Müller细胞反应性胶质化在急性高眼压致大鼠视网膜损伤中作用

 目的 观察Müller细胞反应性胶质化在急性高眼压(AOH)大鼠视网膜中变化及其抑制对视网膜损伤的影响。方法 建立大鼠AOH青光眼模型,分为正常对照(Ctrl)、AOH和AOH+玻璃体内注射胶质毒素α-氨基己二酸(AAA)后再灌注1、3和5d组,以及单纯AAA和AOH+PBS对照组。TUNEL染色检测细胞凋亡,GFAP免疫荧光染色反应Müller细胞反应性胶质化程度,Thy-1染色标记视网膜神经节细胞(RGCs)。结果 AOH可致大鼠视网膜内丛状层和内核层明显变薄、神经节细胞层内细胞排列紊乱和数量减少,并诱发Müller细胞反应性胶质化(GFAP表达增加)。同时,AAA抑制Müller细胞反应性胶质化可明显缓解AOH所致RGCs丢失和凋亡发生。结论 Müller细胞反应性胶质化参与AOH所致视网膜损伤,抑制其反应性胶质化可能是改善高眼压性青光眼视网膜病变的一种有效治疗方法。     

Reaction of Müller cells after increased intraocular pressure in the rat retina

Using light microscopy and immunocytochemistry, we investigated the morphological changes of retinal tissues and the reaction of Müller cells in the ischemic rat retina induced by increasing intraocular pressure. At early stages (from 1 h to 24 h after reperfusion), cells in the ganglion cell layer and in the inner nuclear layer showed some degenerative changes, but at later stages (from 72 h to 4 weeks) marked degenerative changes occurred in the outer nuclear layer (ONL). At 4 weeks after reperfusion, the ONL was reduced to 1 or 2 cell layers. Immunoreactivity for glial fibrillary acidic protein (GFAP) appeared in the endfeet and distal processes of Müller cells as of 1 h after reperfusion. GFAP immunoreactivity in Müller cells increased up to 2 weeks and then decreased at 4 weeks after reperfusion. Our findings suggest that Müller cells are involved in the pathophysiology of retinal ischemia through the expression of GFAP. The degree of GFAP expression in Müller cells closely correlated with that of the degeneration of retinal neurons.     

Reactive changes of retinal astrocytes and Müller glial cells in kainate-induced neuroexcitotoxicity

The aim of this study was to investigate reactive changes of astrocytes and Müller glial cells in rats subjected to kainate treatment, which leads to neuronal degeneration in the ganglion cell layer and the inner border of the inner nuclear layer as confirmed by labelling with Fluoro-Jade B, a marker for degenerating neurons and fibres. Both the astrocytes and the Müller glial cells reacted vigorously to kainate injection as shown by their up-regulated expression of nestin, glial fibrillary acidic protein and glutamine synthetase. A major finding was the induced expression of nestin together with glial fibrillary acidic protein beginning at 1 day post-injection of kainate. The marked nestin expression appeared to be most intense at 1 day and was sustained till 2 weeks as compared with the untreated/normal retina. Western blotting analysis confirmed a marked increase in expression of nestin, glial fibrillary acidic protein and glutamine synthetase as compared with untreated/normal retina. Double labelling study revealed that astrocytes and Müller glial cells expressed the radial glia marker nestin, and incorporated bromodeoxyuridine to re-enter into their cell cycle. The induced expression of these proteins in astrocytes and Müller glial cells indicated an induction of gliotic responses and de-differentiation that may be associated with regenerative efforts after kainate-induced injury. Indeed, with the acquisition of an immature molecular profile as manifested by the induced expression of brain lipid-binding protein and doublecortin in astrocytes and Müller glial cells, the potential of these cells to de-differentiate in retinal neurodegeneration is greatly amplified.     

