Immunohistochemical localization of protein kinase C (PKC) beta I in the pig retina during postnatal development

In order to investigate the expression of protein kinase C (PKC) beta I in the retinas of pigs during postnatal development, we analyzed retinas sampled from 3-day-old and 6-month-old pigs by Western blotting and immunohistochemistry. Western blot analysis detected the expression of PKC beta I in the retinas of 3-day-old piglets and it was increased significantly in the retinas of 6-month-old adult pigs. Immunohistochemical staining showed PKC beta I in the retinas of both groups. Immunohistochemistry of 3-day-old retinas revealed weak PKC beta I reactivity in the ganglion cell layer, inner plexiform layer, inner nuclear cell layer, outer plexiform layer and rod and cone cell layer. In the 6-month-old pig retina, the cellular localization of PKC beta I immunostaining was similar to that of the 3-day-old retina, where PKC beta I was localized in some glial fibrillary acidic protein-positive cells, glutamine synthetase-positive cells, parvalbumin-positive cells, and PKC alpha-positive cells in the retina. This is the first study to show the expression and cellular localization of PKC beta I in the retina of pigs with development, and these results suggest that PKC beta I, in accordance with PKC alpha, plays important roles in signal transduction pathways in the pig retina with development.     

Immunohistochemical localization of heat shock protein 27 in the retina of pigs

The expression of heat shock protein 27 (HSP27) was examined in the retinas of pigs. Western blot analysis detected the expression of HSP27 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 6-month-old adult pigs. Immunohistochemically, HSP27 immunostaining was seen mainly in ganglion cell bodies in the ganglion cell layer, and in some processes of astrocytes in the innermost nerve fiber layer. In 1-day-old piglets, HSP27 was detected weakly in the inner plexiform, inner nuclear cell, outer plexiform, and rod and cone layers. The HSP27 immunoreactivity across the retinal layers was enhanced in the retinas of 6-month-old pigs compared with newborn piglets. The HSP27 immunoreactivity in the radial processes of Müller cells was particularly prominent in adult pig retinas. In summary, this finding suggests that HSP27 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

Immunohistochemical localization of phospholipase D1 in the retina of pigs

The expression of phospholipase D1 (PLD1) was examined in the retinas of pigs. Western blot analysis detected the expression of PLD1 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 2 years old adult pigs. Immunohistochemically, PLD1 was mainly immunostained in ganglion cell bodies in the ganglion cell layer, in some radial processes of Muller cells in the retinal layer and in the inner and outer segments of the rod and cone layer in newborn and adult pigs, but not in astrocytic bundles in nerve fiber layers. The immunoreactivity of PLD1 in the radial processes of Muller cells across the retinal layers was enhanced in adult pig retinas compared to those of newborn piglets. This was the first demonstration to show that PLD1 is constitutively expressed in the retina of pigs, implying that retinal PLD1 expression is enhanced in radial fibers of Muller cells with age. This finding suggests that PLD1 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

Ultramicroscopical immunolocalization of PAX6 in the adult chicken retina

Cell type-specific PAX6 protein expression was examined in all retinal layers of the normal chicken retina. The most intense PAX6 immunostaining was found in the ganglion cell and inner nuclear layers, and in lower amounts in the optic nerve fiber, the inner plexiform and the photoreceptor layers. PAX6 immunostaining was variable in terms of its subcellular localization, even within one cell. PAX6 immunostaining was mainly localized in nuclear heterochromatin of the ganglion cell and inner nuclear layers whereas in the outer nuclear layer, PAX6 immunostaining was only observed in the intercellular space and the cytoplasm. In photoreceptors, the myoid portion of the inner segment showed PAX6 immunostaining, but the ellipsoid portion and the outer segment did not. The ultrastructural distribution pattern of PAX6 in the adult chicken retina suggests that normal expression of PAX6 is variable even in subcellular structures in the same cell type.     

Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development

Brain‐derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age‐dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post‐fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full‐length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.     

