Functional Analysis and Immunochemical Analyses of Ca²⁺ Homeostasis-Related Proteins Expression of Glaucoma-Induced Retinal Degeneration in Rats

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.

Blue-on-Green Flash Induces Maximal Photopic Negative Response and Oscillatory Potential and Serves as a Diagnostic Marker for Glaucoma in Rat Retina

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.

Nestin expressing progenitor cells during establishment of the neural retina and its vasculature / 대한해부학회지

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 Muller 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, Muller 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.

Immunochemical changes of calbindin, calretinin and SMI32 in ischemic retinas induced by increase of intraocular pressure and by middle cerebral artery occlusion / 대한해부학회지

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 Muller 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 Muller 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 Muller glial reaction, whereas MCAO retinas had only a small-scaled axonal transport disturbance.

Microvasculature in the Streptozotocin Induced Diabetic Rat Retina / 대한해부학회지

Diabetic retinopathy is characterized by the pericyte loss, microaneurysms and neovascularization eventually leads to blindness. The present study was examined changes of the microvasculature histochemically and immunochemically in the diabetic rat retina previously documented neuronal alterations, in order to verify the usefulness of the animal model of diabetes for the pathophysiology of angiogenesis. Diabetic condition was induced by a single intravenous injection of streptozotocin in Sprague-Dawley rats aged of 8weeks. The animals showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8, and 12 weeks, respectively. The retinas were processed for Griffonia simplicifolia isolection (GSI) B4 histochmistry, and anti-alpha-smooth muscle actin (alpha-SMA) and anti-NG2 immunochemical techniques. The retinal vasculature was well demarcated by endothelial profiles with GSIB4 histochemistry. alpha-SMA immunoreactivity appeared in the arterioles and the primary capillaries, and NG2 in the arterioles and the whole capillary beds. Changes evoked by diabetes were largely occurred in the capillary. Compared to the retina at normal state, the capillary networks were more complicated, enlarged, and dense. NG2 reactivity was reduced especially under the cytoplasmic processes of the pericytes. In the near periphery of the capillary mainly in the ganglion cell layer of the diabetes, GSIB4 reactive microglia were distributed. These results suggest that the retinal microvasculature showed the precedent events of neovascularization due to diabetes and rat model of diabetes is useful for study of neovascularization mechanism of the diabetic retinopathy.

Pre-synaptic Neuronal Changes of AII Amacrine Cells in the Streptozotocin-induced Diabetic Rat Retina / 대한해부학회지

It has been previously reported that parvalbumin expression was downregulated in AII amacrine cells, while upregulated in a subset of cone bipolar cells electrically synapse with AII amacrine cell in the streptozotocin-induced diabetic rat retina. In the present study, we aimed to trace biochemical changes of pre-synaptic neurons to AII amacrine cells in rat retina following diabetic injury. Diabetic condition was induced by streptozotocin injection into Sprague-Dawley rats aged of 8 weeks. The experimental term of induced diabetes was set at 1, 4, 12 and 24 weeks. Changes of pre-synaptic neurons were evaluated by immunohistochemistry and Western blot analysis with anti-protein kinase C (PKC)-alpha and anti-tyrosine hydroxylase (TH) antibodies. Rod bipolar cells immunolocalized with PKC-alpha antibody extended their enlarged axon terminals into stratum 5 of the inner plexiform layer. In later diabetes, the axon was shorten and its terminals of rod bipolar cell are slightly enlarged. The protein levels of PKC-alpha were slightly increased along with the duration of diabetes. TH immunoreactive neurons are morphologically classified into two subtypes of amacrine cells in the inner nuclear layer: one (type 1) has large soma with long and primary dendrites, classified with dopaminergic, and the other (type 2) has small soma with dendritic arborization. In the outermost inner plexiform layer, ring-like structures being composed of type 1 cell processes were densely distributed. In diabetic retina, the intensity of TH immunoreactivity in type 1 neurons was reduced. In accordance with morphological changes, the protein levels of TH were reduced during diabetes. These results demonstrate that TH immunoreactive dopaminergic amacrine cells are more susceptible to diabetic injury than the rod bipolar cells in the rat retina and may suggest that downregulation of parvalbumin expression in AII amacrine cells of diabetic retina is mainly due to dysfunction of pre-synaptic dopaminergic amacrine cells.