Development of the neonatal rabbit retina in organ culture

Organ cultures from neonatal rabbit retinae grew well over periods of up to 2 weeks in vitro. Proliferation in vitro declined in parallel with the decline seen in vivo, although the rate of proliferation in the explants was slightly reduced. The proliferation of progenitor cells in vitro produced the same cell types produced postnatally in vivo. Postnatally generated cell clones, labeled by means of a retroviral vector, consisted mainly of rods and Müller cells. The layers of the retinae developed as in vivo; an outer plexiform layer occurreed after the first 2 days in vitro. Ultrastructurally, ribbon synapses (outer and inner plexiform layer) and conventional synapses (inner plexiform layer) were observed. The photoreceptor cells grew well-developed inner segments and cilia but no mature outer segments. The cultured retinae contained a well-developed, regular lattice of Müller cells expressing vimentin as in vivo. The neuron-to-Müller cell-ratios were essentially the same as in vivo, viz. about 15 to 16 neurons, among them about 10 to 11 (rod) photoreceptor cells per Müller cell. When the glia cell-specific toxin α-aminoadipic acid (αAAA) was applied, the pattern of vimentin-positive Müller cells became irregular, or even locally missing. In such cases, the tissue became disorganized as indicated by a local disappearance of the regular layering, and development of many rosettes. It is concluded that an intact lattice of Müller cells is necessary for the migration of young neurons, and for correct formation of retinal layers.     

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 demonstration of glial fibrillary acidic protein in normal rat Müller glia and retinal astrocytes

The presence of glial fibrillary acidic protein (GFA)-positive Müller glia and retinal astrocytes were studied immunohistochemically in normal rat retina. Using GFA antiserum both Müller glia and separate star-shaped cells were observed in spread-preparations as well as cryostat sections. The retinal astrocytes were also visualized using two different monoclonal GFA antibodies. These cells were found to be located in the nerve fiber and ganglion cell layers. In contrast, Müller glia were not normally visualized with any of the monoclonal GFA antibodies but could be stained 4 days after an optic nerve crush. Our results demonstrate that normal rat Müller glia expresses GFA-like immunoreactivity.     

Histology of the ferret retina

The retina of the adult ferret, Mustelo furo, was studied with light and transmission electron microscopy to provide an anatomical basis for use of the ferret as a model for retinal research. The pigment epithelium is a simple cuboidal layer of cells characterized by a zone of basal folds, apical microvilli, and pigment granules at various stages of maturation. The distinction between rod and cone photoreceptor cells is based on their location, morphology, heterochromatin pattern and the electron density of their inner segments. The round, light-staining cone cell nuclei occupy the layer of perikarya along the apical border of the outer nuclear layer. The remainder of the outer nuclear layer consists of oblong, deeply-stained rod cell nuclei. Ribbon type synaptic complexes involving photoreceptor cell axons, horizontal cell processes, and bipolar cell dendrites characterize the outer plexiform layer. The inner nuclear layer is comprised of horizontal, bipolar, and amacrine cell perikarya as well as the perikarya of the Müller cells. The light-staining horizontal cell nuclei are prominent along the apical border of the inner nuclear layer. The light-staining amacrine cell nuclei form a more or less continuous layer along the basal border of the inner nuclear layer. Both conventional and ribbon-type synapses characterize the inner plexiform layer. The ganglion cells form a single cell layer. The optic fiber layer contains bundles of axons surrounded by Müller cell processes. Small blood vessels and capillaries are present in the basal portion of the retina throughout the region extending from the internal limiting membrane to the outer plexiform layer. The adult one-year-old retina is compared with the retina at the time of eye opening.     

The rabbit retina: a suitable mammalian tissue for obtaining astroglia-free Müller cell cultures

Monolayer cultures were prepared from two distinct parts of early postnatal rabbit retinae. Cell suspensions obtained from the developing medullary ray (MR) region contained neurons, Müller (glial) cells, and astrocytes, cells obtained from the remainder (peripheral) part of the retina contained neurons and Müller cells, but no astrocytes. Müller cells lack glial fibrillary acidic protein (GFAP) immunolabeling in situ but some of them acquire faint GFAP labeling in both types of cultures. Strongly GFAP-labeled cells, most likely astrocytes, were seen in MR cultures only. We propose that the periphery of the rabbit retina is ideal for obtaining astroglia-free Müller cell cultures to study their functional properties in vitro.     

