RESUMO
The purpose of this study was to investigate the application of various electroretinography (ERG) to the diagnosis of inner retinal dysfunction induced by mild intraocular pressure (IOP) elevation in a rat glaucoma model. For inner retinal function measurements, available photopic ERG protocols were applied under various light conditions including monochromatic combinations, which complement conventional scotopic ERG. Three episcleral veins in the right eyes of Sprague-Dawley rats were cauterized to induce an experimental model of glaucoma, leading to mild IOP elevation. ERG responses were measured before surgery and at 1, 2, 4, and 8 weeks after cauterization. We first confirmed that the amplitude reduction in the standard photopic b-wave was almost comparable to the amplitudes of scotopic a- and b-waves in glaucomatous eyes over time. We have implemented additional photopic ERG protocols under different stimulus conditions, which consisted of a longer duration and different monochromatic combinations. Such a change in the stimulations resulted in more pronounced differences in response between the two groups. Especially in normal animals, blue stimulation on a green background produced the largest b-wave and photopic negative response (PhNR) amplitudes and caused more pronounced oscillatory potential (OP) wavelets (individual components). In glaucomatous eyes, blue stimulation on a green background significantly reduced PhNR amplitudes and abolished the robust OP components. These results, by providing the usefulness of blue on green combination, suggest the applicable photopic ERG protocol that complements the conventional ERG methods of accessing the progression of glaucomatous damage in the rat retina.
RESUMO
The retinal degeneration resulting from elevated intraocular pressure was evaluated through functional and morphological analyses, for better understanding of the pathophysiology of glaucoma. Ocular hypertension was induced via unilateral episcleral venous cauterization in rats. Experimental time was set at 1 and 3 days, and 1, 2, 4, and 8 weeks post-operation. Retinal function was analyzed using electroretinography. For morphological analysis, retinal tissues were processed for immunochemistry by using antibodies against the calcium-sensing receptor and calcium-binding proteins. Apoptosis was analyzed using the TUNEL method and electron microscopy. Amplitudes of a- and b-wave in scotopic and photopic responses were found to be reduced in all glaucomatous retinas. Photopic negative response for ganglion cell function significantly reduced from 1-day and more significantly reduced in 2-week glaucoma. Calcium-sensing receptor immunoreactivity in ganglion cells remarkably reduced at 8 weeks; conversely, protein amounts increased significantly. Calcium-binding proteins immunoreactivity in amacrine cells clearly reduced at 8 weeks, despite of uneven changes in protein amounts. Apoptosis appeared in both photoreceptors and ganglion cells in 8-week glaucomatous retina. Apoptotic feature of photoreceptors was typical, whereas that of ganglion cells was necrotic in nature. These findings suggest that elevated intraocular pressure affects the sensitivity of photoreceptors and retinal ganglion cells, and leads to apoptotic death. The calcium-sensing receptor may be a useful detector for alteration of extracellular calcium levels surrounding the ganglion cells.
RESUMO
PURPOSES: To evaluate changes in mean keratometry and to compare wound repair with corneal lamellar grafts in rabbit eyes using human synthetic tissue adhesives and 10-0 nylon. METHODS: Corneal grafts were made using a 6.0-mm-diameter trephine and blades in the eyes of 15 New Zealand white rabbits. Human fibrin tissue adhesive (Tisseel) was used in group 1, human fibrin tissue adhesive (Beriplast P) was used in group 2, polyethylene glycol adhesive (Coseal) was used in group 3, and 8 bite sutures with 10-0 nylon were used in group 4 (control) for lamellar keratoplasty. Four bite sutures were made with 10-0 nylon in groups 1, 2, and 3. Slit-lamp microscopy and keratometry were performed at 3 days and 1, 2, and 4 weeks after the surgery. Histopathologic and electromicroscopic examinations were performed 4 weeks after the surgery. RESULTS: No inflammation or corneal toxicity was seen in groups 1 and 2. Histologically, a few inflammatory cells were seen in groups 3 and 4. Groups 1, 2, and 3 showed no statistically significant changes in mean keratometry at 4 weeks postoperatively compared with preoperative mean keratometry (Wilcoxon signed-rank test, P = 0.178, 0.208, and 0.889, respectively). The control group showed significant changes in mean keratometry at 4 weeks postoperatively (Wilcoxon signed-rank test, P = 0.018). CONCLUSIONS: Human fibrin tissue adhesives were well tolerated in rabbit eyes, with no apparent corneal toxicity. Polyethylene glycol adhesive showed more inflammation and insufficient wound repair compared with human fibrin tissue adhesives. Therefore, human fibrin tissue adhesives can be used as an alternative to sutures in lamellar keratoplasty.
