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.     

Absence of voltage-dependent calcium channels delays photoreceptor degeneration in rd mice

Retinal degeneration results from the apoptotic cell death of photoreceptors. While mutations in a large number of genes give rise to retinal degeneration, the specific mechanisms are not well understood. One hypothesis involves mediation of apoptosis by high concentrations of intracellular Ca(2+). We used a mouse line that carries the rd mutation but also lacks the major L-type voltage-dependent Ca(2+) channel at the photoreceptor synapse to examine whether this route of Ca(2+) entry plays a role in photoreceptor degeneration. In both experimental and control mice, the photoreceptors degenerate. However, at postnatal days 16, 18, and 21 there is a delay in photoreceptor cell loss in the experimental mice, which lack L-type voltage-dependent Ca(2+) channels, compared to controls. These data indicate that Ca(2+) entry via the L-type voltage-dependent Ca(2+) channel contributes to the mechanisms responsible for photoreceptor cell death in this mouse model of retinitis pigmentosa.     

Remodeling of cone photoreceptor cells after rod degeneration in rd mice

We studied the survival of cone photoreceptors following the degeneration of rods in the rd mouse. Cones were visualized by selective expression of green fluorescent protein (GFP) following transduction with an adeno-associated virus (AAV) vector. As previously reported, many cones survive after the initial degeneration of the rods. Soon after the initial degeneration, they lose their outer segments and all but a vestigial inner segment; and they partially retract or lose their axon and synaptic pedicle. However, they retain many fundamental features of the cone phenotype, and for many weeks show a polarized morphology indicative of substantial regrowth of processes. The cells retain their laminar position, forming a cell row just distal to a much thinned outer plexiform layer. The somata subsequently enlarge. Most of the cells extend bipolar processes, recreating the original bipolar morphology of a photoreceptor cell - though now turned on its side relative to the native position. The cells express short- or middle-wavelength opsins, recoverin and connexin36. One or more of the polarized processes could often be shown to contain synaptic ribbons, as visualized by antibodies against RIBEYE. The cones do not express protein kinase C alpha, Go alpha, ChX10 or calbindin, markers of bipolar or horizontal cells. The partially differentiated cone morphology persists for at least several months, after which the processes begin to retract and there is slow loss of the cells. Thus, during the time following the loss of their rod-dominated microenvironment, the cones achieve a semi-stable state in which much of their normal phenotype is preserved. Cone photoreceptors in retinas of human RP donors appear from their morphology to undergo a similar progression. The therapeutic window for rescue of cone photoreceptors may be longer than would have been thought.     

锂对视网膜色素变性小鼠光感受器细胞凋亡的保护作用

目的 研究锂在视网膜色素变性小鼠模型上对光感受器细胞凋亡的神经保护作用.方法 实验研究.FVB/NJ视网膜色素变性小鼠出生后立刻用含锂的食物喂养,7和14 d时摘除眼球做视网膜冰冻切片,同时取血测血清锂浓度.HE、TUNEL和视杆细胞免疫荧光染色进行视网膜组织形态、光感受器细胞凋亡分析.结果 光镜下可见,对照组外核层可见TUNEL阳性细胞,出生后14 d外核层厚度和细胞层数(3或4层)明显低于7 d时的厚度(9或10层),而锂喂养组出生后14 d外核层的厚度和细胞层数明显高于对照组,与出生7 d时的外核层厚度及细胞层数无明显差异.结论 锂能够保护视网膜色素变性小鼠光感受器细胞免于凋亡.(中华眼科杂志,2008,44:248-252)     

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。     

Retardation of photoreceptor degeneration in the detached retina of rd1 mouse

PURPOSE: To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice. METHODS: RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30. RESULTS: The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD. CONCLUSIONS: RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.     

