Protein In Rat Retina Research Articles

Expressions of nestin and glial fibrillary acidic protein in rat retina after optic nerve transection.

To assess the expression of nestin and glial fibrillary acidic protein (GFAP) in rat retina after optic nerve transection. Rats were randomly divided into normal control group, sham group and operation group, and used for establishing an animal model of optic nerve transection. Retinal specimen of each group was collected at 3, 48h, 7 and 14d postoperative. Nestin and GFAP expressions on sagittal sections were analyzed by immunohistochemical staining, and protein extraction was analyzed by Western blot. Immunohistochemical analysis showed that nestin positive staining was rarely detected in normal control group and sham group, while sham group showed weak positive staining at 3h postoperative, the reaction gradually increased at 48h postoperative, and reached its maximum at 7d postoperative, and then decreased at 14d postoperative. Compared to the expression of GFAP, there was not statistically significant obvious difference among three groups (P>0.05). Result of Western blot method was consistent with that of immunohistochemical method. The expression of nestin increased in a time dependent fashion in Müller cells of retina following optic nerve transection, which was statistically significant, but there was no obvious difference in GFAP expression. The results indicate that an increase in colloid synthesis in retina following optic nerve transection can improve the retinal neurons' environment.

Experimental oral iron administration: Histological investigations and expressions of iron handling proteins in rat retina with aging

Iron is implicated in age-related macular degeneration (AMD). The aim of this study was to see if long-term, experimental iron administration with aging modifies retinal and choroidal structures and expressions of iron handling proteins, to understand some aspects of iron homeostasis. Male Wistar rats were fed with ferrous sulphate heptahydrate (500mg/kg body weight/week, oral; elemental iron availability: 20%) from 2 months of age onward until they were 19.5 month-old. At 8, 14 and 20 months of age, they were sacrificed and serum and retinal iron levels were detected by HPLC. Oxidative stress was analyzed by TBARS method. The retinas were examined for cell death (TUNEL), histology (electron microscopy) and the expressions of transferrin, transferrin receptor-1 [TFR-1], H- and L-ferritin. In control animals, at any age, there was no difference in the serum and retinal iron levels, but the latter increased significantly in 14- and 20 month-old iron-fed rats, indicating that retinal iron accumulation proceeds with progression of aging (>14 months). The serum and retinal TBARS levels increased significantly with progression of aging in experimental but not in control rats. There was significant damage to choriocapillaris, accumulation of phagosomes in retinal pigment epithelium and increased incidence of TUNEL+ cells in outer nuclear layer and vacuolation in inner nuclear layer (INL) of 20 month-aged experimental rats, compared to those in age-matched controls. Vacuolations in INL could indicate a long-term effect of iron accumulation in the inner retina. These events paralleled the increased expression of ferritins and transferrin and a decrease in the expression of TFR-1 in iron-fed rats with aging, thereby maintaining iron homeostasis in the retina. As some of these changes mimic with those happening in eyes with AMD, this model can be utilized to understand iron-induced pathophysiological changes in AMD.

Expression of glucose-regulated protein in rat retina with acute high intraocular pressure and its significance

Background The progressive death of retinal ganglion cells (RGCs) is the primary pathological characteristics of visual defects in glaucomatous eye.Research showed that endoplasmic reticulum stress (ERS) is involved in this progression.Glucose-regulated protein 78 ( GRP78 ) is a special marker of ERS.To understand the change in expression of GRP78 in the retina under the pressure induced is very important for the protection of visual function. Objective The present study was to observe the expression of GRP78 in rat retina with acute high intraocular pressure (IOP) and investigate the possible effect of ERS in acute glaucoma damage. Methods Fiftysix Wistar rats were randomly assigned to normal control group,anterior chamber punctured group and 12 hours,1 day,3,7,14 days groups following acute IOP rising.The acute high IOP models were established in the right eyes of 4.0 Wistar rats by paracentesis of the anterior chamber and infusion of normal saline solution into the anterior chamber.The histopathology changes of the retina were examined by hemotoxylin and eosin staining.The expression of GRP78 protein and mRNA in the retina were detected by immunohistochemistry and real-time fluorescence quantitative PCR. Results The retinal layers and cells were clear with normal alignement in the rats of the normal control group and the anterior chamber punctured group.The edema and thickening of retinas appeared in 12 hours after molding and peaked in 1 day after molding.Then the retina decreased in thickness and atrophied from 3 days through 14 days.The expression levels of GRP78 protein (A value) were 0.195±0.006 in the nounal rats and gradually increased from 12 hours through 3 days (0.268±0.017) following molding with the peak value at 1 day (0.499±0.039 ),showing significant differences in comparison with the normal control group (t =0.098,0.304,0.073,P＜0.05),and the reduction in expression at 7 and 14 days were not significantly different from the normal control group ( t =0.002,0.001,P＞0.05 ).The relative value of GRP78 mRNA ( 2△△C1 ) in the retina was 1.011 ±0.013 in the normal control group and gradually up-regulated from 12 hours through 3 days with the peak value at Iday (2.141±0.171 ) and then down-regulated from the third day onwards.A significant difference was seen in 2-△△C1value between the normal control group and 12 hours group,1 day group or 3 group ( t =0.525,1.130,0.409,P＜0.05 ).However,the 2-△△C1 values of GRP78 mRNA at 7 and 14 days was similar to that of the normal control group (t=0.020,0.004,P＞0.05). Conclusions GRP78 participates in the process of RGCs damage following acute high IOP.The results suggest that interfering with ERS may be helpful for protecting the retina and optical nerve from pressure-induced damage. Key words: Glaucoma/acute ocular hypertension, animal model; Glucose-regulated protein; Retinal ganglion cell; Endoplasmic reticulum stress