Assuntos
Transplante de Córnea/métodos , Adesivo Tecidual de Fibrina/uso terapêutico , Nylons , Polietilenoglicóis/uso terapêutico , Suturas , Adesivos Teciduais/uso terapêutico , Animais , Córnea/efeitos dos fármacos , Topografia da Córnea , Feminino , Adesivo Tecidual de Fibrina/toxicidade , Masculino , Polietilenoglicóis/toxicidade , Coelhos , Técnicas de Sutura , Adesivos Teciduais/toxicidade , CicatrizaçãoRESUMO
In order to test if nestin is a useful marker for various types of progenitor cells, we explored nestin expression in the retina during development. Nestin expression was co-evaluated with bromodeoxyuridine (BrdU) labeling and Griffonia simplicifolia isolectin B4 (GSIB4) histochemistry. Nestin immunoreactivity appears in cell soma of dividing neural progenitor cells and their leading processes in retinas from embryonic day (E) 13 to E20, in accordance with a BrdU-labeled pattern. At postnatal day (P) 5, it is restricted to the end feet of Müller cells. BrdU-labeled nuclei were mainly in the inner part of the inner nuclear layer in postnatal neonates. The retinal vessels demarcated with GSIB4-positive endothelial cells were first distributed in the nerve fiber layer from P3. Afterward the vascular branches sprouted and penetrated deeply into the retina. The endothelial cells positive for GSIB4 and the pericytes in the microvessels were additionally immunoreactive for nestin. Interestingly, the presumed migrating microglial cells showing only GSIB4 reactivity preceded the microvessels throughout the neuroblast layer during vascular sprouting and extension. These findings may suggest that nestin expression represents the proliferation and movement potential of the neural progenitor cells as well as the progenitor cells of the endothelial cell and the pericyte during retinal development. Thus, Müller glial cells might be potential neural progenitor cells of the retina, and the retinal microvasculature established by both the endothelial and the pericyte progenitor cells via vasculogenesis along microglia migrating routes sustains its angiogenic potential.
RESUMO
The reaction of neuroactive substances to ischemic conditions in the rat retina evoked by different methods was immunochemically evaluated in adult Sprague-Dawley rats. Ocular ischemic conditions were unilaterally produced by elevating intraocular pressure (EIOP) or by middle cerebral artery occlusion (MCAO). Two EF-hand calcium binding proteins, calbindin D28K (CB) and calretinin (CR), in the normal retina showed similar immunolocalization, such as the amacrine and displaced amacrine cells, the ganglion cells, and their processes, particularly CB in horizontal cells. CB immunoreactive neurons in the ganglion cell layer in both types of ischemic retinas were more reduced in number than CR neurons compared to those in a normal retina. The CB protein level in both ischemic retinas was reduced to 60-80% of normal. The CR protein level in MCAO retinas was reduced to about 80% of normal but increased gradually to the normal value, whereas that in the EIOP showed a gradual reduction and a slight recovery. SMI32 immunoreactivity, which detects a dephosphorylated epitope of neurofilaments-M and -H, appeared in the axon bundles of ganglion cells in the innermost nerve fiber layer of normal retinas. The reactivity in the nerve fiber bundles appeared to only increase slightly in EIOP retinas, whereas a moderate increase occurred in MCAO retinas. The SMI32 protein level in MCAO retinas showed a gradual increasing tendency, whereas that in the EIOP showed a slight fluctuation. Interestingly, the MCAO retinas showed additional SMI32 immunoreactivity in the cell soma of presumed ganglion cells, whereas that of EIOP appeared in the Müller proximal radial fibers. Glial fibrillary acidic protein (GFAP) immunoreactivity appeared in the astrocytes located in the nerve fiber layer of normal retinas. Additional GFAP immunoreactivity appeared in the Müller glial fibers deep in EIOP retinas and at the proximal end in MCAO retinas. These findings suggest that the neurons in the ganglion cell layer undergo degenerative changes in response to ischemia, although EIOP retinas represented a remarkable Müller glial reaction, whereas MCAO retinas had only a small-scaled axonal transport disturbance.
