Histochemical demonstration of cyclic guanosine 3',5'-monophosphate phosphodiesterase activity in retinal photoreceptor outer segments.

A technique for the histochemical demonstration of cyclic guanosine monophosphate phosphodiesterase in retina is described. Enzyme activity was identified on photoreceptor outer segment lamellae, a finding in agreement with previous biochemical data on isolated outer segment preparations. The distribution of phosphodiesterase activity for cyclic guanosine monophosphate was similar to that found previously in rod outer segments for cyclic adenosine monophosphate, suggesting that the same enzyme may hydrolyze both nucleotides.     

Cyclic nucleotide phosphodiesterases in dystrophic rat retinas: guanosine 3',5' cyclic monophosphate anomalies during photoreceptor cell degeneration.

The kinetic characteristics and enzyme activities of cyclic nucleotide phosphodiesterase (PDE), using cyclic-AMP (cyclic-AMP-PDE) or cyclic-GMP (cyclic-GMP-PDE) as substrate, have been measured in developing retinas of control and Royal College of Surgeons (RCS) rats. The latter are afflicted with an inherited degeneration of the retinal photoreceptor cells. Two classes of cyclic-AMP-PDE are localized in the inner layers and one is in the photoreceptor layer of both control and RCS retinas. In RCS retina, the activity of the photoreceptor-specific cyclic-AMP-PDE surpasses that of the control retina during most of the period when rod outer segment debris accumulates, and falls below that of the control retina when the photoreceptor population is depleted. These data suggest that the accumulated debris has the capacity to hydrolyze cyclic-AMP. One class of cyclic-GMP-PDE is localized in the inner layers and one is in the photoreceptor layer of both control and RCS retinas. The kinetic characteristics of cyclic-GMP-PDE in the layers of the RCS retina are similar to those of control retina for the first 14 days of life but, during the period when debris accumulates, the cyclic-GMP-PDE of the inner layers is not demonstrable and that of the photoreceptor layers shows altered kinetics. Thereafter, the latter disappears due to the depletion of the photoreceptor population, and cyclic-GMP-PDE of the inner layers is again observable. The anomalous kinetics and activities of cyclic-GMP-PDE apparently result from the association of cyclic-GMP-PDE enzymes with a heat-denaturable, non-dialyzable material which is derived from the RCS photoreceptor cells and/or accumulated debris. The specificity of the material for cyclic-GMP-PDE suggests that cyclic-GMP metabolism may be altered, in situ, before the RCS photoreceptor cells degenerate.     

Caspase-3 inhibitor transiently delays inherited retinal degeneration in C3H mice carrying the rd gene

BACKGROUND: The effect of a caspase-3 inhibitor on retinal degeneration in C3H mice carrying the rd gene, a mutation of a rod-specific phosphodiesterase, was investigated. METHODS: A quantity of 2 mg/kg of Ac-DEVD-CHO, as inhibitor, was injected intraperitoneally every other day from 8 days of age, and retinal damage was compared with that in saline-treated C3H mice at 13 days (1 day after the third treatment) and 17 days of age (1 day after the fifth treatment). Retina of ICR mice not carrying rd gene was also evaluated under the same protocol. The efficacy of Ac-DEVD-CHO was evaluated based on total retinal thickness and outer retinal thickness (thickness of outer nuclear layer and photoreceptor layer). An apoptotic index and a cell proliferation index for the photoreceptor cells, at 13 days of age, were calculated based on terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) labeling, respectively. RESULTS: At 13 days of age, total and outer retinal thickness in saline-treated C3H mice were 140.3 microm and 37.5 microm, compared with 160.4 microm and 49.5 microm, respectively, in Ac-DEVD-CHO-treated C3H mice ( P<0.01, respectively). In ICR mice, total and outer retinal thickness were 182.1 microm and 90.9 microm, respectively, in saline-treated mice and 183.8 microm and 89.6 microm in Ac-DEVD-CHO-treated mice (not significant). At this time, the TUNEL index was 23.52 cells/10(4) microm (2) of outer nuclear layer in saline-treated C3H mice; Ac-DEVD-CHO treatment significantly reduced this value to 18.73 cells/10(4) microm(2) ( P<0.05). The TUNEL index in saline- and Ac-DEVD-CHO-treated ICR mice was 0.59 cells/10(4) microm(2) and 0.80 cells/10(4) microm(2), respectively (not significant); Ac-DEVD-CHO treatment had no influence on normally developing retina. The PCNA index was not affected by Ac-DEVD-CHO-treatment. However, at 17 days of age, Ac-DEVD-CHO treatment did not ameliorate retinal degeneration. CONCLUSIONS: The caspase-3 inhibitor was transiently effective in delaying retinal degeneration through inhibition of the apoptosis of photoreceptor cells in rd gene-carrying mice. The use of caspase-3 inhibitors may have therapeutic applications in the treatment of human retinal degeneration.     

