selectin and ICAM-1 were studied at 0.5, 1, 6, 12, and 24 hours after the onset of reperfusion with semiquantitative polymerase chain reaction and Western blot analysis. Immunohistochemical methods were used to detect specific lesions expressing ICAM-1. RESULTS: Significant upregulation of P-selectin and ICAM-1 mRNA (at 6, 12, and 24 hours of reperfusion) were observed, with the expression peaks of both P-selectin and ICAM-1 mRNA occurring at 12 hours after reperfusion. P-selectin protein gradually increased and reached a maximum 6 hours after reperfusion, whereas ICAM-1 protein increased until 24 hours after reperfusion. Immunostaining with ICAM-1 antibodies was positive in endothelial cells after ischemia-reperfusion injury. CONCLUSIONS: Retinal ischemia-reperfusion stimulates P-selectin and ICAM-1 expression. Endothelial ICAM-1 expression in retinal vessels was observed. Upregulation of P-selectin and ICAM-1, which contribute to leukocyte rolling and adhesion, were observed after retinal ischemia.     

Peroxiredoxin6在糖尿病大鼠视网膜中的表达

背景研究表明氧化损伤在糖尿病并发症的发生发展中发挥着重要的作用,Peroxiredoxin6（Prx6）是双功能的蛋白质,其清除磷脂氢过氧化物的活性非常重要,因为细胞质中普遍存在的谷胱甘肽过氧化物酶（GPxl）无此功能。目的观察Prx6在实验性糖尿病大鼠视网膜中的表达,分析随着糖尿病病程的延长其在视网膜的表达情况。方法Wistar大鼠60只,随机分为正常对照组12只和糖尿病组48只。糖尿病组腹腔注射链脲佐菌素（STZ）65mg／kg诱发糖尿病,将造模成功的大鼠随机分为糖尿病1、2、3个月组,每组16只。于各时间点处死大鼠,采用逆转录一聚合酶链反应（RT—PCR）和免疫组织化学法分析Prx6在不同时间点糖尿病组大鼠视网膜中的表达,应用Image—ProPlus6．0图像分析软件测定阳性反应强度,即平均吸光度（A）值。结果免疫组织化学染色结果显示,正常对照组大鼠视网膜内外颗粒层及视网膜、脉络膜血管均有Prx6蛋白的表达,视网膜神经节细胞（RGCs）层有少量Prx6蛋白的表达,表达于细胞质;糖尿病1个月组大鼠视网膜内外颗粒层棕黄色阳性染色细胞增多;2个月组、3个月组大鼠视网膜各层Prx6蛋白的表达均为阴性,各组问总体比较差异均有统计学意义（F=22967．63,P〈0．05）。RT—PCR结果显示,正常对照组大鼠视网膜有Prx6mRNA的表达;糖尿病1个月组视网膜Prx6mRNA表达增高;2个月组Prx6mRNA表达量明显降低,仅有少量表达;3个月组表达阴性,各组问总体比较差异均有统计学意义（F=942．84,P〈0．05）。结论Prx6作为一种抗氧化蛋白存在于视网膜中,可保护组织细胞免受活性氧簇、氧自由基等的损害,随着糖尿病病程的延长,Prx6在视网膜组织和细胞中的表达量逐渐降低,直至消失。     

Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor

PURPOSE. To determine whether intraocular gene transfer of pigment epithelium-derived factor (PEDF) protects the retina from ischemia-reperfusion injury. METHODS. Four days before induction of pressure-induced ischemia, Lewis rats received intravitreous injection of 3 x 10(9) particles of an adenovirus vector expressing PEDF (AdPEDF.11) in one eye and 3 x 10(9) particles of an empty adenovirus vector (AdNull.11) in the contralateral eye. Seven days after reperfusion, eyes were enucleated and processed for morphometric analysis. Apoptotic cells stained by TdT-dUTP terminal nick-end labeling (TUNEL) in the retina were counted 12 hours after initiation of reperfusion. Retina levels of PEDF were measured by enzyme-linked immunosorbent assay. RESULTS. PEDF levels in retinal homogenates from eyes receiving AdPEDF.11 injection were well above the background levels in the untreated baseline and control eyes (P = 0.04). Retinal thickness was preserved in AdPEDF.11-treated eyes. Retinal cell density was significantly greater in the ganglion cell layer (GCL; P = 0.014), inner nuclear layer (INL; P = 0.008), and outer nuclear layer (ONL; P = 0.008) of AdPEDF.11-treated eyes compared with the corresponding layers in AdNull.11-treated eyes. AdNull.11-treated eyes also had significantly more TUNEL-positive cells in these layers than AdPEDF.11-treated eyes (P < 0.05). CONCLUSIONS. Adenoviral vector-mediated intraocular expression of PEDF significantly increases cell survival after ischemia-reperfusion injury of the retina. The protective effect may result from inhibition of ischemia-induced apoptosis. This study provides proof of concept for a gene transfer approach directed at interrupting programmed cell death induced by retinal ischemic insult.     

