Glial fibrillary acidic protein expression in postnatal development of WAG/Rij rat retina
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Introduction: WAG/Rij rat is an experimental model of retinal degenerative diseases intensifying with age.Degeneration begins in the photoreceptor cells layer, followed by disappearance of photoreceptor cells, migration of the pigment epithelium and disorganization of remaining retinal layers.However, the age of onset of the degeneration is unknown.The destroyed neurons of retina are usually replaced by glial cells.A typical marker for several types of glial cells is the glial fibrillary acidic protein (GFAP). Aim of the Work:The aim of the study is to determine postnatal changes of GFAP expression in retina of WAG/Rij rats.Material and Methods: WAG/Rij rats of both sexes and different ages: 1, 10, 15, 20, 30, 60, 180, 360 postnatal days (P) (n =3 for each age) were taken.The immunohistochemical analysis of GFAP expression in the retina of WAG/Rij rats was performed and the expression area was measured.Results: Expression of GFAP in the retina is detected from P1 to P20 as a brown staining in the internal limiting membrane or as a brown lines on the inner retina.On P30 the glial fibrillary acidic protein is already detected in significant quantities in almost all layers of the retina of the experimental rats.From P60 to P360 the expression of GFAP in the retina of the WAG/ Rij rats is more pronounced and increases in direct proportion with the age.Conclusion: It was found that GFAP expression in the retina of WAG/Rij rats increases in direct proportion to the age.The increasing GFAP expression in the retina of the WAG/Rij rats starting from the 30th postnatal day indicates an intensification of destructive processes in retinal neurons with age and their replacement by Müller cells.Keywords:
Inner limiting membrane
GFAP stain
Neuroglia
Abstract Previous reports of increases in glial cell number and expression of glial fibrillary acidic protein (GFAP) in stimulated brain regions or epileptic tissue have implicated a role for increases in extracellular potassium concentration ([K + ] o ) in glial reactions. We examined the effects of altered [K + ] o on DNA and protein syntheses and GFAP expression of cultured glial cells isolated from the posthatch chick brain stem. [K + ] o was varied by adding both KCl and NaCl to K + , NaCl‐free medium to achieve final [K + ] of 1–50 mM. DNA and protein syntheses were measured by incorporation of 3 H‐thymidine and 3 H‐leucine, respectively, into acid‐insoluble material. GFAP expression was measured by a dot‐immunoblotting assay. DNA synthesis in glial cells cultured in high (5–50 mM) K + o was 45–60% less than that of cells cultured in low (1–3 mM) K + o . Protein synthesis per cell was increased 34–44% in cells cultured in high K + as compared to those cultured in low K + . GFAP expression was inversely related to [K + ] o over the 1–10 mM range. Compared to the baseline of 3 mM K + o , GFAP per cell was increased 65% at 1 mM and decreased 45% at 10 mM. These data suggest that increases in glial cell number and GEAP immunoreactivity found in sites of increased neuronal activity and in pathological tissues may not be caused solely by persistent increases in [K + ] o . Instead, these results suggest that neuronal activity, through the release of K + , may have an inhibitory influence on glial proliferation and GFAP expression. In light of work by others implying a relationship between GFAP immunoreactivity and rigidity of astroglial processes together with the data presented here, we suggest that the elevated [K + ] o accompanying neuronal activity, by inhibiting GFAP expression, may facilitate the morphological plasticity of glial cells. Conversely, conditions of low [K + ] o may contribute to rigidity of astrocytic processes.
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GFAP stain
Muller glia
Neuroglia
Gliosis
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Abstract Accumulation of glial fibrillary acidic protein (GFAP) in Müller cells has been observed in retinas of several mammalian species secondary to genetically induced degeneration and neuronal injury. In the present series of experiments, I have examined the effects of the rd (retinal degeneration) mutation on the expression of GFAP in retinas of chicks homozygous for the mutation ( rd / rd ) prior to and following the onset of photoreceptor degeneration, which first appears approximately 7 days posthatch (7dph). Carrier (+/ rd ) and wild‐type (+/+) retinas served as controls. Retinas taken from 1, 7, 21, and 33 dph rd/rd, +/rd, and +/+ chicks were analyzed for the presence of GFAP by immunocytochemical and SDS‐PAGE/Western blot techniques. The following immunocytochemical observations were made: (1) GFAP immunostaining was limited to and located throughout the Müller cells. (2) The intensity of GFAP immunostaining increased with age in all three retina types in tissue sections, as well as on immunoblots. (3) The distribution of GFAP staining within rd/rd Müller cells following the onset of degeneration was slightly different from that observed in +/+ and +/rd retinas and was distinguished by increased staining of the cell bodies and the cell processes forming the outer limiting membrane. The results of these experiments show that Müller cells in chick retina contain GFAP. In addition, they suggest that, in contrast to Müller cells in degenerating mammalian retina, Müller cells in rd chick retina do not accumulate large amounts of GFAP in response to degeneration.
