Differential expression of neuroendocrine-specific protein in form-deprived chick eyes

PURPOSE: To identify genes that are highly expressed in form-deprived retina-retinal pigment epithelium-choroid tissues. Neuroendocrine-specific proteins were found to be highly expressed. METHODS: mRNAs enriched in retina-retinal pigment epithelium-choroid tissues from 3-, 7-, and 14-day form-deprived chick eyes were isolated by differential display technique with cDNA library screening. Neuroendocrine-specific protein A and C were cloned in control and form-deprived eyes. mRNA and protein levels, with respective regional localizations, were examined by Northern blot, Western blot, and immunohistochemical analyses, respectively. RESULTS: The isolated clone included an insert with a sequence homologous to both chick neuroendocrine-specific proteins A and C. The increases in mRNA and protein levels were confirmed by Northern and Western blot analyses, respectively. Immunohistochemical localization of neuroendocrine-specific proteins A and C was detected in the layer of photoreceptor inner segments, presumably in the cone cells. Northern blot analysis using negative lenses showed that levels of neuroendocrine-specific protein A and C mRNAs were not altered using negative lenses. CONCLUSIONS: The expression of both neuroendocrine-specific proteins A and C mRNAs in cone photoreceptor cells was upregulated within 14 days of form deprivation, but not in response to negative spectacle lenses. These data suggest that the increase in induction of neuroendocrine-specific proteins is not a secondary consequence of ocular elongation or myopic refraction. Induction of neuroendocrine-specific proteins in form-deprived eyes may be causally related to the development of myopia or may be an unrelated effect of form deprivation.     

Melatonin receptors in chick ocular tissues: implications for a role of melatonin in ocular growth regulation

PURPOSE: The influences of diurnal rhythms involving a variety of ocular parameters are implicated in the development of myopia. The purpose of this study was to determine the expression of the melatonin receptor subtype proteins in chick ocular tissues and to examine the role of the circadian signaling molecule melatonin in normal ocular growth and the exaggerated ocular growth associated with the development of myopia. METHODS: Expression of the Mel(1a), Mel(1b), and Mel(1c) melatonin receptor proteins in ocular tissues was examined by Western blot analyses, slot blot analyses, and immunocytochemistry. For examining the effect of melatonin on ocular growth, chicks were maintained on a 12-hour light-dark cycle and were monocularly form-vision deprived in one eye with a translucent occluder for 5 days. During the 5-day treatment period, chicks were injected systemically during the early dark period with melatonin (0.6 mg) or 2% ethanol vehicle control. Ocular dimensions of normal and deprived eyes were examined by high frequency A-scan ultrasound. RESULTS: Immunocytochemical analysis of chick ocular tissues revealed the cellular distribution of the Mel(1a) receptor subtype in the cornea, choroid, sclera, and retina. Western blot and slot blot analyses demonstrated that all three receptors were present in these tissues and they demonstrated distinct diurnal rhythms of protein expression in the retina-RPE-choroid, with peak levels of Mel(1a) and Mel(1b) occurring during the night and peak levels of Mel(1c) during the day. Systemic administration of melatonin resulted in significant changes in anterior chamber depth, vitreous chamber depth, and choroidal thickness of form-deprived and/or control eyes. CONCLUSIONS: Results of this study show that all three melatonin receptor subtypes are expressed in retinal and extraretinal ocular tissues of the chick eye. The finding that administration of melatonin alters the growth of several ocular tissues in both control and form-deprived eyes suggests that melatonin, acting through specific melatonin receptors in ocular tissues, plays a role in ocular growth and development. This conclusion suggests that the action of melatonin, combined with expression of melatonin receptors, is involved in the regulation of the diurnal rhythm of ocular growth.     