半胱氨酸天冬氨酸酶3在遗传性视网膜变性中的表达

目的 观察半胱氨酸天冬氨酸酶3（caspase 3)mRNA在RCS大鼠视网膜中的表达，分析caspase 3特异抑制剂Ac-DEVD-CHO对视细胞凋亡的影响。方法 应用逆转录－多聚酶链反应（RT－PCR）检测RCS大鼠出生后不同时间视网膜中caspase 3 mRNA的表达。应用TUNEL方法检测玻璃体内注射Ac-DEVD-CHO后视细胞凋亡的改变。结果 RCS大鼠出生后14天至60天视网膜caspase 3 mRNA均有表达，25天水平明显升高，达到高峰。SD大鼠视网膜15至60天的表达量无明显差异，与RCS大鼠14天水平接近。caspase 3特异抑制剂Ac-DEVD-CHO能显著抑制视细胞凋亡。结论 caspase 3可能在RCS大鼠视网膜变性视细胞凋亡中起关键作用。     

Permeability of retinal capillaries in rats with inherited retinal degeneration.

The permeability of retinal capillaries in RCS rats with inherited retinal degeneration was investigated with horseradish peroxidase used as a tracer. Five-week-old rats showed typical degeneration of photoreceptor cells and accumulation of outer segment debris, but retinal capillaries were not permeable to peroxidase. At 10 weeks of age, capillaries in the inner retina appeared normal, but many in the outer retina were leaky. Peroxidase activity in these latter vessels was demonstrable in the basal laminae of endothelial cells and pericytes and in vesicles on the luminal and abluminal sides of the endothelium. Tracer also permeated the intercellular spaces in the surrounding area. The number of leaky capillaries in the outer retina increased during the course of the dystrophy. The site of the leak in permeable capillaries has not yet been established; it may be due to an alteration of the endothelial cell junctions or of transcellular vesicular transport. In the choriocapillaris, peroxidase permeated Bruch's membrane and the basal infoldings between adjacent pigment epithelial cells; tracer progression along the intercellular spaces was blocked at the zonulae occludens at the apicolateral border. The RCS rat may be a useful model for studying the morphological basis of changes in capillary permeability associated with retinal degeneration.     

米诺环素对视网膜色素变性小鼠光感受器细胞的保护作用

目的 观察米诺环素对视网膜色素变性（RP）的rd小鼠［C3H/HeN (Pde6brd-/rd-)］RP过程的影响。方法 40只新生rd小鼠随机分为10组，5组为实验组，5组为对照组，每组4只小鼠。实验组，出生后每日腹腔注射米诺环素22.5 mg/kg；对照组，出生后每日腹腔注射生理盐水10 ml/kg。在出生后1、7、14、21、28 d各处死一组实验组和对照组小鼠，取眼球做组织学观察并行凋亡细胞检测，并对两组视网膜光感受器细胞数、外核层厚度以及凋亡细胞数目进行统计分析。结果 （1）rd小鼠出生后7 d光感受器细胞开始凋亡，14 d达高峰，28 d光感受器细胞完全消失；（2）出生后7 d实验组光感受器细胞数目和外核层厚度与对照组比较差异无统计学意义；（3）出生后14、21 d实验组光感受器细胞数目和外核层厚度多于对照组相应时间点，差异有统计学意义（14 d：t=-3.03、P=0.016，t=-4.469，P=0.004；21d: t=-8.782、P＜0.001，t=-3.497、P=0.004）；（4）出生后7、14 d实验组外核层凋亡细胞数目少于对照组相应时间点，差异有统计学意义（t=-3.497、P=0.004，t=-8.782、P＜0.0001）。结论 米诺环素在rd小鼠RP早期可以延缓光感受器细胞丢失，但不能完全阻止RP的发生。     

