Glial fibrillary acidic protein and its mRNA: ultrastructural detection and determination of changes after CNS injury.

We have previously demonstrated that glial fibrillary acidic protein (GFAP) containing intermediate filaments in retinal Müller cells undergo both quantitative induction and subcellular reorganization as a response to long-term retinal detachment (an induced CNS degeneration wherein the Müller cells form a multicellular scar). This study demonstrates by RNA blotting analysis that normal retina expresses a low basal level of GFAP mRNA, which is induced approximately 500% within 3 days of retinal detachment. At the cellular level, electron microscopic in situ hybridization analysis readily detects GFAP mRNA in Müller cells of detached retinas, but not in normal retinas. On the other hand, GFAP mRNA was readily detected in retinal astrocytes (which appear to express GFAP mRNA at high, constitutive levels). In both cell types, the ultrastructural localization of GFAP mRNA was the same. In the nuclei, the GFAP mRNA was associated with amorphous, electron-dense regions within the euchromatin. In the cytoplasm, the GFAP mRNA was associated with intermediate filaments near the nuclear pores, along the filaments when no other structures were apparent, and when the filaments appeared to be associated with ribosomes and polysomes. The ultrastructural location of the GFAP mRNA (especially along the intermediate filaments) may be unique to this mRNA or may represent a more generalized mRNA phenomenon.     

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy-induced apoptosis via modulation of retinal glia in vivo

Adenoviral-mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy-induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRalpha) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral-mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter-1 (GLAST-1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRalpha, pSTAT(3), GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection.     

Retinal neurons regulate proliferation of postnatal progenitors and Müller glia in the rat retina via TGF beta signaling

The number of proliferating cells in the rodent retina declines dramatically after birth. To determine if extrinsic factors in the retinal micro-environment are responsible for this decline in proliferation, we established cultures of retinal progenitors or Muller glia, and added dissociated retinal neurons from older retinas. The older cells inhibited proliferation of progenitor cells and Muller glia. When these experiments were performed in the presence of TGF(beta)RII-Fc fusion protein, an inhibitor of TGF(beta) signaling, proliferation was restored. This suggests a retina-derived TGF(beta) signal is responsible for the developmental decline in retinal proliferation. TGFbeta receptors I and II are expressed in the retina and are located in nestin-positive progenitors early in development and glast-positive Muller glia later in development. RT-PCR and immunofluorescence data show TGF(beta)2 is the most highly expressed TGF(beta)ligand in the postnatal retina, and it is expressed by inner retinal neurons. Addition of either TGF(beta)1 or TGF(beta)2 to postnatal day 4 retinas significantly inhibited progenitor proliferation, while treatment of explanted postnatal day 6 retinas with TGF(beta) signaling inhibitors resulted in increased proliferation. Last, we tested the effects of TGF(beta) in vivo by injections of TGF(beta) signaling inhibitors: when TGF(beta) signaling is inhibited at postnatal day 5.5, proliferation is increased in the central retina; and when co-injected with EGF at postnatal day 10, TGF(beta)inhibitors stimulate Muller glial proliferation. In sum, these results show that retinal neurons produce a cytostatic TGF(beta) signal that maintains mitotic quiescence in the postnatal rat retina.     

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy‐induced apoptosis via modulation of retinal glia in vivo

Adenoviral‐mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy‐induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRα) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral‐mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter‐1 (GLAST‐1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRα, pSTAT₃, GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Inhibition of reactive gliosis attenuates excitotoxicity-mediated death of retinal ganglion cells