RESUMO
The AII amacrine cell, a unique rod signal integrator passing through the cone bipolar cell to ganglion cells, uses parvalbumin as a transducer of cytosolic calcium ion signals in the mammalian retina. For clarification of whether AII amacrine cell network contributes to the early neuropathogenesis of diabetic retinopathy, this study first analyzed alteration of parvalbumin expression in experimental diabetic retinas using immunohistochemical methods. Parvalbumin immunoreactivity was found in AII amacrine cells, some amacrine cells of a wide-field type, and displaced amacrine cells of the normal rat retina. During diabetes, cell density of each parvalbumin immunoreactive amacrine cell type showed no large changes despite decrease in immunoreactivity especially in AII amacrine cells. In addition to these parvalbumin immunoreactive amacrine cell types, a type of cone bipolar cells co-expressing glutamate transporter 1b and connecting electrically with AII amacrine cells appeared clearly by 4 weeks of diabetes, and thereafter sharply increased in number to that of AII amacrine cells. Protein levels of parvalbumin throughout the diabetic retinas also showed no large changes, except a transitional slight increase at 4 weeks of diabetes. These results suggest that the parvalbumin expression propagates from AII amacrine cells to a type of cone bipolar cell through electrical synapses due to dysfunction of biased mechanism in calcium ion buffering, caused by diabetic injury, and thus AII amacrine cells are closely involved in neuropathogenesis of ongoing diabetic retinopathy.
Assuntos
Células Amácrinas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Parvalbuminas/biossíntese , Células Bipolares da Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Amácrinas/ultraestrutura , Animais , Western Blotting , Imuno-Histoquímica , Microscopia Confocal , Ratos , Ratos Sprague-Dawley , Células Bipolares da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/ultraestruturaRESUMO
Mammalian neurons express the neural intermediate filament protein neurofilament (NF). In the retina, NFs have been detected primarily in the axons and processes of retinal ganglion and horizontal cells. We found an amacrine cell type that was immunolabeled with an antibody against SMI32, a non-phosphorylated epitope on neurofilament proteins of high molecular weight, in the mouse retina. This type of amacrine cell was non-randomly distributed, and these cells exhibited a central-peripheral density gradient. Most of these cells co-expressed GABA and ChAT, but not glycine or any other amacrine cell marker. These results suggest that some SMI32-immunoreactive amacrine cells belong to a GABAergic population, and that SMI32 can therefore be used as a marker for a subset of amacrine cells in addition to ganglion cells and horizontal cells in the mouse retina.
Assuntos
Células Amácrinas/metabolismo , Proteínas de Neurofilamentos/metabolismo , Retina/citologia , Animais , Colina O-Acetiltransferase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido gama-Aminobutírico/metabolismoRESUMO
Nitric oxide (NO) can play either a neuroprotective or a neurotoxic role in diverse neurodegenerative conditions. This study investigated the differential expression of neuronal nitric oxide synthase (nNOS) in the streptozotocin-induced diabetic rat retina to clarify the involvement of NO produced from neurons in the early pathogenesis of diabetic retinopathy. A decrease in thickness of the outer retina was evident at 12 and 24 weeks after onset of diabetes. nNOS was immunolocalized in two subtypes of amacrine cells, displaced amacrine cells and in some bipolar cells in the normal retinas. The densities of each type of nNOS-expressing neuron showed no significant differences in the diabetic retinas with the exception of the bipolar cells. The numbers of nNOS bipolar cells at 12 weeks of diabetes increased threefold, showing dendritic polarity of nNOS expression. Protein levels of nNOS increased throughout the diabetic retinas reaching a peak value at 24 weeks of diabetes. Thus, diabetes up-regulates the expression of nNOS in bipolar cells, and NO from these cells may aggravate the degeneration of the outer retina in the diabetic retinas.