纤维连接蛋白在形觉剥夺眼和正常眼的差异表达

目的：探讨实验性形觉剥夺性近视眼眼轴增大的影响因素,探讨导致形觉剥夺性近视眼的巩膜改变的分子水平机制。方法：单眼遮盖20只鸡雏4周制备实验性近视模型,然后使用免疫组织化学方法检测遮盖眼和对照眼的巩膜软骨组织的纤维连接蛋白（FN）表达情况。结果：遮盖眼巩膜软骨的软骨细胞质、细胞膜和软骨囊的FN的表达水平比对照眼明显增高（P＜0.01)。结论：FN与实验性近视眼的近视形成机制有关,可能有调节眼球增大的作用。     

豚鼠形觉剥夺及恢复过程中眼压变化

目的 研究豚鼠形觉剥夺性近视形成及恢复过程中眼压的变化情况。方法 20只3周龄花色豚鼠随机分成两组：单眼面罩形觉剥夺组（n=10）和正常对照组（n=10）。形觉剥夺前（0 d）、形觉剥夺后第3、第6、第9、第12、第19、第26 天和恢复后第7天（第33天）测量眼压。形觉剥夺前、形觉剥夺后第26 天和恢复后第7 天测量角膜曲率、屈光度和眼轴及其他各部分长度（前房深度、晶状体厚度和玻璃体腔深度）。将形觉剥夺眼的测量数据与对侧眼和正常眼相比较,采用SPSS 13.0统计软件分析他们之间存在的差异。结果 形觉剥夺前,各组眼的各项生物学参数间的差异无统计学意义。形觉剥夺过程中所有时间点,形觉剥夺眼眼压高于对侧眼和正常眼,差异有统计学意义（P〈0.05）。形觉剥夺去除7 d后,形觉剥夺眼的眼压降低,各组间差异无统计学意义（P〉0.05）。形觉剥夺26 d后,形觉剥夺眼的屈光度、玻璃体腔长度、眼轴与对侧眼和正常眼间差异有统计学意义（P〈0.05）;而角膜曲率、前房深度和晶状体厚度与对侧眼和正常眼间差异无统计学意义。形觉剥夺去除7 d后,形觉剥夺眼与对侧眼和正常眼相比,只有玻璃体腔长度的差异有统计学意义（P〈0.05）。结论 在豚鼠形觉剥夺过程中,形觉剥夺眼的眼压较对侧眼和正常眼高,恢复后眼压又下降至正常。     

N-甲基-D-天冬氨酸受体1在形觉剥夺性近视豚鼠视网膜上的表达

目的对豚鼠行形觉剥夺建立近视动物模型，观察离子型谷氨酸受体N-甲基-D-天冬氨酸受体1（N—methyl—D—aspartate receptor 1．NMDAR1）在近视豚鼠视网膜上的动态表达．探讨其在近视发病机制中的作用。方法60只三色豚鼠随机分为3组：未遮盖组（Ⅰ）、单眼遮盖2周组（Ⅱ）、单眼遮盖3周组（Ⅲ），其中右眼遮盖为实验眼，左眼为自身对照眼。对各组进行视网膜检影和A超测眼轴。分别运用免疫组化及Western Blotting法检测各组豚鼠视网膜NMDAR1蛋白表达。结果Ⅰ组双眼呈轻度远视状态．双眼眼轴差异无显著性（P〉0．05）；Ⅱ组实验眼呈轻度近视（-1.583±1.478）D，自身对照眼呈轻度远视（2.500±1.017）D：实验眼眼轴较自身对照眼轻度延长（P〈0．05）；Ⅲ组实验眼呈中度近视（-3．417±l.169）D，自身对照眼呈轻度远视（1.813±1．072）D；实验眼眼轴较自身对照眼明显延长（P〈0．05）。免疫组化显示NMDAR1主要表达在豚鼠视网膜的内核层细胞及神经节细胞。Ⅰ组实验眼视网膜上NMDAR1蛋白含量为0．338±0．314．Ⅱ组实验眼NMDAB1蛋白含量升高为0．464±0．280．Ⅲ组实验眼视网膜NMDAR1蛋白含量明显上调为0．635±0．037;实验眼视网膜上NMDAR1蛋白含量随遮盖时间延长明显上调．与自身对照眼比较差异有显著性（P〈0．05）。结论形觉剥夺可明显上调豚鼠视网膜NMDAR1的蛋白表达，形觉剥夺产生的异常视觉信号可能通过刺激谷氨酸的释放、NMDAR1过度生成，参与近视的调控。     

