Agonist-Induced Stimulation of Inositol Phosphates in Primary Rabbit Retinal Cultures

Carbachol, noradrenaline, and serotonin stimulated the accumulation of inositol phosphates in a dose-dependent manner and maximally by 172.4%, 71.2%, and 51.6%, respectively, in 3-day-old rabbit retinal cultures. In contrast, dopamine, nicotine, isoproterenol, clonidine, 8-OH-dipropylaminotetralin, and gamma-aminobutyric acid were ineffective. In older cultures identified as containing primarily Müller cells, only carbachol and noradrenaline were able to induce a significant stimulatory response. The carbachol-, noradrenaline-, and serotonin-induced responses were pharmacologically characterized and shown to be mediated by muscarinic, alpha 1-adrenergic, and 5-hydroxytryptamine2 receptors, respectively. The results of the present study show that primary retinal cultures of 3 days of age give results comparable with those of the intact retina. Furthermore, it is demonstrated for the first time that putative Müller cells in cultures possess functional muscarinic and alpha 1-adrenergic receptors.     

Role of Müller glia in neuroprotection and regeneration in the retina

Glial cells are thought to protect neurons from various neurological insults. When there is injury to retina, Müller cells, which are the predominant glial element in the retina, undergo significant morphological, cellular and molecular changes. Some of these changes reflect Müller cell involvement in protecting the retina from further damage. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could have an important role in preventing excitotoxic damage to retinal neurons. Moreover, Müller cells contact to endothelial cells to facilitate the neovascularization process during hypoxic conditions. Finally, recent studies have pointed to a role of Müller cells in retina regeneration after damage, dedifferentiating to progenitor cells and then giving rise to different neuronal cell types. In this article we will review the role of Müller glia in neuroprotection and regeneration after damage in the retina.     

Sonic hedgehog promotes stem-cell potential of Müller glia in the mammalian retina

Müller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Müller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Müller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Müller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Müller glia-derived cells. Together, these results provide evidences that Müller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.     

Mueiller细胞与视网膜功能

Ｍｕｅｌｌｅｒ细胞是视网膜中的主要胶质细胞。除了一般的支持和营养作用外,近年的许多研究表明,在Ｍｕｅｌｌｅｒ细胞和视网膜视风膜神经元之间在着双向的通讯,它们可以直接通过改变细胞外空间神经活性物质的浓度或间接（通过控制神经元的微环境）调制制神经元活动,因此在视网膜功能中起着重要的作用。     

Morphological differentiation of the Müller cell: Golgi and electron microscopy study in the chick retina

The sequence of morphological differentiation of Müller cells in the chick retina was investigated in relation to the differentiation of the retinal neurons using the Golgi method. From the beginning of differentiation, the Müller cell develops spurs and lateral processes. Some of these glial processes become transformed into accessory prolongations of the Müller cell. From the 17th or 18th day of incubation, the morphology of the Müller cells is similar to that of the adult retina. On the basis of their inner prolongation, two types of Müller cells were identified. The first type, with diffuse and abundant descending processes, is identical to that described classically. The second type is a cell characterized by sparse and scanty inner ramifications. This report also describes electron microscopic observations of Müller cells and their enwrapping relationship with the axons of the optic nerve fiber layer.     

Immunohistochemical Localization of GFAP and Glutamate Regulatory Proteins in Chick Retina and Their Levels of Expressions in Altered Photoperiods

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate–aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.     

Temporal and spatial expression of CCN3 during retina development

NOV/CCN3 is one of the founding members of the CCN (Cyr61 CTGF NOV) family. In the avian retina, CCN3 expression is mostly located within the central region of the inner nuclear layer. As retinal development progresses and this retinal layer differentiates and matures, CCN3 expression forms a dorsal–ventral and a central–peripheral gradient. CCN3 is produced by two glial cell types, peripapillary cells and Müller cells, as well as by horizontal, amacrine, and bipolar interneurons. In retinal neurons and Müller cell cultures, CCN3 expression is induced by activated BMP signaling, whereas Notch signaling decreases CCN3 mRNA and protein levels in Müller cells and has no effect in retinal neurons. In Müller cells, the CCN3 expression detected may thus result from a balance between the Notch and BMP signaling pathways. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling

The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.     