Assuntos
Células Amácrinas/enzimologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Óxido Nítrico Sintase Tipo I/metabolismo , Células Bipolares da Retina/enzimologia , Células Amácrinas/patologia , Animais , Western Blotting , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-Dawley , Células Bipolares da Retina/patologia , Regulação para Cima/fisiologiaRESUMO
Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF), which is the neurotrophic factor that influences the plasticity of synapses in the central nervous system. We sought to determine whether BDNF influences the network of dopaminergic amacrine cells in the axotomized rat retina, by immunocytochemistry with an anti-tyrosine hydroxylase (TH) antiserum. In the control retina, we found two types of TH-immunoreactive amacrine cells, type I and type II, in the inner nuclear layer adjacent to the inner plexiform layer (IPL). The type I amacrine cell varicosities formed ring-like structures in contact with AII amacrine cell somata in stratum 1 of the IPL. In the axotomized retinas, TH-labeled processes formed loose networks of fibers, unlike the dense networks in the control retina, and the ring-like structures were disrupted. In the axotomized retinas treated with BDNF, strong TH-immunoreactive varicosities were present in stratum 1 of the IPL and formed ring-like structures. Our data suggest that BDNF affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dopamina/metabolismo , Retina/metabolismo , Células Amácrinas/química , Células Amácrinas/citologia , Células Amácrinas/metabolismo , Animais , Axotomia , Imuno-Histoquímica , Masculino , Parvalbuminas/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/química , Retina/citologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Using immunocytochemistry, a type of amacrine cell that is immunoreactive for aquaporin 1 was identified in the mouse retina. AQP1 immunoreactivity was found in photoreceptor cells of the outer nuclear layer (ONL) and in a distinct type of amacrine cells of the inner nuclear layer (INL). AQP1-immunoreactive (IR) amacrine cell somata were located in the INL and their processes extended through strata 3 and 4 of the inner plexiform layer (IPL) with thin varicosities. The density of the AQP1-IR amacrine cells increased from 100/mm(2) in the peripheral retina to 350/mm(2) in the central retina. The AQP1-IR amacrine cells comprise 0.5% of the total amacrine cells. The AQP1-IR amacrine cell bodies formed a regular mosaic, which suggested that they represent a single type of amacrine cell. Double labeling with AQP1 and glycine, gamma-aminobutyric acid (GABA) or GAD(65) antiserum demonstrated that the AQP1-IR amacrine cells expressed GABA or GAD(65) but not glycine. Their synaptic input was primarily from other amacrine cell processes. They also received synaptic inputs from a few cone bipolar cells. The primary synaptic targets were ganglion cells, followed by other amacrine cells and cone bipolar cells. In addition, gap junctions between an AQP1-IR amacrine process and another amacrine process were rarely observed. In summary, a GABAergic amacrine cell type labeled by an antibody against AQP1 was identified in the mouse retina and was found to play a possible role in transferring a certain type of visual information from other amacrine or a few cone bipolar cells primarily to ganglion cells.
Assuntos
Células Amácrinas/metabolismo , Aquaporinas/isolamento & purificação , Retina/citologia , Células Amácrinas/ultraestrutura , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Aquaporina 1 , Aquaporinas/metabolismo , Western Blotting/métodos , Contagem de Células/métodos , Colina O-Acetiltransferase/metabolismo , Glutamato Descarboxilase/metabolismo , Glicina/metabolismo , Imuno-Histoquímica/métodos , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Microscopia Imunoeletrônica/métodos , Retina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
We have investigated the expression and cellular localization of clusterin in the rat retina following ischemia induced by transiently increasing the intraocular pressure. In the normal retina, weak clusterin immunoreactivity was visible in Müller cell profiles located in the inner nuclear layer. Following ischemia and reperfusion, strong immunoreactivity appeared in Müller cell somata and processes up to 3 days postlesion. Quantitative evaluation by immunoblotting confirmed that clusterin expression continuously increased and showed a peak value at 3 days after ischemic injury (to 1300% of control levels), and then decreased again to 400% of controls at 4 weeks postlesion. Immunocytochemistry using antisera against clusterin or glutamine synthase combined with the TUNEL method or immunocytochemistry using antisera activated caspase 3 and electron microscopy revealed that some clusterin-labeled Müller cells underwent apoptotic cell death. Our findings demonstrate that some Müller cells die by apoptosis, and suggest that clusterin produced and released by Müller cell may play an important role in the pathogenesis of ischemic injury in the rat retina.