BetaB1-crystallin: identification of a candidate ciliary body uveitis antigen

PURPOSE: Perineuclear anti-neutrophil cytoplasmic antibody (pANCA), a marker antibody present in 12% of patients with anterior uveitis, recognizes cytoplasmic antigens in the nonpigmented ciliary body epithelium, a probable site of immunologic reactivity in this inflammatory disease. In this study, a recombinantly isolated pANCA monoclonal antibody was used to identify the corresponding antigenic target(s) in the ciliary body. METHODS: Proteins from microdissected eye bank ocular ciliary body tissue were used to identify the corresponding ANCA antigen. Parallel two-dimensional protein gels were used for simultaneous identification of candidate antigenic protein spots by Western blot analysis and as a source of material for proteomic analysis. Multiple independent methods including Western blot analysis, confocal microscopy, and RT-PCR were used to provide additional characterization of the candidate protein. RESULTS: Proteomic analysis suggested that beta B1 (betaB1)-crystallin is the primary ciliary body antigen. The presence of betaB1-crystallin in the human ciliary body was confirmed by Western blot with a betaB1 specific anti-peptide antibody. Confocal microscopy revealed colocalization of the antigenic reactivity of both anti-betaB1 antibody and monoclonal pANCA. RT-PCR confirmed the presence of betaB1-crystallin RNA in the ciliary body tissues. CONCLUSIONS: This study identified betaB1-crystallin as a new cytoplasmic ciliary body antigenic target of a marker autoantibody associated with uveitis. This characterization of betaB1-crystallin outside the lens raises questions about its extralenticular expression, intracellular role, and potential target of inflammation in uveitis.     

Scleral changes in chicks with form-deprivation myopia

The sclera in myopic regions of chick eyes was studied histologically and compared to the sclera in corresponding regions of normal fellow eyes. Chicks had been monocularly deprived of form vision in the nasal half of the retina from hatching. The fellow control eye and the temporal retina of the deprived eye had normal vision. With this treatment, the resulting form-deprivation myopia and eye enlargement are restricted to the retinal region that had been form deprived. We found that the cartilaginous sclera in the myopic nasal region exhibited several differences from that in the corresponding non-myopic region: it was thicker, its cell density was lower, and the number of chondrocytes and binucleate cells was higher. In contrast, the fibrous sclera was thinner. These changes suggest that form-deprivation myopia causes an increased production of extracellular matrix and an increased level of mitotic activity in the cartilaginous sclera. As expected, the non-myopic temporal regions of experimental and control eyes did not differ in any of these parameters. The findings of the present study suggest that the eye enlargement accompanying form-deprivation myopia is not the consequence of scleral stretching but of abnormal growth.     

Smad1在C57BL/6小鼠形觉剥夺性近视中的作用机制

目的：：通过建立C57 BL/6小鼠形觉剥夺性近视模型,探究Smad1在近视形成中的作用机制。方法：将60只3周龄C57 BL/6小鼠随机分为实验组和正常对照组(NC),包括形觉剥夺3wk组(FDM 3W,n=20),形觉剥夺4wk组(FDM 4W,n=20),FDM3W正常对照组(FDM 3W-NC,n=10)和FDM4W正常对照组(FDM 4W-NC,n=10),实验组右眼遮盖,左眼自然暴露作为自身对照,正常对照组不予任何处理。在实验3 wk及4 wk时检测所有小鼠屈光状态,HE染色观察巩膜及视网膜组织结构变化,免疫组织化学方法及实时荧光定量PCR观察Smad1在视网膜中的表达情况。结果：(1)自身对照眼和正常对照组呈生理性远视化发展,FDM3W、FDM4W实验眼均呈相对近视化发展,差异具有统计学意义(P<0.05);(2)与自身对照组及正常对照组比较,FDM3W、FDM4W实验眼巩膜及视网膜明显变薄;实验眼视网膜中Smad1表达明显下降,差异具有显著统计学意义(P<0.01)。结论：小鼠形觉剥夺性近视眼视网膜(尤其是内核层及内丛状层)中Smad1的表达呈下调趋势, Smad1极有可能通过转导视网膜信号参与了近视发生发展过程。     