Selective blockade of phosphodiesterase types 2, 5 and 9 results in cyclic 3'5' guanosine monophosphate accumulation in retinal pigment epithelium cells

AIM: To investigate which phosphodiesterase (PDE) is involved in regulating cyclic 3'5' guanosine monophosphate breakdown in retinal pigment epithelium (RPE) cells. METHODS: cGMP content in the cultured RPE cells (D407 cell line) was evaluated by immunocytochemistry in the presence of non-selective or isoform-selective PDE inhibitors in combination with the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). mRNA expression of PDE2, PDE5 and PDE9 was studied in cultured human RPE cells and rat RPE cell layers using non-radioactive in situ hybridisation. RESULTS: In the absence of PDE inhibitors, cGMP levels in cultured RPE cells are very low. cGMP accumulation was readily detected in cultured human RPE cells after incubation with Bay60-7550 as a selective PDE2 inhibitor, sildenafil as a selective PDE5 inhibitor or Sch51866 as a selective PDE9 inhibitor. In the presence of PDE inhibition, cGMP content increased markedly after stimulation of the particulate guanylyl cyclase. mRNA of PDE2,PDE5 and PDE9 was detected in all cultured human RPE cells and also in rat RPE cell layers. CONCLUSIONS: PDE2, PDE5 and PDE9 have a role in cGMP metabolism in RPE cells.     

Retinal degeneration following failed photoreceptor maturation in 5A11/basigin null mice

5A11/Basigin is a member of the immunoglobulin gene superfamily which plays an important role in cell-cell interactions in the developing neural retina. These studies were initiated to investigate the distribution of 5A11/Basigin within the mouse retina, as well as the cytoarchitectural and biochemical effects on the retina after the inactivation of the 5A11/Basigin gene in a mouse strain. Immunocytochemical analyses indicated that mouse 5A11/Basigin is located on the surface of Müller cells, the apical and basal surfaces of the retinal pigmented epithelium, and blood vessels. Lower expression levels were found on photoreceptor cell bodies and a portion of the inner segments. Inactivation of the 5A11/Basigin gene in mice resulted in the failure of photoreceptor cells to fully mature. This failed development eventually lead to the degeneration, death and removal of most of the photoreceptors several months after birth. Biochemical analyses indicated that expression of Müller cell specific proteins, including glutamine synthetase and carbonic anhydrase-II, was not effected; however, opsin protein expression never achieved normal adult levels in the 5A11/Basigin null mice. Also, 5A11/Basigin null retinas were considered 'reactive' based on elevated glial fibrillary acidic protein expression. The results presented here suggest that 5A11/Basigin expression on Müller cells and/or the retinal pigmented epithelium is necessary for photoreceptor outer segment biochemical development and structural maintenance. However, the exact role that 5A11/Basigin plays during retinal development remains to be determined.     