Altered Expression of Parvalbumin in Neural Retina of the Insulin-dependent Diabetic Rat / 대한해부학회지

Calcium-binding proteins in the nervous system are functioned in Ca2+ buffering and Ca2+ transport and regulation of various enzyme systems. They potentially have a number of different effects on cells includingaltering the duration of action potentials, promoting neuronal bursting activity and protecting cells against the damaging effects of excessive calcium influx. The present study has been designed to clarify the differential responding patterns of parvalbumin immunoreactive neurons in the rat retina following diabetic injury, for better understandings of role of parvalbumin in the retinal circuitry and in calcium homeostasis. Experimental diabetes was induced by a single intravenous injection of streptozotocin in a dose of 60 mg/kg body weight. Diabetic rats showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8, 12 and 24 weeks, respectively. The retinas at each time point were processed for immunohistochemistry and Western blotting using antiparvalbumin antibody. In the rat retina at normal, parvalbumin immunoreactivity appeared in AII amacrine cells, amacrine cells of a widefield type and displaced amacrine cells. A few bipolar cells are also showed the reactivity. During diabetes, the intensity of parvalbumin immunoreactivity is decreased especially in the AII amacrine cells. The cell number of parvalbumin immunoreactive neurons has showed no large changes throughout the diabetes, except that of bipolar cells. That population of parv immunoreactive of bipolar cells has increased remarkably at later diabetic periods. The protein levels of parvalbumin have showed transiently a slight increase at earlier diabetic periods, and then decreased to lower than that of normal. Parvalbumin immunoreactive bipolar cells at diabetes are co-localized not with PKC-alpha or recoverin, but with glutamate transporter Glt-1b. AII amacrine cell processes were joined with each other and with axon terminals of presumed cone bipolar cells by gap junction. These results suggest that the calcium buffering activity of parvalbumin is shifted from AII amacrine cells to a certain type of cone bipolar cells, in response to diabetes. This event may be occurred through electrically coupled gap junction in between these cell processes.

Expression of Substance P in the Experimental Diabetic Rat Retina / 대한해부학회지

Substance P (Sub P) being composed of 11 amino acids sequence is a kind of tachykinin family peptides. It has been known that this substance plays a role of neurotransmitter and/or neuromodulator and is a very potent vascular growth factor in the nervous system. This study has been investigated expression pattern of Sub P in the rat retina at normal and alteration of Sub P expression following diabetic injury using immunohistochemistry. Diabetic condition was induced by a single injection of streptozotocin in Sprague-Dawley rats aged 8 weeks. The animals showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8 and 12 weeks, respectively. The whole-mounted or sectional preparations of the retinas were used for Sub P immunohistochemistry. Sub P immunoreactivity has been localized in subsets of amacrine cells in the inner nuclear layer (INL) and displaced amacrine cells in the ganglion cell layer (GCL) in the normal retina. The dendrites from amacrine cells in the INL were ramified with strata 1 and 3, and those from displaced amacrine cells in the GCL with strata 5 of the inner plexiform layer. Sub P immunoreactive neurons in both the INL and the GCL were more densely populated in the superior half of the retina. During diabetes, the cell number of Sub P immunoreactive neurons was decreased to one third of the normal value at 4 weeks of diabetes and then slightly increased to half of the normal value at 12 weeks of diabetes. In addition, Sub P mRNA levels were reduced at 4 weeks but reincreased at 12 weeks. These results suggest that Sub P in the rat retina at normal state may function differentially in the superior or the inferior halves and Sub P synthetic pathway in the retinal neurons maybe irradiated in earlier stages of diabetic retinopathy.

Morphological Changes of the Dopaminergic Network in the Rat Retina after Axotomy / 대한해부학회지

In the retina, dopaminergic cells express the receptor for brain-derived neurotrophic factor (BDNF), which is known to be retrogradely transported from higher center to the retina. This study was conducted to identify the effect of optic nerve transaction on the dopaminergic cells in the rat retina by immunocytochemistry using antityrosine 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 (INL) 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. Our data suggest that retrogradely transported neurotrophic factor affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system.