形觉剥夺性和光学离焦性近视豚鼠视紫红质表达变化研究

目的：研究形觉剥夺性和光学离焦性近视豚鼠视紫红质的表达变化，探讨视紫红质表达与实验性近视眼之问的关系。方法：40只出生后1周的豚鼠随机分为形觉剥夺组和光学离焦组（n=20），形觉剥夺组单眼戴半透明（半透明薄膜贴于平镜表面）平光硬性角膜接触镜片（Rigid gass—permeable contactlens，RGP），光学离焦组单眼戴-4．0DRGP镜片，另一只眼为对照组。戴镜干预后1、2周各组分别测量屈光度、眼轴长度、玻璃体腔深度，并于上午10～12点钟取材，实时荧光定量PCR观察视紫红质mRNA的变化，Western—blot观察视紫红质的变化，进行对比比较，统计分析。结果：实验干预后1周，形觉剥夺组和光学离焦组与对照组相比各项指标无显著性差异（除形觉剥夺组屈光度以外）。实验干预后2周，与对照组相比较，形觉剥夺组和光学离焦组明显发生近视、眼轴延长、玻璃体腔加深（t：22．20、18．32、19．65、15．78、6．18、11．20，P〈0．01）；形觉剥夺组视紫红质及其mRNA表达均增加（t=17．489、14．31，P〈0．05），光学离焦组视紫红质及其mRNA表达无明显变化。结论：视紫红质的表达可能参与了形觉剥夺性近视眼的形成，而在光学离焦性近视眼中作用有限，     

形觉剥夺下雏鸡后极部巩膜中MMP-2 TIMP-2及RARβ表达变化

目的 分析雏鸡形觉剥夺性近视眼及形觉剥夺性近视恢复眼后极部巩膜中基质金属蛋白酶2(matrix metallopmteinaae 2,MMP-2)、组织金属蛋白酶抑制剂2(tissue inhibitor of matrix metallopro-teinase 2,TIMP-2)及视黄酸受体-β(retinoic acid receptor-β,RARβ)的水平及其相关性.方法 选用新孵出家鸡75只,半透明眼罩遮盖的方法对左眼进行形觉剥夺,分为形觉剥夺组,遮盖14d;形觉剥夺恢复组,遮盖11d后,去遮盖3d.右眼为对照眼.取直径8mm的后极部眼组织块,分离出巩膜纤维层和软骨层.RT-PCR检测MMP-2、TIMP-2及RARβ mRNA的相对含量,并将MMP-2、TIMP-2 mRNA水平分别与RARβ的mRNA水平作线性相关分析.结果 形觉剥夺14d后,纤维层中MMP-2的表达增强(P<0.01),TIMP-2的表达减弱(P<0.01);去剥夺3d后,MMP-2的表达减弱(P<0.01),而TIMP-2的表达恢复到接近对照眼的水平(P>0.05).软骨层表达没有明显变化.形觉剥夺14d后,RARβ的表达均增强,但纤维层中的表达更为强烈(P<0.01).去剥夺3d后,RARβ的表达均明显下降(P<0.01).MMP-2与RARβmRNA水平呈正相关(P<0.05,r=0.944);TIMP-2与RARβmRNA水平呈负相关(P<0.05,r=-0.863).结论 雏鸡形觉剥夺性近视眼及形觉剥夺性近视恢复眼后极部巩膜纤维层中MMP-2、TIMP-2及RARβ的水平均发生明显变化,并有相关性.RARβ参与了巩膜纤维层细胞外基质的降解过程.     