NADPH氧化酶抑制剂对遗传性视网膜色素变性感光细胞凋亡的抑制作用

背景 我们先前的研究表明,rd小鼠遗传性视网膜色素变性（RP）过程中小胶质细胞活化与感光细胞的凋亡密切相关.研究显示,小胶质细胞中烟酰胺二磷酸腺苷（NADPH）氧化酶的活化在小胶质细胞活化及神经元损伤中发挥重要作用,但NADPH氧化酶在RP过程中作用机制及其抑制剂的作用有待探讨.目的 探讨rd小鼠发生RP过程中NADPH氧化酶产生活性氧簇（ROS）的活化反应及其抑制剂对感光细胞的保护作用. 方法 按抛掷硬币法将60只SPF级rd小鼠随机分为香荚兰乙酮注射组和PBS对照组,香荚兰乙酮注射组小鼠于出生后9d（P9）腹腔内注射NADPH氧化酶抑制剂香荚兰乙酮10 mg/kg（0.01 ml/kg）,每日1次,连续5d（至P13）;PBS对照组rd小鼠以同样方式注射等容量的PBS;C57 BL/6N小鼠10只不注射任何药物作为rd小鼠的野生对照鼠.各组小鼠于出生后14 d（P14）处死并制备视网膜冰冻切片,采用二氢乙锭（DHE）荧光染色法检测3个组小鼠视网膜中ROS的表达;采用实时定量PCR（real-time PCR）法测定2个组rd小鼠视网膜感光细胞中视紫红质mRNA的定量表达;采用苏木精-伊红染色法检查2个组rd小鼠视网膜外核层厚度.结果 DHE荧光染色表明,小鼠视网膜中ROS表达呈红色荧光,注药组小鼠视网膜外核层中ROS的红色荧光明显强于C57BL/6N野生鼠,但明显弱于PBS对照组.Real-time PCR检测表明,香荚兰乙酮注射组小鼠感光细胞中视紫红质mRNA相对表达量为4.21 ±0.33,明显低于PBS对照组的0.93±0.24,差异有统计学意义（t=2.360,P=0.000）;香荚兰乙酮注射组小鼠视网膜外核层厚度为（35.95±1.63） μm,明显厚于PBS对照组的（23.17±1.38） μm,差异有统计学意义（t=3.850,P=0.016）.结论 在rd小鼠视网膜感光细胞变性过程中,NADPH氧化酶生成ROS的活化反应明显增强;香荚兰乙酮能够延缓rd小鼠感光细胞的凋亡过程.     

Adenosine 3',5'-monophosphate analogue increases the outflow facility of the primate eye.

Our previous studies showed that cyclic-AMP increases the outflow facility of the eye of the rabbit. In this investigation, an analogue of cyclic-AMP, the 8-methylthio derivative, perfused into the anterior chamber of the eye of the vervet monkey, increased outflow facility twofold. This observation supports the hypothesis that cyclic-AMP mediates the action of adrenergic agents to reduce intraocular pressure in the primate eye.     

补肾益精方对先天性视网膜色素变性RCS大鼠感光细胞凋亡的抑制作用

目的　探讨补肾益精方对先天性视网膜色素变性RCS大鼠感光细胞凋亡的影响。方法　将先天性视网膜色素变性模型RCS大鼠24只随机分为补肾益精方组及蒸馏水组,分别给予补肾益精方药液及蒸馏水灌胃,12只健康SD大鼠常规饲养作为正常组。在给药7 d及28 d后HE染色观察各组大鼠视网膜组织病理学变化,TUNEL法检测细胞凋亡情况,实时荧光定量PCR检测视网膜中睫状神经营养因子、脑源性神经营养因子以及碱性成纤维细胞生长因子表达情况。结果　HE染色切片光学显微镜下正常组SD大鼠视网膜结构清晰,层次分明,各层细胞排列整齐。蒸馏水组大鼠视网膜内核层及外核层均较正常组明显变薄,细胞排列稀疏,可见空泡样改变,感光细胞数减少,且随鼠龄增加减少日益显著。补肾益精方组大鼠视网膜内核层及外核层亦较正常组变薄,但与蒸馏水组相比增厚,感光细胞数较后者增多,细胞排列也较为整齐。给药7 d及28 d时补肾益精方组每个高倍视野感光细胞数分别为140±9和80±9,蒸馏水组分别为113±8和44±6,补肾益精方组较蒸馏水组明显增多,差异有统计学意义（均为P<0.05）。TUNEL检测结果显示给药7 d及28 d时补肾益精方组感光细胞凋亡率分别为31.67±5.39%及29.68±4.31%,蒸馏水组分别为50.34±5.21%及44.02±7.17%,补肾益精方组较蒸馏水组均明显降低,差异均有统计学意义（均为P<0.05）。实时荧光定量PCR结果显示给药7 d及28 d补肾益精方组视网膜睫状神经营养因子表达均较蒸馏水组增加（均为P<0.05）,给药7 d时补肾益精方组视网膜脑源性神经营养因子表达较蒸馏水组明显增加（P<0.05）,28 d时两组差异无统计学意义（P>0.05）;给药7 d及28 d两组碱性成纤维细胞生长因子的表达差异均无统计学意义（均为P>0.05）。结论　补肾益精方对RCS大鼠感光细胞凋亡有明显的抑制作用,其作用机制可能与补肾益精方促进视网膜分泌神经营养因子有关。     

Adenosine 3',5'-monophosphate increases the outflow of aqueous humor from the rabbit eye.