Mechanism of protection from light-induced retinal degeneration by the synthetic antioxidant phenyl-N-tert-butylnitrone

PURPOSE: A prior study demonstrated that pretreatment with phenyl-N-tert-butylnitrone (PBN), a synthetic antioxidant and free radical trapping agent, protects rats from light-induced photoreceptor cell death. The objective of the present study was to elucidate the molecular mechanism of PBN neuroprotection. METHODS: Sprague-Dawley rats (5-6 weeks old) raised in dim (5 lux) cyclic light (12 hours ON/OFF) from birth were injected intraperitoneally with PBN or water 30 minutes before exposure to three columns of fluorescent light (approximately 2700 lux intensity) for 0, 3, 6, 12, or 24 hours. mRNA levels were measured by RNase protection assay and DNA fragmentation by TUNEL assay. Activator protein (AP)-1 complex was determined by electrophoretic mobility shift assay. Immunocytochemistry and Western blots were used to measure changes in c-fos levels. RESULTS: Typical apoptotic features (TUNEL staining and DNA laddering) were seen in rat retinas after 24 hours of continuous exposure to light, but not in PBN-injected rats. FasL, Bax, and caspase-3 were upregulated in a time-dependent manner. PBN treatment markedly inhibited caspase-3 gene expression, but neither PBN nor bright light exposure had any effect on caspase-3 activity. AP-1 activation by exposure to light was inhibited by PBN. Western blot analysis showed that the c-fos protein level increased in the nuclear fraction after a 6-hour exposure to light, but was decreased in PBN-treated rats. CONCLUSIONS: Inhibition of c-fos activation by PBN may be the key event in protection. The involvement of oxygen free radicals has been suggested in c-fos activation and the action of PBN could be through its antioxidant activity.     

Cellular responses to photoreceptor death in the rd1 mouse model of retinal degeneration

PURPOSE: Retinal degeneration is a disease that typically involves the loss of photoreceptors. Murine models have been established for such degenerations, and a variety of methods have been used to follow the progression of the disease. In the present study in situ hybridization was used to analyze gene expression responses in the different retinal cell types during the period of cone death in the rd1 mouse model. METHODS: A preliminary microarray analysis led to the selection of 169 candidate genes that might change in level of expression during degeneration. Probes corresponding to these genes were used for in situ hybridization on tissue during the period of cone death. Expression values were assigned to the intensities of in situ hybridization signals and were compared between mutant and wild-type tissue. RESULTS: During the peak of cone death, the in situ hybridization signals were typically higher in the mutant. This signal change was often true of genes with a wild-type pattern of expression in ganglion cells, bipolar cells, and/or Müller glia. In such cases, the upregulation was highest in bipolar cells and/or Müller glia. CONCLUSIONS: All retinal cell types responded during the process of retinal degeneration, as revealed by changes in gene expression. Genes that showed changes in the in situ hybridization signals during the period of cone death were typically higher in the mutant, with many of them expressed in both the ganglion cell layer and the inner nuclear layer.     

NOX2基因缺陷对rd1小鼠感光细胞凋亡的保护作用

目的 观察NADPH氧化酶2(NOX2)基因缺陷对遗传性视网膜变性小鼠1(rd1)感光细胞的保护作用。设计 实验研究。研究对象 出生后14天的NOX2基因缺陷rd1小鼠(实验组)6只及同龄NOX2基因缺陷的C57BL/6N小鼠(对照组1)、无NOX2基因缺陷的rd1小鼠(对照组2)、C57BL/6N野生正常小鼠(对照组3)各6只(共24只)。方法 对照组1与对照组2小鼠多次交配获得实验组小鼠并进行基因型鉴定。取该实验鼠及对照组小鼠眼球,对视网膜进行HE染色并测量视网膜外核层厚度,TUNEL染色并计算凋亡细胞占外核层细胞总数百分比、免疫荧光法CD1 1b抗体标记小胶质细胞并检测NOX2主要亚单位gp91^pbox蛋白的表达。主要指标 视网膜外核层厚度,感光凋亡细胞百分比,gp91^pbox蛋白的表达量,小胶质细胞活化情况。结果 与同龄对照组2相比,实验组小鼠视网膜内外核层排列整齐,其外核层厚度(36.18±2.59)μm明显大于对照组2小鼠(21.45±1.33)μm(t=8.77,P=0.001)。实验组小鼠视网膜凋亡细胞主要出现于外核层,但数量...     