Localization of glial aquaporin-4 and Kir4.1 in the light-injured murine retina

Excessive light causes damage to photoreceptor and pigment epithelial cells, and a local edema in the outer retina. Since Müller glial cells normally mediate the osmohomeostasis in the inner retina (mainly via channel-mediated transport of potassium and water), we determined whether retinal light injury causes an alteration in the retinal localization of glial water (aquaporin-4) and potassium (Kir4.1) channels, and in the potassium conductance of Müller cells. Mice were treated with bright white light (intensity, 15,000lx) for 2h. Light treatment results in Müller cell gliosis as indicated by the enhanced staining of the glial fibrillary acidic protein and an increase in the cell membrane area reflecting cellular hypertrophy. In light-injured retinas, the immunostaining of the photoreceptor water channel aquaporin-1 disappeared along with the degeneration of the outer retina, and the outer nuclear layer contained large spherical bodies representing photoreceptor nuclei which were fused together. The immunostainings of the aquaporin-4 and Kir4.1 proteins were increased in the outer retina after light treatment. Since the amplitude of the potassium currents of Müller cells remained largely unaltered, the increase in the Kir4.1 immunostaining is supposed to be caused by a redistribution of the channel protein. The data indicate that Müller glial cells respond to excessive light with an alteration in the localization of Kir4.1 and aquaporin-4 proteins; this alteration is thought to be a response to the edema in the outer retina and may support the resolution of edema.     

Expression of aquaporin-1 immunoreactivity by photoreceptor cells in the mouse retina

Aquaporin water channels play a crucial role in the maintenance of ionic and osmotic homeostasis in the neural tissue. In the sensory retina, aquaporin-4 is expressed by Müller glial cells, predominantly in the inner retina, while aquaporin-1 is expressed mainly in the outer retina. However, it is unknown whether aquaporin-1 expression occurs in Müller cells or photoreceptor cells. By using immunohistochemical staining of retinal slices from rds mice, we show that the immunoreactivity for aquaporin-1 disappears along with the photoreceptor cell degeneration. In suspensions of dissociated retinal cells from control mice, photoreceptor cells expressed aquaporin-1 immunoreactivity while Müller cells were largely devoid of staining. The data suggest that photoreceptor cells, but not Müller cells, express aquaporin-1 in the murine retina.     

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.     

Blood-retinal barrier disruption and ultrastructural changes in the hypoxic retina in adult rats: the beneficial effect of melatonin administration

Reactive changes in astrocytes and Müller cells in the retina of adult rats subjected to hypoxia were investigated. Along with this, the integrity of the blood-retinal barrier (BRB) was assessed using fluorescent and electron-dense tracers. In hypoxic rats, mRNA and protein expression of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQ4) were significantly increased. AQ4 immunoreactive cells were identified as astrocytes and Müller cells by double immunofluorescence labelling. Another alteration in the hypoxic retina was marked reduction in melatonin content compared to controls. In this connection, administration of exogenous melatonin reduced the tissue concentration of vascular endothelial growth factor (VEGF) and nitric oxide (NO); both were elevated in hypoxic rats. A major structural change in the hypoxic retina was swelling of astrocyte and Müller cell processes but this was noticeably attenuated after melatonin administration. Following an intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in the retina in hypoxic rats but not in the controls, indicating that the functional integrity of the BRB is compromised in hypoxia/reoxygenation. It is suggested that enhanced tissue concentration of VEGF and NO production in the hypoxic retina contribute to increased permeability of the retinal blood vessels. The concurrent up-regulation of AQ4, a water-transporting protein, in astrocytes and Müller cells in hypoxia suggests its involvement in oedema formation. Since melatonin effectively reduced the vascular permeability in the retina of hypoxic rats, as evidenced by reduced leakage of RhIC, we suggest that its administration may be of potential benefit in the management of retinal oedema associated with retinal hypoxia.     