Altered Expression of Metabotropic Glutamate Receptors mGluR1, mGluR5, and mGluR2/3 in the Diabetic Rat Retina / 대한해부학회지

An excitatory neurotransmitter glutamate is engaged in slow transmission by activating the secondary signal transduction pathway through metabotropic receptors of the target cells. The present study has been investigated the localization of group I (mGluR1 and mGluR5) and II (mGluR2/3) metabotropic glutamate receptors in the retina and their altered expression patterns following long-term diabetes using immunohistochemistry, in order to clarify the involvement of the slow transmission of glutamate in diabetic retinopathy. Insulin-dependent diabetes was induced by a single intravenous injection of streptozotocin. Experimental periods were set at 1, 4, 12 and 24 weeks after the onset of diabetes. MGluR1 and mGluR5 were expressed in the outer plexiform layer, 1, 3, and 5 strata of the inner plexiform layer, and the photoreceptor layer in the retina at normal state. In the early periods of diabetes, the expression pattern of group I receptors was no large change. The expression level in the photoreceptor layer of 12 and 24 weeks diabetic retinas was increased, while that in the IPL was decreased. MGluR2/3 was expressed in the amacrine cells, in the displaced amacrine cells, and in two bands in the inner plexiform layer at normal retina. In later diabetic periods, the expression level of mGluR2/3 was increased in the two bands especially. These results demonstrate that diabetes induces the activation of I and II mGluRs in the retina, and may suggest the involvement of slow transmission of glutamate via metabotropic receptors in progression of diabetic retinopathy.

Immunocytochemical Localization of GABA Transporter-3 in the Guinea Pig Retina / 대한해부학회지

The cellular localization of the GABA transporter-3 (GAT-3) was examined in the guinea pig retina by immunocytochemistry, using antisera against GAT-3. GAT-3 immunoreactivity was localized to cell bodies in the inner nuclear layer, and labeled processes were densely distributed in the inner plexiform layer (IPL) close to the ganglion cell layer. All GAT-3 labeled cells exhibited GAD65 immunoreactivity. In addition, 67% of GAT-3 labeled amacrine cells showed carbohydrate epitope CD15 immunoreactivity. These results indicate that GAT-3 is involved in modulating the rod pathway in the IPL of the guinea pig retina via presumptive A17 amacrine cells.

Altered Expression of GLAST and Glutamine Synthetase in the Streptozotocin-induced Diabetic Rat Retina / 대한해부학회지

Diabetic hyperglycemia induces transient ischemia in the neural retina. High level of extracellular glutamate resulting from ischemia, in turn, influences on glutamate homeostasis. The present study has been conducted to clarify the alteration of the glutamate homeostasis-associated substances in the retinal Muller cells in response to a diabetic injury by streptozotocin injection. Young adult Sprague -Dawley rats were injected with streptozotocin (60 mg/kg body weight in 0.05 M sodium citrate buffer, pH 5.5) under anesthesia with 4% chloral hydrate. Animals above 300 mg/dl in blood glucose level were cared for 1, 4, 12 and 24 weeks, respectively. At each time-point, the retinas were dissected out and processed for immuno-histochemical and immunoblotting analyses by using guinea pig anti -GLAST and mouse anti-glutamine synthetase (GS) antibodies. In the normal retina, GLAST and GS were immuno-localized in the Muller cells, the outer plexiform layer (OPL), the border between the inner nuclear layer and the inner plexiform layer (IPL), a band in the middle of the IPL, and the border between the IPL and the ganglion cell layer. The expression of both proteins was decreased remarkably in the OPL by 12 weeks of diabetes and increased slightly in the end feet of the Muller cells from 4 weeks onwards. Immunoblotting results of the two proteins in the diabetic retinas were largely consistent with those of immuno-histochemistry. These results suggest that the alteration of glutamate homeostasis in the diabetic state is initiated mainly in the OPL by decreasing the uptake of glutamate via down-regulated GLAST.