Glutathione depletion induces differential apoptosis in cells of mouse retina, in vivo

Oxidative stress affects numerous intracellular macromolecules, and may result in cell death unless precisely regulated. Unregulated oxidative stress can be controlled by various cellular defense mechanisms such as glutathione (GSH) which can critically counteract the damaging effects of oxidative stress in mammalian cells. We determined the effects of unregulated oxidative stress induced by GSH depletion on cells in mouse retina. Mice were intraperitoneally injected with buthionine sulphoximine (BSO) at 1.5 g/kg. After 0, 1, 4, and 7 days of BSO administration, retinas were excised and sections were subjected to GSH assay and terminal uridine deoxynucleotidyl nick end labeling (TUNEL) analysis. After 4 days of BSO administration, the number of TUNEL positive cells was significantly increased. However, after 7 days, TUNEL positive cells returned to the basal level. The retinal region most affected by the BSO treatment appeared to be the outer nuclear layer where the photoreceptor cells reside. Different from cells in other regions, retinal cells in the inner nuclear layer increased in their apoptosis even after the first day of BSO injection, and the increase was further potentiated after 4 days. Taken together, our studies suggested that GSH depletion may cause unregulated oxidative stress to the cells in the retina and indeed increased cell death in the retina. The cells in the inner nuclear layer seemed to be affected earlier than the cells in other layers of the retina. The GSH level in the retina may be a crucial therapeutic target in preventing blindness.     

Intermediate filament complement of the normal and gliotic canine retina.

Intermediate filament expression of various cell types in the adult canine normal and gliotic retina was determined by an immunoperoxidase method of using monoclonal antibodies on aldehyde-fixed tissues. In the normal retina, vimentin was present in astrocytes in the nerve fibre layer, horizontal cell processes, and Müller cell fibres from the internal limiting membrane to the outer nuclear layer. Neurofilamentous axons were noted in the nerve fibre, inner plexiform layer, and outer plexiform layer, although the degree of staining intensity varied among the three molecular weight neurofilament antisera used. Glial fibrillary acidic protein (GFAP) staining was confined to the nerve fibre and ganglion cell layer; this was interpreted as representing fibrous astrocytes. Astrocyte density varied according to retinal topography with an increased number around retinal blood vessels and in the peripapillary retina. Quantitative, but not qualitative differences in staining for vimentin and the neurofilaments were noted in degenerative, gliotic retinas. In common with several other mammalian species previously studied, the canine Müller cells accumulate or express GFAP under pathological conditions involving a gliotic response.     

人胎视网膜细胞凋亡与小白蛋白免疫阳性神经元的发育

目的：观察人胎视网膜细胞凋亡与小白蛋白Parvalbumin,PV)免疫阳性神经元的分布与发育。方法：不同孕龄的人胎16例,TUNEL法标记凋亡细胞,ABC免疫细胞化学方法观察PV免疫阳性神经元的发育。结果：（1）细胞凋亡观察：12周人胎视网膜未见凋亡细胞;15周、17周凋亡细胞较多,大小不一,分布于视网膜的全局;20周凋亡细胞主要集中在内核层,数量减少;28周凋亡细胞仅见于内核层,呈指环样外观,着色较深,数量较20周减少明显。（2）PV免疫阳性神经元发育：12周人胎视网膜未见PV免疫阳性神经元;15周、17周视网膜节细胞层和内核层有弱阳性的PV免疫阳性神经元的分布与20周相似,但内核层的数量减少,呈整齐的带状排列,着色增强。结论：在胚胎28周视网膜神经元之间的突触联系已基本建立,而PV免疫阳性神经元发育和细胞凋亡在时间和数量变化上的一致性提示Ca^2 在视网膜的发育中起重要作用。     

Expression profile of heat shock protein 108 during retinal development in the chick

In the developing chick retina, heat shock protein 108 (HSP108), which exhibits transferrin binding activity, has been demonstrated at the mRNA level, while transferrin shows two expression peaks. Here, we investigated the expression profile of HSP108 in the developing chick retina at the protein level. The localization of HSP108 in embryonic days 15 (E15), E18, and postnatal day 2 (P2) chick retina was examined immunohistochemically using monoclonal antibody 9G10 specific for chick HSP108, while the expression levels of HSP108 in developing chick retina from E12 to P2 and adult were measured by Western blot analysis. HSP108 was expressed in the ganglion cell layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segments of photoreceptors and retinal pigment epithelium. Two peaks of HSP108 expression were found at around E13 and E18, respectively. Since the two HSP108 peaks appeared to be correlated with the transferrin expression peaks during retinal development, HSP108 may be associated with iron metabolism during the development of the retina.     