Inhibition of cyclooxygenase-2 expression by zinc-chelator in retinal ischemia

The zinc ion (Zn2+) is abundant in neurons. However, excessive Zn2+ can induce neuronal cell death. This study examined the role of Zn2+ in transient retinal ischemia in adult male rats. The rats were sacrificed 4-24 h after retinal ischemia by high intra-ocular pressure, and the retinas were prepared for microscopic examination of retinal cell degeneration, and fluorescence microscopy using zinquin ethyl ester as the zinc ion-specific probe. Moreover, COX-2 expression was observed by Western blotting. In control retinas, there was a low Zn2+ concentration in the inner plexiform layer (IPL), a high Zn2+ concentration in the outer plexiform layer (OPL), and no detectable Zn2+ in either the ganglion cell layer (GCL) or the inner nuclear layer (INL). In contrast, in the retinas exposed to ischemia without the administration of the zinc ion chelators (Ca2+-EDTA and TPEN), Zn2+ deposits were found in the IPL and INL beginning 4 h after ischemia and degeneration of neurons was found in the GCL and INL. Less Zn2+ accumulation in the IPL and INL and less neuronal degeneration in the GCL and INL were found in the retinas treated with Ca2+-EDTA or TPEN before ischemia. Furthermore, the COX-2 protein levels increased 4-8 h after retinal ischemia, and chelation of zinc ion inhibited this effect. These results suggest that the accumulation of Zn2+ following an ischemic insult can cause retinal degeneration and induce abnormal COX-2 expression.     

In vivo evaluation of leukocyte dynamics in the retinal and choroidal circulation

We have developed a new method to visualize leukocytes and evaluate their kinetics in the chorioretinal microcirculation of the living eyes. Nuclear staining dyes and a scanning laser ophthalmoscope were used to image leukocytes in the fundus. Acridine orange was used to visualize leukocytes in the retinal microcirculation. For imaging leukocytes in the choroid, indocyanine green was injected intravenously. Dynamics of leukocytes in the capillaries of the retina and choroid were quantitatively estimated in monkeys and rats. This method also allowed evaluation of leukocyte-endothelial interactions, such as rolling or firm adhesion, in vivo. Acridine orange leukocyte fluography was used to study leukocyte dynamics in the following experimentally induced microcirculatory disturbances of the retina: 1) interferon-associated retinopathy, 2) ischemia-reperfusion injury of the retina, and 3) experimental diabetes mellitus. 1) Interferon-associated retinopathy Systemic administration of interferon alpha enhanced leukocyte-endothelial interactions in the retina, which resulted in leukocyte rolling and entrapment in the retinal capillary beds. Leukocyte accumulation was also detected in the lung. The entrapment or accumulation of leukocytes in the microcirculation was inhibited by simultaneous administration of corticosteroids or other agents. These results suggested that leukocytes play a major role in the development of adverse effects of interferon, such as retinopathy or interstitial pneumonia. 2) Ischemia-reperfusion injury of the retina During reperfusion period after transient (60 min) retinal ischemia by optic nerve ligation, the rolling of leukocytes in the retinal veins was prominent and numerous leukocytes were trapped in the retinal capillaries. The number of rolling leukocytes was at a maximum 12 hours after reperfusion. Leukocyte entrapment peaked at 24 hours after reperfusion. By blocking adhesion molecules on the vascular endothelium, these leukocyte-endothelial interactions were effectively inhibited. Postischemic retinal atrophy was also inhibited by blocking adhesion molecules. These results suggested that leukocytes may be major players in the pathophysiology of ischemia reperfusion injury of the retina. 3) Experimental diabetes mellitus Leukocyte dynamics in the retina were studied in streptozotocin-induced diabetes and spontaneous diabetes (OLETF rats). In both diabetic models, leukocyte entrapment in the retinal capillaries was increased even in the early stages of diabetes. Fluorescein angiography revealed that trapped leukocytes disturbed the regional capillary blood flow in the downstream. Enhanced expression of adhesion molecules was observed in the capillary endothelium of the retina in the diabetic rats. Leukocyte entrapment in the retinal capillaries might cause microvascular occlusions and dysfunction, in turn causing diabetic retinopathy.     