Regulatory aspects of the in vitro development of retinal Müller glial cells

Utilizing immunochemical and biochemical methods we have examined the maturation of retinal Müller cells in vitro both in monolayer cultures of dissociated tissue as well as rotation-mediated suspension culture of reaggregated embryonic retina cells. We have manipulated heterotypic cell-cell interactions through the use of such cell surface probes as plant lectins and monoclonal antibodies. In this report we show that the succinylated derivative of Con-A is capable of blocking neuronal-glial interactions in reaggregation cultures resulting in neuronal-glial segregation and failure of glial maturation. Furthermore, we describe a new monoclonal antibody which also inhibits glial maturation in vitro. This antibody recognizes an antigen which is present on retinoblast cells in general early in development, but becomes gradually restricted to Müller cells and to a much lesser extent photoreceptor cells during tissue maturation. The results further substantiate the regulatory influence of heterotypic cell-cell interactions in the development of retinal Müller cells and establishes probes for the analysis of the molecular basis of this phenomenon.     

Regional adaptation of Müller cells in the chick retina. A Golgi and electron microscopical study

We report the morphological differences of Müller cells in relation to their topography, using the Golgi method. Müller cells in the central retina are long and slender, with numerous inner prolongations. In the peripheral retina, the morphology of the Müller cells adapts to the reduced thickness of the retinal layers. In this zone, they are short and have thick inner prolongations which end in a large foot in the internal limiting membrane. In the optic disc margin, Müller cells have a particular morphology characterized by thick, arched prolongations that in general form a glial network between the retina and optic nerve. The ultrastructure of these cells is also described. The results are discussed with respect to the nature of Müller cells.     

NPY in rat retina is present in neurons, in endothelial cells and also in microglial and Müller cells

NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y(1), Y(2), Y(4) and Y(5) receptors. Retina endothelial cells express all NPY receptors except NPY Y(5) receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina.     

Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

低亲和性神经营养素受体p75在人胚胎视网膜中的表达

p75神经营养素受体在视网膜的发育以及再生过程中发挥着重要的作用,而在人类视网膜中的分布状况尚未被研究. 利用免疫组织化学方法,在光镜水平下确定了p75在人胚胎发育5、6和7个月的视网膜中的分布情况. 在视网膜神经节细胞层出现最强的p75免疫阳性反应,在其他各层也有较弱的免疫阳性反应. 在胚胎6、7月的视网膜中,主要由Müller细胞的终足构成的内界膜上出现了比较强的p75表达. p75在人胚胎视网膜中的分布情况与大鼠视网膜中很类似,主要表达在Müller细胞, 在神经节细胞上也可能有表达.     

microRNA-320a prevent Müller cells from hypoxia injury by targeting aquaporin-4

Müller cells are closely related to diabetic retinopathy (DR). Aquaporin-4 (AQP4) can effectively promote the diffusion of water across cellular membranes. However, the dynamic balance of water plays key role in many diseases, such as cerebral edema. Meanwhile, the unusual expression and distribution of AQP4 in the retina are the significant causes of ocular hypertension and reperfusion injury. To explore the functional significance between microRNA-320a (miR-320a) and AQP4 in pathological hypoxia-induced DR related retinal edema, we hypothesized that miR-320a regulates AQP4 expression and internalization to relieve the edema of Müller cells under the pathological retinal hypoxia stress by targeting AQP4, thereby attenuate the damage of Müller cells. Results demonstrated that miR-320a mimics inhibited the expressions of AQP4 in Müller cells. Furthermore, overexpression miR-320a protected Müller cells by suppressing superoxide anion. In addition, overexpression miR-320a markedly attenuated hypoxia-induced injury, significantly increased the cell viability, and promoted the internalization of AQP4. Furthermore, miR-320a can also regulate the stable anchoring of AQP4 on the cell membrane. Our study indicated that miR-320a may be a potential modulator which can mediate AQP4 expression and attenuate the hypoxia damage of Müller cells. In conclusion, miR-320a may be a potential target for DR therapy by targeting AQP4.     

Comparative study of glial fibrillary acidic protein (GFAP)-like immunoreactivity in the retina of some representative vertebrates.

A polyclonal glial fibrillary acidic protein (GFAP) antiserum was used to study the distribution of GFAP-like immunoreactivity in the retina of adult vertebrates (teleosts, amphibians, reptiles, birds and mammals). GFAP-positive Müller cells were demonstrated in all the species studied, although with different degrees and patterns of immunoreactivity. In nonmammalian vertebrates, Müller cells were the only immunoreactive retinal elements. The staining was located throughout the retina of the species examined, with the exception of the rabbit, which exhibited regional variability in the expression of GFAP. The data indicate that GFAP expression in retinal Müller cells is a common feature of a wide variety of adult vertebrate species.