Strain differences in sensitivity to light-induced photoreceptor degeneration in albino mice

The sensitivity to light-induced photoreceptor degeneration was examined in 7 different inbred strains of albino mice. The mice were exposed to 3 weeks of constant fluorescent light at an illuminance level of 115-130 ft-c (ca. 1,265-1,430 lux), after which the eyes were examined histologically. The degree of light-induced photoreceptor degeneration was compared to that found in BALB/cByJ (BALB/c) albino mice, which have previously been described as sensitive to the damaging effects of light, and to C57BL/6J-c2J albino mice, which have been shown to be resistant to light-induced damage. Mice of the A/J, AKR/J and NZW/LacJ strains were indistinguishable from BALB/c mice in light sensitivity, as measured by mean outer nuclear layer thickness and the presence or absence of outer segment membranes. Mice of the Ma/MyJ and RF/J strains were somewhat more sensitive to light than BALB/c mice, and those of the RIIIs/J were far more sensitive than all of the other strains. The LG/J strain differed from other strains by individual mice displaying one of two degrees of light sensitivity, those similar to the light-sensitive Ma/MyJ and RF strains and those remarkably more resistant to light, with photoreceptor outer segment integrity even greater than that of the light-resistant C57BL/6J-c2J strain. These findings demonstrate that different inbred strains of a given species may exhibit a wide range of sensitivities to constant light exposure and that most albino mouse strains examined thus far are highly sensitive to the damaging effects of light.     

低氧预适应对小鼠视网膜光感受器细胞光损伤的防护作用

目的研究低氧预适应对光损伤后小鼠视网膜光感受器细胞的保护作用,并探讨其可能的基因调控机制。方法将54只BALB／c小鼠随机分成单纯光照组、低氧预适应组和正常对照组,并将前两组动物在自制光照箱中连续光照3h,制成光损伤动物模型。应用光镜、核苷酸末端转移酶介导的dUTP缺口标记法观察各组小鼠视网膜组织结构的改变;应用免疫组织化学方法检测各组小鼠视网膜c-fos和caspase-1的表达。结果单纯光照组小鼠视网膜组织学改变出现的早且明显,光照后视网膜出现不同程度的病理学改变,且损伤主要发生在光感受器细胞层。随着光照后时间的延长,光感受器细胞凋亡数增加,视网膜光损伤逐渐加重,c-fos和caspase-1在该层出现了不同程度的阳性表达：低氧预适应组同单纯光照组相比,各时间段损伤均较轻,视网膜光感受器细胞层明显得到了保护,同单纯光照组比较,caspase-1的阳性表达明显减少,而c-fos无明显变化;正常对照组小鼠视网膜组织结构层次清楚,外核层排列规则,无c-fos和caspase-1的阳性表达。结论低氧预适应通过抑制凋亡相关基因的表达而对光感受器细胞有神经保护作用,且caspase-1参与了低氧预适应的保护机制。(中华眼科杂志,2005,41：631-635)     

Intraocular penetration of systemically administered antifungal agents

Amphotericin B, 5-flucytosine (5-FC), and ketoconazole levels were estimated in vitreous and aqueous samples taken from four patients undergoing therapeutic vitrectomy for fungal endophthalmitis. The levels of amphotericin B in the vitreous of three patients were low (.04 - .17 microgram/ml). However, 5-FC was present in a concentration of 22.2 micrograms/ml in one patient. In another case the aqueous level of ketoconazole was 0.35 microgram/ml. The vitreous in the same patient contained 0.71 microgram/ml of the drug.     