Distribution Pattern and Synaptic Circuitry of Cholinergic Neurons in the Rat Retina / 대한해부학회지

The role of acetylcholine as an excitatory neurotransmitter is well established, and cholinergic neurons appear to play an important role in the mammalian retinae. Though it has been reported that certain conventional and displaced amacrine cells are consistently labeled with anti-choline acetyltransferase antiserum in the mammalian retinae, little has been studied on the synaptic circuitry of cholinergic neurons to clarify mechanism of its action in the visual processing of the mammalian retinae. This study was conducted to localize cholinergic neurons and to define their synaptic circuitry in the rat retina by immunocytochemical method using anti-choline acetyltransferase antiserum. The results were as follows: 1. Cholinergic neurons of the rat retina were conventional amacrine cells located in the inner nuclear layer and displaced amacrine cells in the ganglion cell layer. 2. Cholinergic amacrine cells were branched in the middle of the sublamina a of the inner plexiform layer, and cholinergic displaced amacrine cells branched in the sublamina b, forming one prominent band, respectively. 3. Presynaptic processes to cholinergic amacrine cell processes were axon terminals of invaginating and flat cone bipolar cells, and unlabelled amacrine cell processes in the inner plexiform layer. Postsynaptic dyads at the ribbon synapses of axon terminals of cone bipolar cells were cholinergic amacrine cell process and dendrite of ganglion cell, cholinergic amacrine cell process and unlabelled amacrine cell process and cholinergic amacrine cell process and cholinergic amacrine cell process. In addition, cholinergic amacrine cell process formed postsynaptic monoad at the ribbon synapse. 4. Cholinergic amacrine cell processes made output conventional chemical synapses onto the dendrites of ganglion cells, unlabelled amacrine cell processes and cholinergic amacrine cell processes in the inner plexiform layer. These results demonstrate that (1) cholinergic neurons are conventional amacrine cells and displaced amacrine cells of which somata are located in the inner nuclear layer and ganglion cell layer, respectively, (2) cholinergic conventional amacrine cells are involved in OFF pathway, and cholinergic displaced amacrine cells play an important role in ON pathway in visual processing of lightness, and (3) acetylcholine released from cholinergic neurons by light excites directly ON and OFF ganglion cells or indirectly ON and OFF ganglion cells via non-cholinergic amacrine cells.

Synaptic Diversity in the Neostriatum of the Cat / 대한해부학회지

An attempt has been made to discriminate synaptic diversity in the neostriatum of the cat with emphasis on the characteristic structures of axon terminals and postsynaptic profiles. The differentiation of the axon terminals was based on the size and shape of synaptic vesicles in the axoplasm. Three types of axon terminals could be differentiated: Type I, the terminals contained small round (45 nm in diameter) vesicles; type II, the terminals with large pleomorphic (50 nm) vesicles; and type III, the terminals contained flattened (45 x 25 nm) vesicles. The type I terminals were making asymmetrical or symmetrical synapses in contact with the somata, dendrites and dendritic spines of neurons in the neostriatum, and a few type I terminals making asymmetrical or symmetrical contact with axons were also observed. The type II and III terminals were making symmetrical contact with the somata and dendrites of neostriatal neurons. A few type II terminals formed at the node of Ranvier of myelinated nerve fibers were making symmetrical contact with large dendrites. Additionally, dendro-dendritic and serial syanpses were rarely found in the neostriatum. In the serial synapses composed of axo-dendritic and dendro-dendritic synapses, the type I terminals making asymmetrical contact and the type II making symmetrical contact were identified.

Topographic distribution, ultrastructure and synaptic organization of dopaminergic neurons in the retina of redents II. ultrastructure and synaptic organization / 대한해부학회지

No abstract available.

Totpgraphic distribution. ultrastructure and synaptic organization of dopaminergic neurons in the retinae of rodents: I. topographic distribution / 대한해부학회지

No abstract available.

Lysozyme immunoreactivity of paneth cells of human, rat and mouse / 대한해부학회지

No abstract available.