Roller organ cultures of the retina from postnatal rats

Whole retinas of 2–14-day-old rats were cultured in a roller device for 2–14 days. Floating retinas of 7–14-day-old rats formed hole spheroid structures (spheroids) with the wall completely retaining the linear structure and layer-by-layer cellular and fibrous architecture, including the outer nuclear, outer plexiform, inner nuclear, inner plexiform layers, layers of ganglion cells and nerve fibers. The retina obtained at earlier terms of development often formed folds, with pyknotic nuclei of dead neurons in their deep compartments. In organ cultures of the retina isolated from rats at early postnatal periods, rosettes were formed in sites of local injury to the outer nuclear layer and pigmented epithelium. Roller organ cultures can be used for in vitro studies of the development and experimental diseases of the retina. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 10, pp. 471–474, October, 2006     

Developmental expression of calretinin immunoreactivity in the human retina and a comparison with two other EF-hand calcium binding proteins.

This paper reports the localization pattern of calretinin, a calcium-binding protein, in the human retina during development, as studied by immunohistochemistry. A comparison is made of the cellular distribution of calretinin with two other calcium-binding proteins, calbindin and parvalbumin, recently reported by us in the human retina, and by parallel labeling with both antisera in the same tissues. At 11-12 weeks of gestation, calretinin immunoreactivity was expressed in many prospective ganglion cells of the central inner neuroblastic zone. At 16-17 weeks of gestation, the immunoreactivity was localized in the ganglion cell layer, inner plexiform layer, and in most differentiated amacrine, horizontal and cone cells located in the central (1-2 mm temporal from optic disc) to midperipheral parts of the retina. By midgestation (20-21 weeks), calretinin immunoreactivity was strongly developed in the cone photoreceptors. Parallel labeling with calbindin and parvalbumin antisera revealed that the calretinin-positive horizontal cells were somewhat smaller and less frequent and less intense than the calbindin- and parvalbumin-positive counterparts, at 16-21 weeks of gestation. No horizontal cells were calretinin immunopositive in the postnatal (four-month-old infant) and adult retinas examined. Also, at both stages, a few bipolar and cone cells were weakly immunoreactive. These observations suggest a critical role for calretinin in the development and maturation of a select class of horizontal cells. The widespread expression of immunoreactivity in the early ganglion cells indicates that calretinin may be involved in their differentiation. The weak immunoreactivity pattern noted in the adult photoreceptor and bipolar cells, and an apparent lack of immunoreactivity in the mature horizontal cells, tends to indicate that, unlike calbindin and parvalbumin, calretinin plays little role in the transport and physiological buffering of Ca2+ in these neurons of the human retina. It appears, however, that calretinin is predominantly involved in both processes in amacrine cells.     

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.     

Nitric oxide synthase expression in the transient ischemic rat retina: neuroprotection of betaxolol

Betaxolol is a β-adrenergic blocker but its neuroprotective action is generally thought to be due to its calcium channel blocking properties. In this study, we investigated neuronal cell damage and changes in the expression of neuronal nitric oxide synthase (nNOS) immunoreactivity in the ischemic retina and its relationship to the neuroprotection of betaxolol treatment after ischemic injury. Using the retina after ischemia, the expression of nNOS was studied by immunocytochemistry. In control retinas, two types of amacrine cells and a class of displaced amacrine cells were nNOS-labeled. After ischemia/reperfusion, the number of nNOS immunoreactive cells increased in both the ganglion cell layer and the inner nuclear layer compared to the control retinas. However, when experiments were carried out on animals that had been treated with betaxolol twice daily after ischemia/reperfusion, the number of nNOS immunoreactive cells decreased compared to the untreated ischemic retinas. These results suggest that an increase in nNOS expression could be associated with the degenerative changes in the ischemic retina, and that betaxolol treatment appears to play a role in protecting retinal tissue from ischemic damage.     