Soluble retinal proteins associated with photoreceptor cell death in the rd mouse

In the mouse, the homozygous presence of the rd gene results in the genetically programmed death of the photoreceptor cells of the retina. Using congenic strains of mice and a novel, sensitive, immunological approach for visualizing unique retinal proteins, we identified four bands of protein whose concentrations are regulated by the homozygous presence of the gene for retinal degeneration. Since these proteins (with apparent molecular weights of 23, 33, 55, and 69 kD) are present in normal adult mouse retinas and absent from rodless retinas, and from other mouse non-retinal tissues including brain, heart, kidney and liver, the data support the identification of these proteins as being retina specific. These proteins are not peculiar to the normal mouse retina; but rather, all four (23, 33, 55 and 69 kD) are common to rat retina; three (23, 33, and 55 kD) are common to bovine retina; and presently at least two, 23 and 69 kD, are clearly detectable in normal, adult human retina. The temporal appearance and disappearance of the four retinal specific protein bands coincide with the morphological maturation and degeneration of the photoreceptor cell population. Collectively, the present data suggest that one or more may be photoreceptor specific. These observations present the first step in the identification and characterization of specific soluble proteins correlated with the biochemical phenotype of the rd gene and the death of photoreceptor cells of the retina.     

Oxidative stress retards vascular development before neural degeneration occurs in retinal degeneration rd1 mice

Purpose

To investigate the role of reactive oxygen species (ROS) in retinal development during the early postnatal stage of rd1 mice.

Methods

Development of the three retinal vascular layers of C57BL/6 J (WT) and C3H/HeN (rd1) mice was evaluated from 9th postnatal day (P9) to P21. Retinal ROS production was semi-quantitatively measured using dihydroethidium fluorescence. Mice were treated with intraperitoneal injections of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) at a dose of 275 mg/kg body weight, and PBS as the control from P3 to P8.

Results

Rd1 mice showed retardation of retinal vascular development in the deep layer at P9. No significant difference was observed in the outer nuclear layer thickness of rd1 and WT mice. ROS production in the outer nuclear layer of rd1 mice was significantly higher than that in the outer nuclear layer of WT mice at P9, P13, and P17 (P?<?.05). TEMPOL facilitated the development of the deep vascular layer when compared with injection of PBS.

Conclusions

Retardation of retinal vascular development is observed in rd1 mice; ROS is partially responsible for this finding. When using rd1 mice, we should be aware of this difference in comparison to other retinal degeneration animal models and human pathophysiological changes.     

Synergism between environmental lighting and taurine depletion in causing photoreceptor cell degeneration

Experiments were conducted to examine the possible interaction between retinal taurine depletion and environmental lighting in causing photoreceptor cell degeneration. Albino rats were raised from birth in either dim (2 lx) or relatively bright (300 lx) cyclic light. Beginning at weaning, half the animals from both light environments were taurine-depleted by treating them for 10 weeks with guanidinoethyl sulfonate as a 1% solution in their drinking water. The remaining animals were given ordinary tap-water and served as controls. In the 2 lx light environment, taurine depletion caused a decrease in electroretinogram (ERG) a- and b-wave amplitude of 36 and 46%, respectively; however, no photoreceptor cells were lost in this group. Tap-water controls kept in the 300 lx light environment had a 59- and 43% decrease in ERG a- and b-wave amplitude, respectively, and a 21% reduction in the number of photoreceptor cells. In contrast to the other groups, animals that were taurine-depleted in the 300 lx environment showed a marked retinal degeneration. ERG a- and b-wave amplitude was decreased by 94- and 89% respectively and there was a 62% loss of photoreceptor cells. The greatest cell loss occurred in the central superior region of the retina, in which the outer nuclear layer was typically reduced to one to two rows of nuclei. The results of a two-way analysis of variance applied to the data indicated that the effects of taurine depletion on the retina were greater in the 300 lx as compared with the 2 lx environment in terms of loss of photoreceptor cells and reduction in log ERG a- and b-wave amplitude. These findings demonstrate a synergism between environmental lighting and taurine depletion in causing photoreceptor cell degeneration.