Assuntos
Glicoproteínas/metabolismo , Pressão Intraocular , Isquemia/metabolismo , Chaperonas Moleculares/metabolismo , Neuroglia/metabolismo , Retina/metabolismo , Doenças Retinianas/metabolismo , Animais , Caspase 3 , Caspases/metabolismo , Clusterina , Modelos Animais de Doenças , Glutamato-Amônia Ligase/metabolismo , Imuno-Histoquímica , Isquemia/fisiopatologia , Masculino , Microscopia Eletrônica , Neuroglia/ultraestrutura , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Retina/fisiopatologia , Retina/ultraestrutura , Doenças Retinianas/fisiopatologia , Regulação para CimaRESUMO
Disabled 1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by Reelin, which controls cell positioning in the developing brain. It has been localized to AII amacrine cells in the mouse and guinea pig retinas. This study was conducted to identify whether Dab1 is commonly localized to AII amacrine cells in the retinas of other mammals. We investigated Dab1-labeled cells in human, rat, rabbit, and cat retinas in detail by immunocytochemistry with antisera against Dab1. Dab1 immunoreactivity was found in certain populations of amacrine cells, with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smooth dendritic tree in the inner half of the IPL. Double-labeling experiments demonstrated that all Dab1-immunoreactive amacrine cells were immunoreactive to antisera against calretinin or parvalbumin (i.e., other markers for AII amacrine cells in the mammalian retina) and that they made contacts with the axon terminals of the rod bipolar cells in the IPL close to the ganglion cell layer. Furthermore, all Dab1-labeled amacrine cells showed glycine transporter-1 immunoreactivity, indicating that they are glycinergic. The peak density was relatively high in the human and rat retinas, moderate in the cat retina, and low in the rabbit retina. Together, these morphological and histochemical observations clearly indicate that Dab1 is commonly localized to AII amacrine cells and that antiserum against Dab1 is a reliable and specific marker for AII amacrine cells of diverse mammals.
Assuntos
Células Amácrinas/química , Proteínas do Tecido Nervoso/análise , Animais , Gatos , Contagem de Células/métodos , Humanos , Imuno-Histoquímica , Coelhos , Ratos , Ratos Sprague-Dawley , Proteína Reelina , Especificidade da EspécieRESUMO
The FVB/N mouse is a model of retinitis pigmentosa which shows a rapid loss of photoreceptors during early postnatal (P) life. We investigated the cellular localization of glycine transporter 1 (GlyT-1) in the developing FVB/N mouse retina. In control retinas, the developmental pattern of GlyT-1-immunoreactive amacrine cells was well in accordance with a previous report. However, in the FVB/N mouse retina, some GlyT-1-labeled amacrine cells sent their processes into the outer plexiform layer (OPL) from P14 onward. From P21 onward, GlyT-1-labeled cells were visible in the OPL. These cells were further characterized by double-label immunofluorescence experiments with an antiserum against disabled 1 (Dab-1), and showed Dab-1 immunoreactivity, indicating that these cells are putative AII amacrine cells. These results clearly demonstrate that AII amacrine cells have the potential capacity to respond to photoreceptor degeneration by migrating or sprouting their processes into the OPL in the developing FVB/N mouse retina.
Assuntos
Células Amácrinas , Coristoma/patologia , Retina/patologia , Doenças Retinianas/patologia , Retinose Pigmentar/patologia , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Animais Recém-Nascidos , Coristoma/metabolismo , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Proteínas da Membrana Plasmática de Transporte de Glicina , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Retina/crescimento & desenvolvimento , Doenças Retinianas/metabolismo , Retinose Pigmentar/metabolismoRESUMO
Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.
Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Imuno-Histoquímica , Lipoproteínas , Proteínas do Tecido Nervoso , Retina/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Western Blotting , Calbindinas , Proteínas de Ligação ao Cálcio/metabolismo , Carbocianinas/metabolismo , Células Cultivadas , Proteínas do Olho/metabolismo , Feminino , Junções Comunicantes/ultraestrutura , Cobaias , Hipocalcina , Masculino , Microscopia Confocal/métodos , Microscopia Imunoeletrônica/métodos , Recoverina , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteína delta-2 de Junções ComunicantesRESUMO
Disabled-1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by reelin, that controls cell positioning in the developing brain. It localizes to selected neurons in the nervous system, including the retina, and Dab1-like immunoreactivity is present in AII amacrine cells in the mouse retina. This study was conducted to characterize Dab1-labeled cells in the guinea pig retina in detail using immunocytochemistry, quantitative analysis, and electron microscopy. Dab1 immunoreactivity is present in a class of amacrine cell bodies located in the inner nuclear layer adjacent to the inner plexiform layer (IPL). These cells give rise to processes that ramify the entire depth of the IPL. Double-labeling experiments demonstrated that these amacrine cells make contacts with the axon terminals of rod bipolar cells and that their processes make contacts with each other via connexin 36 in sublamina b of the IPL. In addition, all Dab1-labeled amacrine cells showed glycine transporter 1 immunoreactivity, indicating that they are glycinergic. The density of Dab1-labeled AII amacrine cells decreased from about 3,750 cells/mm(2) in the central retina to 1,725 cells/mm(2) in the peripheral retina. Dab1-labeled amacrine cells receive synaptic inputs from the axon terminals of rod bipolar cells in stratum 5 of the IPL. From these morphological features, Dab1-labeled cells of the guinea pig retina resemble the AII amacrine cells described in other mammalian species. Thus, the rod pathway of the guinea pig retina follows the general mammalian scheme and Dab1 antisera can be used to identify AII amacrine cells in the mammalian retina.
Assuntos
Células Amácrinas/química , Proteínas do Tecido Nervoso/análise , Retina/química , Células Amácrinas/ultraestrutura , Animais , Feminino , Cobaias , Imuno-Histoquímica , Masculino , Microscopia Confocal , Microscopia Imunoeletrônica , Proteína Reelina , Retina/ultraestruturaRESUMO
This study investigated the expression and cellular localization of neuronal nitric oxide synthase in the rat retina following optic nerve transection (ONT). In the normal rat retina, nNOS immunoreactivity was localized to amacrine cells and displaced amacrine cells. A few bipolar cells were also labeled. In the axotomized retina, ganglion cells showed nNOS immunoreactivity from 3 days after ONT, and these cells increased in number, peaking 5 days after ONT. Quantitative evaluation using immunoblotting confirmed that nNOS expression showed a peak value (255% of control levels) 5 days after ONT and decreased to 137% of controls by 28 days. These findings suggest that axotomized ganglion cells degenerate via NO-mediated excitotoxicity.
Assuntos
Degeneração Neural/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Traumatismos do Nervo Óptico/enzimologia , Células Ganglionares da Retina/enzimologia , Células Amácrinas/citologia , Células Amácrinas/enzimologia , Animais , Axotomia , Contagem de Células , Corantes Fluorescentes , Imuno-Histoquímica , Masculino , Degeneração Neural/fisiopatologia , Óxido Nítrico Sintase Tipo I , Traumatismos do Nervo Óptico/fisiopatologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/citologia , Estilbamidinas , Regulação para Cima/fisiologiaRESUMO
Doublecortin is 40 kDa microtubule-associated phosphoprotein required for neuronal migration and differentiation in various regions of the developing central nervous system. We have investigated the expression and cellular localization of doublecortin in the developing rat retina using immunocytochemistry and Western blot analysis. The expression of doublecortin was high from embryonic day 18 (E18) until E20 and was low during the postnatal period. The doublecortin immunoreactivity first appeared in a few radially orientated cells in the mantle zone of the primitive retina at E15. From E16 onward, the immunoreactivity appeared in two different regions: the inner part of the retina and middle of the neuroblastic layer. In the inner part, the somata of cells in the ganglion cell layer, in the distal row of the neuroblastic layer and profiles in the inner plexiform layer showed doublecortin immunoreactivity up to postnatal day 1 (P1). Afterwards, the doublecortin immunoreactivity persisted in the inner plexiform layer until P15, although the intensity decreased gradually with the maturation of the retina. In the middle of the neuroblastic layer, doublecortin immunoreactivity appeared in the radially orientated cells. These cells transformed into horizontal cells. The doublecortin immunoreactivity persisted in these cells up to P21. Given these results, doublecortin may play an important role in the migration and differentiation of specific neuronal populations in developmental stages of the rat retina.