Reactive gliosis is a hallmark of many retinal neurodegenerative conditions, including glaucoma. Although a majority of studies to date have concentrated on reactive gliosis in the optic nerve head, very few studies have been initiated to investigate the role of reactive gliosis in the retina. We have previously shown that reactive glial cells synthesize elevated levels of proteases, and these proteases, in turn, promote the death of retinal ganglion cells (RGCs). In this investigation, we have used two glial toxins to inhibit reactive gliosis and have evaluated their effect on protease-mediated death of RGCs. Kainic acid was injected into the vitreous humor of C57BL/6 mice to induce reactive gliosis and death of RGCs. C57BL/6 mice were also treated with glial toxins, alpha-aminoadipic acid (AAA) or Neurostatin, along with KA. Reactive gliosis was assessed by immunostaining of retinal cross sections and retinal flat-mounts with glial fibrillary acidic protein (GFAP) and vimentin antibodies. Apoptotic cell death was assessed by TUNEL assays. Loss of RGCs was determined by immunostaining of flat-mounted retinas with Brn3a antibodies. Proteolytic activities of matrix metalloproteinase-9 (MMP-9), tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) were assessed by zymography assays. GFAP-immunoreactivity indicated that KA induced reactive gliosis in both retinal astrocytes and in Muller cells. AAA alone or in combination with KA decreased GFAP and vimentin-immunoreactivity in Mϋller cells, but not in astrocytes. In addition AAA failed to decrease KA-mediated protease levels and apoptotic death of RGCs. In contrast, Neurostatin either alone or in combination with KA, decreased reactive gliosis in both astrocytes and Mϋller cells. Furthermore, Neurostatin decreased protease levels and prevented apoptotic death of RGCs. Our findings, for the first time, indicate that inhibition of reactive gliosis decreases protease levels in the retina, prevents apoptotic death of retinal neurons, and provides substantial neuroprotection.     

Heterogeneity of Glia in the Retina and Optic Nerve of Birds and Mammals

We have recently described a novel type of glial cell that is scattered across the inner layers of the avian retina [1]. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and up-regulate the nestin-related intermediate filament transitin. These changes in glial activity correspond with increased susceptibility of neurons to excitotoxic damage. This novel cell-type has been termed the Non-astrocytic Inner Retinal Glia-like (NIRG) cells. The purpose of the study was to investigate whether the retinas of non-avian species contain cells that resemble NIRG cells. We assayed for NIRG cells by probing for the expression of Sox2, Sox9, Nkx2.2, vimentin and nestin. NIRG cells were distinguished from astrocytes by a lack of expression for Glial Fibrilliary Acidic Protein (GFAP). We examined the retinas of adult mice, guinea pigs, dogs and monkeys (Macaca fasicularis). In the mouse retina and optic nerve head, we identified numerous astrocytes that expressed GFAP, S100β, Sox2 and Sox9; however, we found no evidence for NIRG-like cells that were positive for Nkx2.2, nestin, and negative for GFAP. In the guinea pig retina, we did not find astrocytes or NIRG cells in the retina, whereas we identified astrocytes in the optic nerve. In the eyes of dogs and monkeys, we found astrocytes and NIRG-like cells scattered across inner layers of the retina and within the optic nerve. We conclude that NIRG-like cells are present in the retinas of canines and non-human primates, whereas the retinas of mice and guinea pigs do not contain NIRG cells.     

Aging-related changes in astrocytes in the rat retina: imbalance between cell proliferation and cell death reduces astrocyte availability

The aim of this study was to investigate changes in astrocyte density, morphology, proliferation and apoptosis occurring in the central nervous system during physiological aging. Astrocytes in retinal whole-mount preparations from Wistar rats aged 3 (young adult) to 25 months (aged) were investigated qualitatively and quantitatively following immunofluorohistochemistry. Glial fibrillary acidic protein (GFAP), S100 and Pax2 were used to identify astrocytes, and blood vessels were localized using Griffonia simplicifoli a isolectin B4. Cell proliferation was assessed by bromodeoxyuridine incorporation and cell death by TUNEL-labelling and immunolocalization of the apoptosis markers active caspase 3 and endonuclease G. The density and total number of parenchymal astrocytes in the retina increased between 3 and 9 months of age but decreased markedly between 9 and 12 months. Proliferation of astrocytes was detected at 3 months but virtually ceased beyond that age, whereas the proportion of astrocytes that were TUNEL positive and relative expression of active caspase 3 and endonuclease G increased progressively with aging. In addition, in aged retinas astrocytes exhibited gliosis-like morphology and loss of Pax2 reactivity. A small population of Pax2⁺/GFAP⁻ cells was detected in both young adult and aged retinas. The reduction in the availability of astrocytes in aged retinas and other aging-related changes reported here may have a significant impact on the ability of astrocytes to maintain homeostasis and support neuronal function in old age.     