Direct administration of cyclic-AMP into the anterior chamber increases the outflow facility of the eye for aqueous humor. This is consistent with the hypothesis that catecholamines lower the intraocular pressure of the rabbit eye, at least in part, by a cyclic-AMP-mediated mechanism. This mechanism is active in the outflow channels and increases the rate at which aqueous humor leaves the anterior chamber.     

Selective neovascularization of the retinal pigment epithelium in rat photoreceptor degeneration in vivo.

Photoreceptor cell degeneration in rodents from a variety of causes results in neovascularization of the retinal pigment epithelium as a late stage phenomenon. Even though the vessels within the pigment epithelium arise from the retinal circulation, they can manifest the choroidal endothelial cell phenotype of fenestrated endothelial cells. In order to study the detailed cellular events which result in incorporation of retinal vessels within the retinal pigment epithelium, a morphological and morphometric analysis of the RPE and vasculature was performed in rats. Urethane, given subcutaneously to newborn rats, results in a photoreceptor degeneration but does not affect the RPE, choroid or inner retinal layers. Retinas were studied from rats of 8 to 24 weeks of age, the time period when vascularization of the RPE occurs. Loss of retinal vessels is first seen at 12 weeks, primarily in substantial dropout of vessel profiles in the outer plexiform layer (OPL) vessel bed. There is a gradient of loss from the OPL bed to the nerve fiber layer (NFL) bed and from the central to peripheral region. Total vessel density of the experimental retinas is greater than controls at 8 and 12 weeks. This occurs because there is marked loss of retinal thickness, due to photoreceptor degeneration, without a comparable loss of vessel profiles. The total retinal vessel density decreases from 8 to 20 weeks, and appears to stabilize at 20 and 24 weeks. Analysis of the separate vessel beds shows that this apparent stabilization is due to continued loss of vessels within the sensory retina, and increased presence of vascular profiles within the RPE. Total absence of the photoreceptor cell is necessary for incorporation of vessels within the RPE. Since new vessel profiles develop in the RPE but not the adjacent sensory retina, we speculate that the RPE may stimulate neovascularization of the RPE. A model of the cellular events leading to RPE neovascularization is proposed.     

Cyclic GMP and visual cell degeneration in the inherited disorder of rd mice: a progress report

Cyclic GMP accumulates in visual cells of rd (retinal degeneration) mice before the onset of morphological pathology. Observations are presented which support the hypothesis that elevated levels of cyclic GMP initiate visual cell degeneration in some early-onset disorders causing blindness. The accumulation of cyclic GMP in rd visual cells results apparently from defective mechanisms that regulate cyclic GMP metabolism.     

Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium

Experiments were conducted to evaluate the role played by photoreceptor cells in the accumulation of age pigment, or lipofuscin, in the retinal pigment epithelium (RPE). The age-related accumulation of RPE lipofuscin was compared between rats with hereditary photoreceptor degeneration (RDY) and congenic rats with normal retinas. In the RDY animals, the age-related increase in RPE lipofuscin content was substantially less than in normal controls. This suggests that the photoreceptor cells play a significant role in RPE lipofuscin deposition, although they may not be the sole contributors to RPE lipofuscin formation. Evidence that outer-segment components may be converted into lipofuscin fluorophores was provided by the discovery that in young RDY rats, fragments of outer segments from degenerating photoreceptor cells had fluorescence properties similar to those of RPE lipofuscin. Chloroform-methanol extraction of retina-RPE tissue from young normal and dystrophic rats, and analysis of the chloroform fractions by thin-layer chromatography, revealed three distinct fluorescent components associated with the lipofuscin-like fluorescence of the outer-segment fragments in the RDY rats.