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Inner limiting membrane
Neuroglia
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GFAP stain
Schwann cell
Neuroglia
Galactocerebroside
Immunofluorescence
Peripheral Nervous System
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Glial fibrillary acidic protein (GFAP) is normally found in astrocytes. In the normal rat retina at all ages, only astrocytes stain for GFAP. This staining pattern is also found in RCS rats with inherited retinal dystrophy younger than 38 days. Beginning on day 38, when about 61% of the photoreceptors have degenerated, a few GFAP-positive fibers span the retina from the inner limiting membrane to the external limiting membrane. By day 41 and at all later ages examined, the radial fibers of Müller cells is a response to photoreceptor necrosis or might be a direct effect of the mutant gene, we induced photoreceptor degeneration in normal, adult Sprague-Dawley rats by exposing them to constant light for variable periods of time. After 3 days in constant light, there is a 20% reduction in the number of photoreceptors and many Müller cells are positive for GFAP. Immunoblot studies confirmed that the anti-GFAP reacted with a single protein from retina that corresponded in molecular weight and Triton-insolubility to GFAP. The immunoblots also corroborated the results from anti-GFAP immunostaining of control and experimental retinas. These results indicate that Müller cells express GFAP immunoreactivity in response to experimentally as well as genetically induced photoreceptor degeneration.
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Inner limiting membrane
Photoreceptor cell
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GFAP stain
Gliosis
Inner limiting membrane
Polyclonal antibodies
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Müller cells in the mammalian retina normally express low levels of glial fibrillary acidic protein (GFAP); however, its expression is upregulated in response to the loss of retinal neurons. The change in expression of GFAP is one of the earliest indicators of retinal damage and is correlated with the time course of disease. The aim of this study was to investigate the time course of degeneration and the expression of GFAP in the retina of mer knockout mice.A total of 30 mer knockout mice, aged from 15 - 20 days to 1 year and 32 age-matched wild type mice as controls were tested. Immunohistochemistry was used to show the expression of GFAP in the central and peripheral retina of mer knockout and control mice at postnatal age of 15 days (P15d), 20 days (P20d), 4 weeks (P4w), 6 weeks (P6w), 8 weeks (P8w), 3 months (P3m), 6 months (P6m) and 1 years (P1y).The expression of GFAP in the central and peripheral retina of wild type mice was limited to the retinal ganglion cell and nerve fiber layers. In the central retina of mer knockout mice, GFAP expression was upregulated at P4w and GFAP immunolabelling penetrates across the entire thickness of the retina at P8w; whereas in the peripheral retina, the GFAP expression was upregulated at P20d and GFAP immunolabelling penetrates the entire retina after P4w.Increased expression of GFAP in Müller cells of mer knockout mice occur at P20d in the peripheral retina and P4w in the central retina. GFAP expression in Müller cells appears to be a secondary response to the loss of retinal neurons. Increased expression of GFAP may occur prior to any detectable morphological changes in the retina. This study suggests that the loss of retinal neurons may begin in the early stages of retinitis pigmentosa, prior to the discovery of any morphological changes in the retina.
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Conditional gene knockout
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GFAP stain
Neuroglia
Carbonic anhydrase II
Primary culture
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Reactive changes of retinal astrocytes and Müller glial cells in kainate‐induced neuroexcitotoxicity
Abstract 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.
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Neuroglia
Kainate receptor
Muller glia
Ganglion cell layer
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Low intensity diffuse white fluorescent light (1,000 1x for 2 h) exclusively induced photoreceptor damage in the inferior retina of albino rats; the temporal retina showed extensive damage, whereas the nasal retina revealed threshold lesions prior to recovery. To expand our morphological data, further experiments were undertaken to determine if glial fibrillary acidic protein (GFAP) expression was associated with the regions of photoreceptor damage. To follow the time course of GFAP expression, immunoblot analysis was carried out on retinal homogenates of dark-adapted (control) rats and light-exposed rats returned to cyclic light for 0 h, 1, 2, 3 and 6 days. A significant twofold increase in GFAP immunoreactivity over controls was observed in the retinas of light-exposed rats returned to cyclic light for 6 days. Using an indirect immunohistochemical method, retinal sections of the control and light-exposed rats allowed to recover for 6 days were stained for GFAP. GFAP immunoreactivity was localised to the astrocytes and Müller cells. Moreover, GFAP staining in Müller cells in the retinas of control animals was uniformly restricted to rare end-feet. In contrast, a gradient of GFAP immunoreactivity was observed in experimental rats, rising from the superior retina to the inferior temporal quadrant; the GFAP staining in the inferior nasal quadrant was intermediate. Thus, GFAP immunoreactivity was proportional to photoreceptor damage. Interestingly, no GFAP induction could be demonstrated in the pineal glands of light-exposed rats.
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