Impaired retinal vascular development in anencephalic human fetus

This study was to investigate the effect of the absence of ganglion cells on the development of human retinal vasculature. Anencephaly (AnC) and age-matched control eyes derived from each three spontaneously aborted fetus (ranging from 15 to 20 weeks gestation) were subjected to immunofluorescence staining for HIF-1α, Thy-1, glial fibrillary acidic protein (GFAP) and platelet/endothelial cell adhesion molecule (PECAM) and apoptosis assay. In developing mouse retina, Western blotting for hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) was performed. Under hypoxic condition (O₂ < 1%), cellular proliferation and VEGF mRNA expression in astrocytes were measured. Apoptotic cells in AnC retina were primarily localized in the ganglion cell layer (GCL), whereas apoptotic cells in normal retina were distributed in the retinoblastic layer. With increase of apoptotic cells in GCL of AnC retina, HIF-1α expression were severely distinguished in avascular retina and GFAP expression in junctional area between avascular and vascular retina was much reduced, accompanied by decrease of PECAM expression compared to normal retina. In developing mouse retina, HIF-1α and VEGF expression were high in hypoxic retina of early stage with incomplete vascular development and then progressively decreased with regression to arborous pattern of matured vascular networks. In hypoxic condition, a significant increase in cellular proliferation and VEGF mRNA expression was observed in astrocytes. Therefore, our results suggest that vascular attenuation in AnC retina could be closely related to the absence of ganglion cells as the metabolic demander to induce retinal vascular development.     

Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.     

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.     

Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma.

The presence of astrocyte precursor cells (APCs) and time course and topography of astrocyte differentiation during development were investigated by triple-label immunohistochemistry with intact fetal and adult human retinas. Throughout retinal development and adulthood, expression of Pax2 was restricted to cells of the astrocytic lineage. Three distinct stages of astrocytic differentiation were identified during development: i) Pax2+/vimentin+/GFAP- APCs; ii) Pax2+/vimentin+/GFAP+ immature perinatal astrocytes; and iii) Pax2+/vimentin-/GFAP+ mature perinatal astrocytes. In adult, cells with the antigenic phenotype of mature perinatal astrocytes were restricted to a region surrounding the optic nerve head (ONH), whereas cells at a fourth stage of differentiation, adult astrocytes (Pax2-/vimentin-/GFAP+), were apparent throughout the vascularized retina. APC appearance was centered around the ONH and preceded the appearance of perinatal astrocytes. A cluster of Pax2+ somas was also present in a small region surrounding the ONH at the ventricular surface of the developing retina, which suggests the existence of two distinct sites of astrocytic differentiation. The coincidence in the location of APCs and perinatal astrocytes at the ventricular zone with that of optic nerve colobomas, together with the association of Pax2 gene mutations with this condition, suggests that coloboma formation may result from impaired astrocyte differentiation during development.     

Biocompatibility of subretinal parylene-based Ti/Pt microelectrode array in rabbit for further artificial vision studies

To evaluate the biocompatibility of subretinal implanted parylene-based Ti/Pt microelectrode arrays (MEA). Eyes were enucleated 3 months after MEAs were implanted into the subretinal space of rabbits. Morphological changes of the retinas were investigated by H&;E staining. Immunohistochemical staining for glial fibrillary acidic protein and opsin were performed to evaluate changes in Muller cells and photoreceptors in the retinas. Retina tissue around the array remained intact. Photoreceptor degeneration and glial cell activation were observed in the retina overlaying the MEA implant. However, the cells in the inner retinal layers were preserved. Photoreceptor degeneration and glial cell activation at the MEA–retina interface are expected to be a normal reaction to implantation. Material used in this experiment has good biocompatibility within the subretinal environment and is expected to be promising in the further retinal prosthesis studies.     

Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes.