Assuntos
Proteínas Associadas aos Microtúbulos , Neuropeptídeos/biossíntese , Retina/embriologia , Retina/crescimento & desenvolvimento , Animais , Western Blotting , Calbindinas , Diferenciação Celular , Movimento Celular , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Imuno-Histoquímica , Neurônios/citologia , Neurônios/metabolismo , Ratos , Retina/citologia , Proteína G de Ligação ao Cálcio S100/biossíntese , Tubulina (Proteína)/biossínteseRESUMO
We investigated the effect of L-glutamate on horizontal cell growth after postnatal photoreceptor degeneration in the developing FVB/N mouse retina, using immunocytochemistry with antisera against calbindin D-28 K (calbindin) or neurofilament 200 NE14. The numbers of horizontal cells and amount of axonal arborization in the outer plexiform layer were unchanged in FVB/N mice injected with L-glutamate. Instead, more numerous processes emerging from horizontal cells descended into the inner plexiform layer (IPL) and formed a loose network in stratum 1. Our results clearly demonstrate that horizontal cells are resistant to excitotoxicity by excessive glutamate, and that sprouting of horizontal cell axons into the IPL is potentiated by excessive glutamate in FVB/N mice as they mature.
Assuntos
Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Retina/crescimento & desenvolvimento , Animais , Calbindinas , Contagem de Células , Ácido Glutâmico/toxicidade , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Proteínas de Neurofilamentos/análise , Neurônios/efeitos dos fármacos , Células Fotorreceptoras/patologia , Retina/efeitos dos fármacos , Proteína G de Ligação ao Cálcio S100/análiseRESUMO
Neuropeptide Y (NPY), an inhibitory neuropeptide expressed by a moderately dense population of wide-field amacrine cells in the rat retina, acts through multiple (Y1-y6) G-protein-coupled receptors. This study determined the cellular localization of Y1 receptors and the synaptic connectivity of Y1 processes in the inner plexiform layer (IPL) of the rat retina. Specific Y1 immunoreactivity was localized to horizontal cell bodies in the distal inner nuclear layer and their processes in the outer plexiform layer. Immunoreactivity was also prominent in cell processes located in strata 2 and 4, and puncta in strata 4 and 5 of the IPL. Double-label immunohistochemical experiments with calbindin, a horizontal cell marker, confirmed Y1 immunostaining in all horizontal cells. Double-label immunohistochemical experiments, using antibodies to choline acetyltransferase and vesicular acetylcholine transporter to label cholinergic amacrine cell processes, demonstrated that Y1 immunoreactivity in strata 2 and 4 of the IPL was localized to cholinergic amacrine cell processes. Electron microscopic studies of the inner retina showed that Y1-immunostained amacrine cell processes and puncta received synaptic inputs from unlabeled amacrine cell processes (65.2%) and bipolar cell axon terminals (34.8%). Y1-immunoreactive amacrine cell processes most frequently formed synaptic outputs onto unlabeled amacrine cell processes (34.0%) and ganglion cell dendrites (54.1%). NPY immunoreactivity in the rat retina is distributed primarily to strata 1 and 5 of the IPL, and the present findings, thus, suggest that NPY acts in a paracrine manner on Y1 receptors to influence both horizontal and amacrine cells.
Assuntos
Ratos , Receptores de Neuropeptídeo Y/análise , Retina/química , Transmissão Sináptica , Animais , Feminino , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/imunologia , Retina/fisiologiaRESUMO
The effects of L-arginine, a nitric oxide synthase (NOS) substrate, on cell proliferation in the developing postnatal rat retina were studied by immunocytochemistry using anti-bromodeoxyuridine (BrdU) antiserum, and by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Densitometric analysis by immunoblotting confirmed that neuronal NOS expression significantly increased in the L-arginine-treated retinas in comparison with the control retinas at postnatal day (P) 5 to P10. In the retinas of the control and L-arginine-treated rats, BrdU-labeled cells were only seen in the neuroblastic layer of the retinas up to P7. BrdU-labeled cells were significantly more numerous in the retinas of L-arginine-treated rats than in control retinas at P5 in the central retina and at P5 and P7 in the peripheral retina. In addition, TUNEL-positive apoptotic cells were more numerous in the retinas of L-arginine-treated rats at P5 and P7. Our results suggest that NO might play an important role in retinal maturation through regulating proliferative phases in the early stages of rat postnatal development.