急性高眼压后大鼠视网膜谷氨酰胺合成酶的表达变化

为了探索谷氨酰胺合成酶在青光眼视网膜中的表达变化及其可能作用,本实验用急性高眼压模型,结合免疫组织化学染色和Western blot检测了急性高眼压后大鼠视网膜中谷氨酰胺合成酶的表达。结果显示:正常视网膜中,谷氨酰胺合成酶免疫组织化学染色主要见于Muller细胞胞体;0 d组中,Muller细胞胞体染色稍淡,而内网层中表达增加,且呈明显的点状分布; 1d组、3 d组中Muller细胞胞体染色进一步变淡,但内网层中呈现弥散染色。至再灌第7 d、14 d,Muller细胞胞体又出现浓的染色。平均灰度值显示:与正常组相比,0 d组中谷氨酰胺合成酶表达有增加但差异无显著性;1 d组中表达显著增加,3 d组、7 d组表达逐渐减少,至第14 d时基本恢复正常。Western blot显示谷氨酰胺合成酶为一分子量约为45 kD的单一蛋白带,与其它组相比,1 d组中表达显著增加。提示:急性高眼压导致的视网膜缺血再灌早期,Muller细胞中谷氨酰胺合成酶的快速重新分布和表达上调可能加速了胞外谷氨酸的代谢,对缺血再灌条件下的视网膜特别是节细胞起到保护作用。     

Rhodopsin和S-opsin在MNU诱导的大鼠视网膜损伤过程中的表达变化

目的:观察在N-甲基-N亚硝酸脲(MNU)诱导的大鼠视网膜外核层细胞损伤过程中视紫质(rhodopsin)和蓝光视蛋白(blue-sensitive opsin/S-opsin)在视网膜中的表达变化,分析其与MNU诱导的视网膜损伤的关系。方法:将30只SPF级50 d龄雌性SD大鼠随机分为正常对照组和MNU模型1、3、7、10 d组,每组各6只大鼠。模型组以腹腔注射MNU(40 mg/kg)建立MNU模型;正常对照组大鼠腹腔注射生理盐水(5 mL/kg)。右眼冰冻切片行苏木素-伊红(HE)染色判断视网膜损伤程度。通过逆转录-聚合酶链反应(RT-PCR)和免疫荧光检测各组视网膜中rhodopsin和S-opsin的mRNA及蛋白表达情况。结果:病理检测确定造模结果与以往实验一致,各组病理分级之间的差异显著(2=16.838,P0.01)。RT-PCR检测结果表明,与正常对照组相比,各MNU模型组rhodopsin和S-opsin的mRNA表达水平随MNU作用时间增加而逐渐降低且差异均显著(rhodopsin 1 d组P0.05;S-opsin 1 d组P0.01;3、7、10 d组rhodopsin和S-opsin均P0.01)。免疫荧光检测结果显示,在正常大鼠视网膜rhodopsin主要在光感受器细胞外段表达,MNU作用后rhodopsin主要在外核层表达,少量在内核层表达,并随MNU作用时间增加而逐渐表达降低。S-opsin在正常视网膜各层均有表达,在各模型组随MNU作用时间增加S-opsin的表达逐渐降低,在外核层和光感受器细胞内、外段尤为明显。结论:MNU诱导的视网膜外核层细胞损伤可降低rhodopsin和S-opsin的表达,并与MNU选择性引起光感受器细胞丧失有关。     

Dopamine- and adenosine-3':5'-monophosphate (cAMP)-regulated phosphoprotein of Mr 32,000 (DARPP-32) in the retina of cat, monkey and human.

The cellular localization of a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) was investigated in cat, monkey and human retina by immunohistochemistry. In cat, DARPP-32-immunoreactive cell bodies identified as Müller cells were demonstrated in the inner nuclear layer (INL) with processes closely surrounding the cell soma of photoreceptors in the outer nuclear layer. Some DARPP-32-IR cells were also seen in the nerve fiber layer (NFL) sending processes to the inner plexiform layer. In monkey and human retina, DARPP-32-IR cell bodies were also demonstrated in the INL, with few cells located in the NFL.     