BACKGROUND: Chronic diabetes causes structural changes in the retinal capillaries of nearly all patients with a disease duration of more than 15 years. Acellular occluded vessels cause hypoxia, which stimulates sight-threatening abnormal angiogenesis in 50% of all type I diabetic patients. The mechanism by which diabetes produces acellular retinal capillaries is unknown. MATERIALS AND METHODS: In this study, evidence of programmed cell death (PCD) was sought in the retinas of early diabetic rats, and the effect of nerve growth factor (NGF) on PCD and capillary morphology was evaluated. RESULTS: Diabetes induced PCD primarily in retinal ganglion cells (RGC) and Muller cells. This was associated with a transdifferentiation of Muller cells into an injury-associated glial fibrillary acidic protein (GFAP)-expressing phenotype, and an up-regulation of the low-affinity NGF receptor p75NGFR on both RGC and Muller cells. NGF treatment of diabetic rats prevented both early PCD in RGC and Muller cells, and the development of pericyte loss and acellular occluded capillaries. CONCLUSIONS: These data provide new insight into the mechanism of diabetic retinal vascular damage, and suggest that NGF or other neurotrophic factors may have potential as therapeutic agents for the prevention of human diabetic retinopathy.     

Regeneration of the retina: toward stem cell therapy for degenerative retinal diseases

Degenerative retinal diseases affect millions of people worldwide, which can lead to the loss of vision. However, therapeutic approaches that can reverse this process are limited. Recent efforts have allowed the possibility of the stem cell-based regeneration of retinal cells and repair of injured retinal tissues. Although the direct differentiation of pluripotent stem cells into terminally differentiated photoreceptor cells comprises one approach, a series of studies revealed the intrinsic regenerative potential of the retina using endogenous retinal stem cells. Muller glial cells, ciliary pigment epithelial cells, and retinal pigment epithelial cells are candidates for such retinal stem cells that can differentiate into multiple types of retinal cells and be integrated into injured or developing retina. In this review, we explore our current understanding of the cellular identity of these candidate retinal stem cells and their therapeutic potential for cell therapy against degenerative retinal diseases. [BMB Reports 2015; 48(4): 193-199]     

Methamphetamine exacerbates the toxic effect of kainic acid in the adult rat retina

The recreational use of the psychoactive drug, methamphetamine has increased markedly over the last three decades. It has long been known that this drug has detrimental effects upon the mammalian brain monoaminergic system, but the long- or short-term effects on the retina, a neurological extension of the central nervous system, have received little attention. The aim of this study was, therefore, to determine whether intraocular injection of methamphetamine (MA) is toxic to the healthy adult rat retina and to analyse its effects on the compromised retina after an injection of the ionotropic glutamate receptor agonist, kainate, which is known to cause retinal neuropathology. The equivalent of 1 mM (in the vitreous humour) MA and/or kainate (40 μM) were injected intravitreally. Flash electroretinograms (ERGs) were recorded before and 2 and 4 days after treatment. Five days after treatment, animals were killed and the retinas analysed either for the immunohistochemical localisation of various antigens or for electrophoresis/Western blotting. Some animals were kept for 19 days after treatment and the retinas analysed for tyrosine hydroxylase immunoreactivity. No differences could be found between vehicle- and MA-treated retinas with respect to the nature or localisation of either tyrosine hydroxylase immunoreactivity after 5 or 19 days or other antigens after 5 days. Moreover, the normal ERG and GFAP and calretinin protein antigens were unaffected by MA. Kainate treatment, however, caused a change in the ERGs after 2 and 4 days, an alteration in every antigen localised by immunohistochemistry and an increase in the retinal levels of calretinin and GFAP proteins. Significantly, the changes seen in the b-wave amplitude and implicit time of the ERG after 4 days and the increased level of GFAP protein after 5 days following kainate treatment were enhanced when MA was co-injected. Intravitreal injection of methamphetamine had no detectable detrimental effect on the normal adult rat retina but exacerbated the damaging effects of kainic acid. Such data suggest that a neurotoxic effect of MA may be more obviously illustrated when the tissue is already compromised as occurs in, for example, ischemia.     