The structure of rabbit retinal Müller (glial) cells is adapted to the surrounding retinal layers

Summary Radial glial (Müller) cells of the rabbit retina were studied by various techniques including Golgi impregnation, scanning electron microscopy, horseradish peroxidase application, and staining of enzymatically isolated cells. This combination of methods produced detailed information on the specialized morphology of the Müller cells within the different topographical regions of the retina, and of the Müller cell processes within the various retinal layers. As a general rule, the retinal periphery contains short thick Müller cells with big endfeet, whereas the thick central retina is occupied by long slender cells with small endfeet. Independent of their location within the retina, Müller cell processes were found to be adapted to the structure of the surrounding retinal layers. Within the outer and inner nuclear layers, Müller cell processes (and somata) extend thin cytoplasmic bubbles ensheathing the neuronal somata, as do the velate astrocytes in the brain. In the plexiform layers, Müller cells extend many fine side branches between the neuropil, comparable to the protoplasmic astrocytes of the brain. In the thick myelinated nerve fibre layer of the central retina the Müller cell processes are rather smooth, similar to those of fibrous astrocytes. It is concluded that the neuronal microenvironment determines the morphology of a given glial process, or even of a part of a glial process running through a specialized neuronal compartment.     

Retinal degeneration in experimental Creutzfeldt-Jakob disease

Mice with experimental Creutzfeldt-Jakob disease (CJD) develop a progressive retinal degeneration after a prolonged incubation period. Sections of the eyes stained with hematoxylin and eosin revealed pathologic changes in the optic nerve and a marked degeneration of photoreceptor cell inner and outer segment areas. Both peripheral and central retina, normally 10 cells thick, were reduced to one photoreceptor cell or less in thickness. Ultrastructural analysis revealed total loss of outer segment and most inner segment elements. Only Müller cell microvilli and macrophages remained in the subretinal space. Macrophages were also visible in the remnant photoreceptor cell layer. The inner nuclear layer and pigment epithelial cell layers appeared normal. Müller cell hypertrophy was evident but was not accompanied by spongiform vacuolation. Several of the degenerative changes of the eye in mice with experimental Creutzfeldt-Jakob disease differ from those observed for scrapie in rodents. The pathologic similarities between the retinal degenerations occurring in mice with experimental Creutzfeldt-Jakob disease and those found in some forms of human retinal degeneration are provocative. These similarities raise the question whether or not other retinal degenerative diseases might be caused by infectious agents such as prions or slow viruses.     

Expression of natriuretic peptides in rat Müller cells

Natriuretic peptides (NPs) have been shown to modulate neuronal activities. By immunohistochemistry and confocal microscopy, we examined expression of atrial NP (ANP), brain NP (BNP) and C-type NP (CNP) in rat retina. Our results showed that these peptides were differentially expressed in the neural retina. While strong ANP-, BNP- and CNP-immunoreactivity (IR) was clearly seen in the outer and inner plexiform layers and on numerous neurons in the inner nuclear layer, BNP- and CNP-, but not ANP-IR, was present in some ganglion cells. Furthermore, ANP, BNP and CNP were expressed in Müller cells with distinct profiles, as shown by double labeling of NPs and vimentin. Labeling for BNP was rather strong in the main trunks, major processes, but hardly detectable in the endfeet. The expression profile for ANP was similar, but with a much lower level. On the contrary, the endfeet and major processes in the inner retina were strongly CNP-positive, with the main trunks and other major processes in the outer retina much less labeled. These results raise a possibility that NPs, when released from Müller cells, may perform layer dependent functions.     

Expression of developmentally defined retinal phenotypes in the histogenesis of retinoblastoma.