Human ganglion cells express the alpha-2 adrenergic receptor: relevance to neuroprotection

BACKGROUND/AIM: Alpha-2alpha adrenergic receptor (alpha(2)-AR) agonists are thought to be neuroprotective, preventing retinal ganglion cell death independent of pressure reduction. Previous studies have identified alpha(2)-ARs in rat retina. The authors aimed to demonstrate the presence and localisation of alpha(2)-ARs in human and rat retina and on the rat retinal ganglion cell line, RGC-5. METHODS: Seven postmortem human and three postmortem rat eyes were paraformaldehyde fixed and frozen. RGC-5 cells were also paraformaldehyde fixed. The expression of alpha(2A)-ARs was determined by antibody immunofluorescence. RESULTS: alpha(2A)-AR expression was identified in the human retina, on ganglion cells, and cells in the inner and outer nuclear layers (INL, ONL). Differential alpha(2A)-AR staining patterns in the INL and ONL suggest a further restriction to as yet unidentified neuronal subclasses. The RGC-5 cell line also expressed alpha(2A)-ARs in undifferentiated cells and an increased expression upon fully differentiated cells. CONCLUSION: alpha(2)-AR agonists in addition to their pressure lowering effects in the eye, may act directly upon retinal neurons, including retinal ganglion cells. The presence of alpha(2)-ARs on the RGC-5 cell line allows future investigation of these possible direct effects using in vitro glaucoma model systems.     

Apoptosis and caspases after ischemia-reperfusion injury in rat retina

PURPOSE: Extensive cell loss in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) was noted in a rat model of retinal ischemia-reperfusion injury by transient elevated intraocular pressure (IOP). The possible involvement of apoptosis and caspases was examined in this model of neuronal loss. METHODS: Transient elevated IOP was induced in albino Lewis rats through the insertion of a needle into the anterior chamber connected to a saline column. Elevated IOP at 110 mm Hg was maintained for 60 minutes. Groups of animals were euthanatized at various times after reperfusion, and their retinas were evaluated by morphology, agarose gel electrophoresis of DNA, in situ terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL), immunohistochemistry of caspases II (ICH1) and III (CPP32), and morphometry. YVAD.CMK, a tetrapeptide inhibitor of caspases, was used to examine the involvement of caspases. RESULTS: A marked ladder pattern in retinal DNA gel analysis, typical of internucleosomal DNA fragmentation and characteristic of apoptosis, was present 12 and 18 hours after reperfusion. Labeling of nuclei in the RGCL and the inner nuclear layer (INL) by TUNEL was noted between 8 and 18 hours after reperfusion. Histologic and ultrastructural features typical of apoptosis were also observed in the inner retina after ischemia. YVAD.CMK administered during the ischemic period inhibited apoptotic fragmentation of retinal DNA and ameliorated the tissue damage. When administered intravitreally 0, 2, or 4 hours after reperfusion, YVAD.CMK was also effective in preserving the inner retina but had no significant effect when administered 6 or 8 hours after reperfusion. The inner retina showed transient elevated immunoreactivity of caspases II and III 4 and 8 hours after reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion after transient elevated IOP induced apoptosis of cells in the retinal ganglion cell layer and the INL. Caspases may have a pivotal role in the early events of the apoptotic pathway(s). Rescue by using anti-apoptotic agents after ischemia-reperfusion is feasible.     

自动化分割算法分析RTVue100 OCT黄斑区视网膜各层厚度的重复性和再现性研究

背景视网膜各层厚度的检测和评估对多种眼科疾病的诊断、治疗及预后的监测至关重要,频域光学相干断层扫描（OCT）是目前常用的测量工具。以往的测量方法采用软件分析法,但关于视网膜自动分层软件可靠性的研究较少。目的评估自动化分割算法分析RTVue100 OCT测量黄斑区视网膜各层厚度的重复性和再现性。方法采用横断面研究设计,纳入正常受试者18人18眼,用RTVue100 OCT拍摄受试者右眼以黄斑中心凹为中心6mm扫描区的视网膜各层厚度图像,采用自动化分割算法将视网膜图像分割成神经纤维层（RNFL）、神经节细胞层和内丛状层（GCL＋IPL）、内核层（INL）、外丛状层（OPL）、外核层（ONL）、光感受器内节（IS）、光感受器外节（OS）、视网膜色素上皮（RPE）层,并用Matlab软件进行分析,通过将自动探测得到的相邻两条边界相对应的位置相减获得结果。每个受检眼先由一位操作者连续拍摄2次,然后由另一位操作者拍摄1次,采用组内相关系数（ICC）和重复性系数（COR）分析测得结果的重复性和再现性。结果RTVue100 OCT测量得到水平方向视网膜全层厚度为（303．22±14．10）μm,垂直方向为（306．68±13．32）μm。视网膜各层结构中,最厚的为GCL＋IPL以及ONL。同一检查者测量2次的重复性结果和不同检查者之间的再现性结果均表明,无论在水平方向还是在垂直方向,OPL、IS和OS的ICC和COR均〈0．60,而RNFL、GCL＋IPL、INL、ONL和RPE层的ICC和COR均〉0．70。结论自动化分割算法分析RTVue OCT图像是一种可靠的工具,可提供重复性的OCT视网膜厚度资料,有利于视网膜疾病的诊断和监测。     