Heterogeneous immunoreactivity of glial cells in the mesencephalon of a lizard: A double labeling immunohistochemical study

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star-shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear-shaped cells that are therefore astrocytes with unusual morphology. Other round or pear-shaped cells, also scattered in the tegmentum and the tectum, display only GS. Electron microscopy reveals that these cells may be oligodendrocytes. In this lizard, the GS is expressed in some oligodendrocytes while this does not occur in the central nervous system of mammals in situ. These results confirm that the cellular specificity of GS is different in various species and suggest that ependymal cells are also immunoreactive for GS but they do not contain GFAP. J. Morphol. 235:109–119, 1998. © 1998 Wiley-Liss, Inc.     

Astrocyte invasion and vasculogenesis in the developing ferret retina

We studied the time course of astrocyte invasion and blood vessel formation in the developing ferret retina using glial fibrillary acidic protein (GFAP)-immunohistochemistry for astrocytes and isolectin B4 histochemistry for blood vessels. As in other mammals, strongly GFAP positive astrocytes invade the ferret retina from the optic nerve. At birth, strongly GFAP positive astrocytes have reached about 22% of the distance between optic disc and outer retinal edge whereas weakly GFAP positive processes already extend to the edge of the retina. At postnatal days P30–P37 about 82% of the distance between optic disc and outer retinal edge and in the adult 88% of this distance is covered with strongly labelled astrocytes. Superficial blood vessels form from the optic disc. They reach up to about 24% of the retinal radius at birth and grow radially across the retina during further development. At P30–P37, the whole retina is covered with superficial blood vessels. The deep vascular layer forms later (around P30) through sprouting from superficial vessels. The radial pattern of astrocyte and vessel growth from the optic disc is not affected by the formation of the area centralis and visual streak.     

Comparison of dark- and light-adapted carp retinas with NADPH diaphorase staining

The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids of inner segments of both rods and cones and some horizontal cells were heavily stained. Staining was also found in subpopulations of amacrine cells and ganglion cells. In addition, Muller cells were strongly positive for NADPH diaphorase. When light-adapted for 5h, ellipsoids of photoreceptors and ganglion cells were less intensely stained, whereas Muller cells and horizontal cells became negative for NADPH diaphorase. Furthermore, rod ON-center bipolar cells were clearly stained. The difference of staining of amacrine cells between dark- and light-adapted retinas was not significant. The differences in diaphorase-staining pattern between dark- and light-adapted retinas suggest that Muller cells, some horizontal cells and rod ON-center bipolar cells contain inducible nitric oxide synthase,     

“Super p53” Mice Display Retinal Astroglial Changes

Tumour-suppressor genes, such as the p53 gene, produce proteins that inhibit cell division under adverse conditions, as in the case of DNA damage, radiation, hypoxia, or oxidative stress (OS). The p53 gene can arrest proliferation and trigger death by apoptosis subsequent to several factors. In astrocytes, p53 promotes cell-cycle arrest and is involved in oxidative stress-mediated astrocyte cell death. Increasingly, astrocytic p53 is proving fundamental in orchestrating neurodegenerative disease pathogenesis. In terms of ocular disease, p53 may play a role in hypoxia due to ischaemia and may be involved in the retinal response to oxidative stress (OS). We studied the influence of the p53 gene in the structural and quantitative characteristics of astrocytes in the retina. Adult mice of the C57BL/6 strain (12 months old) were distributed into two groups: 1) mice with two extra copies of p53 (“super p53”; n = 6) and 2) wild-type p53 age-matched control, as the control group (WT; n = 6). Retinas from each group were immunohistochemically processed to locate the glial fibrillary acidic protein (GFAP). GFAP+ astrocytes were manually counted and the mean area occupied for one astrocyte was quantified. Retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p53 availability. The mean GFAP+ area occupied by one astrocyte in “super p53” eyes was significantly higher (p<0.05; Student’s t-test) than in the WT. In addition, astroglial density was significantly higher in the “super p53” retinas than in the WT ones, both in the whole-retina (p<0,01 Student’s t-test) and in the intermediate and peripheral concentric areas of the retina (p<0.05 Student’s t-test). This fact might improve the resistance of the retinal cells against OS and its downstream signalling pathways.