Retinoblastoma, the most common intraocular tumor of childhood, is a malignant neoplasm that arises during retinal development. The embryonal cell target for neoplastic transformation is not yet clearly defined. To better understand the histogenetic potential of this tumor, the expression of photoreceptor and glial cell-associated proteins were examined in 22 primary retinoblastomas. Interphotoreceptor retinol-binding protein (IRBP), cone and rod opsins were selected as the photoreceptor specific proteins due to their different temporal patterns of expression during normal retinal development. Neoplastic Müller cell differentiation, and non-neoplastic reactive astrocytes were identified using cellular retinaldehyde binding-protein (CRAlBP), and glial fibrillary acidic protein (GFAP), respectively. Photoreceptor proteins were present in 16 cases and showed different cellular patterns of expression. IRBP and cone opsin were usually abundant. Although rod opsin was clearly identified in eight tumors, its expression was more restricted than either IRBP or cone opsin. This differential pattern of expression, opposite to the normal pattern of photoreceptor gene expression in the adult retina, corresponded to a marked decrease in mRNA for rod opsin. Cone opsin and IRBP colocalized in fleurettes demonstrating that neoplastic human cone cells are capable of IRBP synthesis. Müller cell differentiation was present in 12 of the 16 cases in which photoreceptor proteins were detected. In contrast, GFAP was only present in reactive, stromal astrocytes associated with blood vessels. Our data suggest that the retinoblastoma has the histogenetic potential of the immature neural retinal epithelium which can give rise to both photoreceptor and Müller cell lineages. The differential expression of cone and rod phenotypes in retinoblastoma is consistent with the "default" mechanism of cone cell differentiation.     

Endothelin receptors in the detached retina of the pig

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes hypoperfusion of the neurosensory retina. We investigated immunohistochemically the expression of the receptors for ET-1, ET(A) and ET(B), in control and locally detached retinas of the pig. Immunoreactivity for ET(A) was expressed in the innermost retinal layers and in the outer plexiform layer in control retinas, and was additionally strongly expressed by retinal blood vessels at 7 days after detachment of the sensory retina from the pigment epithelium. Immunoreactivity for ET(B) was expressed by the innermost retinal layers, by ganglion cell somata, and by Müller glial cells in the control tissue, and was not altered in its expression after detachment. The vascular expression of ET(A) may suggest a hypoperfusion of the retina after detachment.     

Early postnatal Müller cell death leads to retinal but not optic nerve degeneration in NSE-Hu-Bcl-2 transgenic mice

Topographically localized over-expression of the human Bcl-2 protein in retinal glial Müller cells of a transgenic mice (line 71) leads to early postnatal apoptotic Müller cell death and retinal degeneration. Morphological, immunohistological and confocal laser microscopic examination of transgenic and wild-type retinas were achieved on paraffin retinal sections, postnatally. Apoptosis occurs two to three days earlier in the internal nuclear layer of transgenic retinae, than in wild-type littermates. In parallel there was a progressive disappearance of transgenic Hu-Bcl-2 over-expression, as well as of the Müller cell markers, cellular retinaldehyde-binding protein and glutamine synthetase. This phenomenon led to retinal dysplasia, photoreceptor apoptosis and then retinal degeneration and proliferation of the retinal pigment epithelium. The optic nerve, however, remains intact. Two complementary observations confirm the pro-apoptotic action of Bcl-2 over-expression in Müller cells: (i) in the peri-papillary and peripheral regions where the transgene Bcl-2 is not expressed, cellular retinaldehyde-binding protein or glutamine synthetase immunostaining persist and Müller glia do not die; and (ii) the retina conserves a normal organisation in these two regions inspite of total retinal degeneration elsewhere.We conclude that retinal dysplasia and degeneration are linked to primary Müller cell disruption. Besides its generally accepted anti-apoptotic function, over-expression of Bcl-2 also exerts a pro-apoptotic action, at least in immature Müller glia. One may suppose that Bcl-2 translocation resulting in its over-expression in retinal Müller cells could be a putative mechanism for early retinal degeneration.