人胎视网膜超氧化物歧化酶和波形蛋白免疫阳性细胞的发育

目的：观察人胎视网膜超氧化物歧化酶（SOD）和波形蛋白（VIM）免疫阳性细胞的分布发育。方法：不同孕龄的人胎16例,ABC免疫细胞化学方法显示视网膜SOD和VIM免疫阳性细胞,结果：（1）SOD免疫阳性细胞：E15w节细胞层开始出现SOD免疫阳性细胞;D20W和E28W SOD免疫阳性细胞排列较整齐,分布于视网膜的外核层,内核层,节细胞层,其数量增多,其中内核层SOD免疫阳性细胞增多明显。（2）VIM免疫阳性细胞的发育：E15w内界膜开始出现Muller细胞的VIM免疫阳性终足,并见VIM免疫阳性突起伸向外界膜;E20 人VIM免疫阳性物质集中于内界膜,并见VIM免疫阳性突起伸向外界膜;E28wVIM免疫阳性物质的数量较E20w以前各孕龄明显增多,除色素上皮和视杆视锥层外均有VIM免疫阳性物质出现,除伸向外界膜的VIM免疫阳性突起外,还内网层,内核层,节细胞层和神经纤维层还可见水平走行的细胞突起,结论：（1）视网膜发育基本成熟后,视网膜SOD可能主要来源于内核层的SOD免疫阳性细胞。（3）视网膜神经纤维髓鞘是从内向外逐渐形成的。     

Mg2+ reduces N-methyl-D-aspartate neurotoxicity in embryonic chick neural retina in vitro

N-Methyl-D-aspartate (NMDA) is a potent neurotoxin that affects cells in the inner layers of the embryonic chick retina exposed in vitro. After exposure of the embryonic day 12 neural retina to 0.5-10.0 mM NMDA for 30 min, 50-80% of the cells in the inner region of the inner nuclear layer and 50-100% of the cells in the ganglion cell layer were hypochromatic. When retinas were incubated with Mg2+ (0.5-10.0 mM) for 15 min and then incubated with Mg2+ and NMDA (0.5 mM) for 30 min, the NMDA effect in the inner layers was dramatically reduced but not abolished. Removal of Mg2+ before NMDA exposure produced retinas as seriously affected as retinas not exposed to Mg2+. Studying the effects of NMDA inhibitors, such as Mg2+, may help elucidate the mechanism of the cytotoxic events that occur in the retina in response to certain excitatory acidic amino acids.     

Spatial and temporal distribution patterns of enkephalinergic neurons in adult and developing retinas of the preproenkephalin-green fluorescent protein transgenic mouse

Enkephalin (ENK) peptides are present in the retina of several vertebrate species and play a crucial role in establishing specific circuits during retinal development. However, there is no information available concerning the development of ENKergic neurons in the mouse retina. To address this question, we used preproenkephalin-enhanced green fluorescent protein (GFP) transgenic mice, in which ENKergic neurons are revealed by GFP. Our results showed that most GFP-positive cells were located in the proximal part of the inner nuclear layer with a scattering of GFP-immunoreactive cells in the ganglion cell layer (GCL) in the adult retina. Double immunostaining with syntaxin indicates that GFP expression was restricted to a population of amacrine cells. The proportions of glycine transporter-1 and γ-aminobutyric acid-positive cells among ENKergic neurons were 57.3 ± 2.4% and 10.1 ± 1.8%, respectively. We then injected retrograde tracer into the superior colliculus and observed that none of the ENKergic neurons in the GCL were retrogradely labeled with the tracer. GFP-positive cells were first observed at embryonic day (E) 15 in the inner neuroblastic layer at only very low levels, which gradually increased until E18. After birth, there was a steep rise in GFP expression levels, reaching maximal activity by postnatal day (P) 7. The distribution and intensity of GFP-positive cells at P15 were similar to those of adult retina. It was found that immunoreactive processes in the inner plexiform layer formed strongly stained patches. The present results provide detailed morphological evidence of the cell type and spatial and temporal distribution of ENKergic neurons in the retina.