蛋白酪氨酸激酶抑制剂对血-视网膜屏障损害的影响及其作用机制

背景白介素1p（IL-1β）等细胞因子与血一视网膜屏障损害关系密切,细胞因子的信号传导主要由蛋白酪氨酸激酶受体传导通路介导。目的研究蛋白酪氨酸激酶（PTK）抑制剂-Genistein对IL-1β诱导血一视网膜屏障损害的影响,探讨PTK信号转导通路在血一视网膜屏障损害中的作用和机制。方法健康sD大鼠24只玻璃体腔内注射2mg／LIL-1β10ng建立血一视网膜屏障损害的动物模型,再随机分为IL-1β模型组和0．2、1、5P,gGenistein干预组。Genistein干预组大鼠于造模后立即用1μl各剂量Genistein行玻璃体腔内注射,IL-1β模型组应用1μ1DMSO以同样的方法注射。分别于注射后3h和47h每组各取2只鼠经颈静脉10S内快速注射Evansblue（45ing／kg）并收集动脉血,检测血一视网膜屏障通透性的变化;分别于玻璃体注射后4h和48h收集视网膜,用苏木精一伊红染色观察视网膜的组织学变化,RT-PCR法观察视网膜中IL．8和单核细胞趋化蛋白-1（MCP-1）mRNA表达的变化,用免疫组织化学法染色观察视网膜中MCP-1蛋白表达的变化。结果大鼠玻璃体腔注射Genistein后,视网膜与血浆Evansblue比值明显下降,各组间的总体比较差异有统计学意义（F4h=7．510,P=0．010;F48h=5．960,P=0．019）;随着玻璃体腔注射Genistein的剂量增加,Genistein各剂量组的视网膜与血浆Evansblue比值逐渐下降,与IL-1β比较差异均有统计学意义（P〈0．05）。视网膜苏木精一伊红染色结果表明,IL-1β在玻璃体腔注射后4h视网膜血管扩张并有炎性细胞浸润,注射48h后上述症状减轻,而Genistein干预组视网膜血管反应轻,无明显炎性细胞浸润。RT-PCR法结果显示,各剂量Genistein组玻璃体腔注射后视网膜中IL-8和MCP-1mRNA的表达量均明显减少,与IL-1β组比较差异均有统计学意义（P〈0．05）。免疫组织化学染色提示Genistein大鼠神经视网膜中MCP-1蛋白表达与IL-1β组比较明显减弱。结论PTK抑制剂Genistein通过抑制IL-1β诱导的视网膜趋化因子的分泌及减少白细胞在视网膜中的浸润,进而减轻IL-1β诱导的血-视网膜屏障损害。Genistein的作用强度呈剂量依赖性。     

Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish

Teleost fish regenerate retinal cells from a population of inner nuclear layer (INL) stem cells. To characterize photoreceptor regeneration in zebrafish (Danio rerio), adult albino fish were subjected to constant intense light to cause photoreceptor cell death. Retinal morphometry was performed on histological sections of control and light-lesioned albino retinas to compare the extent of light damage in the ventral, central and dorsal retinal regions. In addition, opsin immunohistochemistry and TUNEL were used to compare photoreceptor cell death in these different retinal areas, while PCNA immunolabeling quantified the cell proliferation that precedes the photoreceptor regeneration. Transgenic albino; Tg(alpha1-tubulin:egfp) zebrafish were also exposed to the intense light in order to examine regeneration-related gene expression changes. The light-lesioned retinas are characterized by extensive rod and cone photoreceptor cell death in the central and dorsal regions. In contrast, many of the rods and cones survive in the ventral retina. The highest levels of INL cell proliferation, which occurs subsequent to photoreceptor death, correspond to the retinal regions that suffer the greatest levels of photoreceptor damage. In the ventral retina, where photoreceptor cell death is minimal, cell proliferation is confined to the ONL. In addition, EGFP expression from the alpha1-tubulin promoter is increased in Müller glial cells in the light-damaged central and dorsal retina, while transgene expression in the ventral retina is restricted to small, round INL cells. Furthermore, expression of the HuC/D neuronal antigen is detected in a subpopulation of the Müller cells in the light-damaged superior retinal region. These data demonstrate that adult albino zebrafish display retinal regional differences in photoreceptor cell death and in the regeneration-related INL cell proliferation response. The high levels of INL cell proliferation and alpha1-tubulin:egfp transgene expression in the Müller cells may be graded in response to the degree of photoreceptor cell death. This suggests that the levels of photoreceptor damage may directly influence cell responses in the underlying retinal layers.