Expression of sigma receptor 1 mRNA and protein in rat retina

Sigma receptor (σR), known as a unique nonopiate, nonphencyclidine brain receptor, can bind diverse classes of psychotropic drugs, neurosteroids and other synthetic compounds, such as (+)pentazocine, etc. Two types of σRs have been identified: σR1 and σR2. In this work, we examined the expression of σR1 in rat retina by reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunofluorescence double labeling. RT-PCR analysis showed that σR1 mRNA was present in rat retina. Furthermore, labeling for σR1 was diffusely distributed in the outer and inner plexiform layers. The σR1-immunoreactivity (IR) was also observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina σR1 was expressed in all horizontal cells labeled by calbindin. In contrast, no σR1-IR was detected in several subtypes of bipolar cells, including rod-dominant ON-type bipolar cells, types 2, 3, 5 and 8 bipolar cells, labeled by protein kinase C (PKC), recoverin and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) respectively. In the inner retina, most of GABAergic amacrine cells, including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, expressed σR1. Some glycinergic amacrine cells were also labeled by σR1, but glycinergic AII amacrine cells were not labeled. In addition, σR1-IR was seen in almost all somata of the ganglion cells retrogradely labeled by fluorogold. These results suggest that σR1 may have neuromodulatory and neuroprotective roles in the retina.

Potassium channel Kir4.1 macromolecular complex in retinal glial cells

A major role for Müller cells in the retina is to buffer changes in the extracellular K+ concentration ([K+]o) resulting from light-evoked neuronal activity. The primary K+ conductance in Müller cells is the inwardly rectifying K+ channel Kir4.1. Since this channel is constitutively active, K+ can enter or exit Müller cells depending on the state of the [K+]o. This process of [K+]o buffering by Müller cells ("K+ siphoning") is enhanced by the precise accumulation of these K+ channels at discrete subdomains of Müller cell membranes. Specifically, Kir4.1 is localized to the perivascular processes of Müller cells in animals with vascular retinas and to the endfeet of Müller cells in all species examined. The water channel aquaporin-4 (AQP4) also appears to be important for [K+]o buffering and is expressed in Müller cells in a very similar subcellular distribution pattern to that of Kir4.1. To gain a better understanding of how Müller cells selectively target K+ and water channels to discrete membrane subdomains, we addressed the question of whether Kir4.1 and AQP4 associate with the dystrophin-glycoprotein complex (DGC) in the mammalian retina. Immunoprecipitation (IP) experiments were utilized to show that Kir4.1 and AQP4 are associated with DGC proteins in rat retina. Furthermore, AQP4 was also shown to co-precipitate with Kir4.1, suggesting that both channels are tethered together by the DGC in Müller cells. This work further defines a subcellular localization mechanism in Müller cells that facilitates [K+]o buffering in the retina.

Molecular cloning and characterization of a new neuron-specific homologue of rat polypyrimidine tract binding protein.

Cancer-associated retinopathy (CAR) is a rare form of retinal degeneration and also one of the paraneoplastic neurologic disorders. Sera of CAR patients usually contain high titers of antibodies against retinal proteins, and CAR is believed to be an autoimmune disease. Using serum from a CAR patient as a molecular probe, a homologue of the polypyrimidine tract-binding protein (PTB) was isolated from a cDNA library of rat neonatal retina. This homologue, named PTB-like protein (PTBLP), encodes a 532 amino acid residue protein and has 73.5 and 68.8% homology with PTB and with a regulator of differentiation 1, respectively. Functional domains in the PTB, such as nuclear localization signals and four RNA recognition motifs (RRMs), were highly conserved. The expression of PTBLP mRNA was observed in the retina and brain but not in liver, kidney, spleen, or lung. The expression of PTBLP protein in rat retina was distributed in most of the cells in the ganglion cell layer and some cells in the inner nuclear layer. The PTBLP protein was localized in the nuclei of these cells. These results suggest that PTBLP is a new member of the PTB gene family and a neuron-specific homologue.