豚鼠形觉剥夺性近视发生及恢复期眼球生物参数变化的研究

目的 研究青少年豚鼠不同形觉剥夺时期,眼球屈光不正度数和玻璃体腔长度恢复的情况.方法 实验研究.将96只3周龄豚鼠随机分为两组,即剥夺组和正常组各48只.剥夺组再随机分为4个组,每组12只,分别予头套法形觉剥夺1、2、4、6周,单眼剥夺后每组6只予去头套恢复3 d.另6只恢复7 d;正常组亦分为4个组,分别与剥夺组形成对照.在剥夺前后及恢复期后测量豚鼠屈光不正度数和玻璃体腔长度的变化,并比较实验眼、自身对照眼和正常对照眼之间的差异.采用单因素方差分析来比较实验眼、自身对照眼及正常对照眼的眼轴长度、屈光不正度数及玻璃体腔长度之间的差异,进一步两两比较采用LSD检验分析.结果 实验眼形觉剥夺2、4、6周时屈光不正度数(F=12.504,15.003,6.829)及玻璃体腔长度(F=6.108,28.222,19.195)与自身对照眼的差异有统计学意义(P<0.05),两眼屈光不正度数之间的差值分别为-2.36、-3.64、-3.68 D,两眼玻璃体腔长度的差值分别为0.08、0.19、0.22 mm.在去除头套后,实验眼(形觉剥夺恢复后)较对照眼向远视方向发展.剥夺2周的实验眼在恢复3 d时,两组实验眼和对照眼屈光不正度数和玻璃体腔长度之间已无统计学意义(F=0.032,0.280;P>0.05),恢复7 d时,实验眼屈光不正度数及玻璃体腔长度可恢复到对照眼水平.剥夺4周和6周的实验眼在恢复3 d时,两组实验眼和对照眼屈光不正度数和玻璃体腔长度之间差异有统计学意义(F=7.658,8.415,5.150,6.061;P<0.01);而剥夺4周的实验眼在恢复7 d时两眼屈光不正度数无统计学意义,玻璃体腔长度差异有统计学意义(F=4.020,P<0.05),剥夺6周的实验眼在恢复7 d时两眼屈光不正度数和玻璃体腔长度差异仍有统计学意义(F=4.108,6.317;P<0.05).结论 豚鼠形觉剥夺可使实验眼形成明显的近视,实验眼在去除剥夺3 d内恢复速度较快,3至7 d时速度减慢.近视的恢复以及恢复程度取决于剥夺时间的长短,剥夺时间越长,近视越难恢复.     

豚鼠形觉剥夺性近视发生及恢复期眼球生物参数变化的研究

目的 研究青少年豚鼠不同形觉剥夺时期,眼球屈光不正度数和玻璃体腔长度恢复的情况.方法 实验研究.将96只3周龄豚鼠随机分为两组,即剥夺组和正常组各48只.剥夺组再随机分为4个组,每组12只,分别予头套法形觉剥夺1、2、4、6周,单眼剥夺后每组6只予去头套恢复3 d.另6只恢复7 d;正常组亦分为4个组,分别与剥夺组形成对照.在剥夺前后及恢复期后测量豚鼠屈光不正度数和玻璃体腔长度的变化,并比较实验眼、自身对照眼和正常对照眼之间的差异.采用单因素方差分析来比较实验眼、自身对照眼及正常对照眼的眼轴长度、屈光不正度数及玻璃体腔长度之间的差异,进一步两两比较采用LSD检验分析.结果 实验眼形觉剥夺2、4、6周时屈光不正度数(F=12.504,15.003,6.829)及玻璃体腔长度(F=6.108,28.222,19.195)与自身对照眼的差异有统计学意义(P<0.05),两眼屈光不正度数之间的差值分别为-2.36、-3.64、-3.68 D,两眼玻璃体腔长度的差值分别为0.08、0.19、0.22 mm.在去除头套后,实验眼(形觉剥夺恢复后)较对照眼向远视方向发展.剥夺2周的实验眼在恢复3 d时,两组实验眼和对照眼屈光不正度数和玻璃体腔长度之间已无统计学意义(F=0.032,0.280;P>0.05),恢复7 d时,实验眼屈光不正度数及玻璃体腔长度可恢复到对照眼水平.剥夺4周和6周的实验眼在恢复3 d时,两组实验眼和对照眼屈光不正度数和玻璃体腔长度之间差异有统计学意义(F=7.658,8.415,5.150,6.061;P<0.01);而剥夺4周的实验眼在恢复7 d时两眼屈光不正度数无统计学意义,玻璃体腔长度差异有统计学意义(F=4.020,P<0.05),剥夺6周的实验眼在恢复7 d时两眼屈光不正度数和玻璃体腔长度差异仍有统计学意义(F=4.108,6.317;P<0.05).结论 豚鼠形觉剥夺可使实验眼形成明显的近视,实验眼在去除剥夺3 d内恢复速度较快,3至7 d时速度减慢.近视的恢复以及恢复程度取决于剥夺时间的长短,剥夺时间越长,近视越难恢复.     

Regulatory elements in the rat betaB2-crystallin promoter.

The suggested common regulator of the eye lens crystallin genes is c-Maf. Maf responsive elements have been detected in a number of crystallin promoters including that of the rat betaB2-crystallin gene. The betaB2-crystallin gene is active in the post-natal lens and its mRNA reaches its maximal level in the rat lens 6 months after birth. Yet c-Maf has been reported to be present in the rat lens only up to 3 months of age. This discrepancy prompted an investigation into the role of the Maf responsive element (MARE) in the regulation of activity of the rat betaB2-crystallin promoter in rat lens fiber cells. Although betaB2 promoter activity is enhanced by c-Maf in both in vitro differentiating rat lens fiber cells and CHO cells, deletion of the betaB2 MARE, which was mapped to -143/-123, does not decrease betaB2 promoter activity in lens fiber cells. Furthermore, a dominant negative c-Maf construct did not inhibit activity of the betaB2 promoter in lens fiber cells. The data suggest that the betaB2 MARE does not play a major role in regulating activity of the betaB2 promoter. Rather, a putative Sox binding site at -164/-159 and a positive element at -14/-7 seem to be the prime regulatory elements.     

豚鼠形觉剥夺性近视发生及恢复期眼球生物参数变化的研究

目的 研究青少年豚鼠不同形觉剥夺时期,眼球屈光不正度数和玻璃体腔长度恢复的情况.方法 实验研究.将96只3周龄豚鼠随机分为两组,即剥夺组和正常组各48只.剥夺组再随机分为4个组,每组12只,分别予头套法形觉剥夺1、2、4、6周,单眼剥夺后每组6只予去头套恢复3 d.另6只恢复7 d;正常组亦分为4个组,分别与剥夺组形成对照.在剥夺前后及恢复期后测量豚鼠屈光不正度数和玻璃体腔长度的变化,并比较实验眼、自身对照眼和正常对照眼之间的差异.采用单因素方差分析来比较实验眼、自身对照眼及正常对照眼的眼轴长度、屈光不正度数及玻璃体腔长度之间的差异,进一步两两比较采用LSD检验分析.结果 实验眼形觉剥夺2、4、6周时屈光不正度数(F=12.504,15.003,6.829)及玻璃体腔长度(F=6.108,28.222,19.195)与自身对照眼的差异有统计学意义(P<0.05),两眼屈光不正度数之间的差值分别为-2.36、-3.64、-3.68 D,两眼玻璃体腔长度的差值分别为0.08、0.19、0.22 mm.在去除头套后,实验眼(形觉剥夺恢复后)较对照眼向远视方向发展.剥夺2周的实验眼在恢复3 d时,两组实验眼和对照眼屈光不正度数和玻璃体腔长度之间已无统计学意义(F=0.032,0.280;P>0.05),恢复7 d时,实验眼屈光不正度数及玻璃体腔长度可恢复到对照眼水平.剥夺4周和6周的实验眼在恢复3 d时,两组实验眼和对照眼屈光不正度数和玻璃体腔长度之间差异有统计学意义(F=7.658,8.415,5.150,6.061;P<0.01);而剥夺4周的实验眼在恢复7 d时两眼屈光不正度数无统计学意义,玻璃体腔长度差异有统计学意义(F=4.020,P<0.05),剥夺6周的实验眼在恢复7 d时两眼屈光不正度数和玻璃体腔长度差异仍有统计学意义(F=4.108,6.317;P<0.05).结论 豚鼠形觉剥夺可使实验眼形成明显的近视,实验眼在去除剥夺3 d内恢复速度较快,3至7 d时速度减慢.近视的恢复以及恢复程度取决于剥夺时间的长短,剥夺时间越长,近视越难恢复.