Ciliary Neurotrophic Factor and Stress Stimuli Activate the Jak-STAT Pathway in Retinal Neurons and Glia

Ciliary neurotrophic factor (CNTF) is pleiotrophic for central, peripheral, and sensory neurons. In the mature retina, CNTF treatment enhances survival of retinal ganglion and photoreceptor cells exposed to otherwise lethal perturbation. To understand its mechanism of action in vivo, the adult rat retina was used as a model to investigate CNTF-mediated activation of Janus kinase/signal transducer and activator of transcription (Jak-STAT) and ras-mitogen activated protein kinase (ras-MAPK). Intravitreal injection of Axokine, an analog of CNTF, phosphorylates STAT3 and MAPK and produces delayed upregulation of total STAT3 and STAT1 protein in rat retina. Activated STAT3 is predominantly localized in nuclei of retinal Müller (glial) cells, ganglion cells, and astrocytes, but not in photoreceptors. Although CNTF alpha-receptor (CNTFRalpha) mRNA and protein are localized predominantly if not exclusively in retinal neurons, coincident CNTF-mediated STAT3 signaling was observed in both glia and neurons. CNTF-induced activation of Jak-STAT signaling prompted us to investigate STAT3 phosphorylation after a variety of stress-mediated, conditioning stimuli. We show that STAT3 is activated in the retina after exposure to subtoxic bright light, mechanical trauma, and systemic administration of the alpha(2)-adrenergic agonist xylazine, all of which have been shown previously to condition photoreceptors to resist light-induced degeneration. These results demonstrate that CNTF directly stimulates Jak-STAT and ras-MAPK cascades in vivo and strongly suggest that STAT3 signaling is an underlying component of neural responsiveness to stress stimuli. The observation that CNTF activates STAT3 in ganglion cells, but not in photoreceptors, suggests that Jak-STAT signaling influences neuronal survival via both direct and indirect modes of action.

Reticalmin: A novel calcium/calmodulin-dependent protein kinase IV-like protein in rat retina

Western blot analysis of 100000g supernatant of rat retina using a polyclonal anti-Ca 2+/ calmodulin-dependent protein kinase IV (CaM-kinase IV) antibody revealed an immunoreactive mass of 35 kDa, termed reticalmin. Lower amount of a isoform of CaM-kinase IV was also expressed in rat retina. Reticalmin did not react with anti-CaM-kinase IV C-terminal peptide antibody which recognized a and β isoforms of CaM-kinase IV and calspermin. Immunohisto-chemically reticalmin was shown to be localized mainly in the outer segment of photo-receptor cells, and in dendrites of inner plexiform layers and may be in nuclei of ganglion cells and some inner nuclear layer cells.

Immunolocalization of cellular retinol-, retinaldehyde- and retinoic acid-binding proteins in rat retina during pre- and postnatal development

Cellular retinol-, retinaldehyde- and retinoic acid-binding proteins were localized in rat retina during pre- and postnatal development by indirect immunofluorescence. Cryostat tissue sections were prepared daily from embryonic day 11 until the day of birth (E11-22) and from postnatal days 1-32 (P1-32). Cellular retinaldehyde- and retinol-binding proteins were first detected in retinal pigment epithelium on E13 and E18, respectively, and in Müller cells at P1 and P15. Parallel studies showed that in adult retina cellular retinoic acid-binding protein is present in a subpopulation of GABAergic amacrine cells. During retinal differentiation, cellular retinoic acid-binding protein was first detected at E18 in cells sclerad to the developing inner plexiform layer, suggesting that this binding protein is expressed in amacrine cells very early during differentiation. During early ocular morphogenesis, cellular retinoic acid-binding protein was present in mesenchymal cells enveloping the eye (E12-15), in the neuroblastic layer of the retina (E13-15), in the nerve fibre layer (E14-15), and the developing optic nerve (E15). Our results suggest that retinoic acid, the natural ligand of cellular retinoic acid-binding protein, may be involved in neuronal differentiation in the inner retina. The studies further support a role for cellular retinoic acid-binding protein in mediating the effects of retinoic acid on developing neural crest cells and raise new questions about the role of cellular retinaldehyde-binding protein in the visual cycle and during development.

Immunohistochemical localization of guanine nucleotide-binding protein in rat retina

The localization of a guanine nucleotide-binding protein, Go, in rat retina has been immunohistochemically examined by use of affinity-purified antibody against the alpha-subunit of Go. Dense Go-immunoreactive products are localized in the inner and outer plexiform layers which are considered as the region of synaptic interplay between neuronal components of the retina. Weak Go-immunoreactivity is also found in the neuropil of the ganglion cell layer and the inner nuclear layer. The selective enrichment of Go in synaptic zones of the retina suggests its major role in neuronal transmission within the retina.