Myung-Hoon Chun

Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina

Aims/hypothesisNeurodegenerative changes in the diabetic retina occurring before diabetic retinopathy could be inevitable by the altered energy (glucose) metabolism, in the sense that dynamic image-processing activity of the retinal neurons is exclusively dependent on glucose. We therefore investigated the morphological changes in the neural retina, including neuronal cell death, of a streptozotocin-induced model of diabetes.MethodsStreptozotocin was intravenously injected. Rats were maintained hyperglycaemic without insulin treatment for 1 week and 4, 8, 12, and 24 weeks, respectively. Diabetic retinas were processed for histology, electron microscopy, and immunohistochemistry using the TUNEL method.ResultsA slight reduction in the thickness of the inner retina was observed throughout the diabetic retinas and a remarkable reduction was seen in the outer nuclear layer 24 weeks after the onset of diabetes. The post-synaptic processes of horizontal cells in the deep invaginations of the photoreceptors showed degeneration changes from 1 week onwards. A few necrotic ganglion cells were observed after 4 weeks. At 12 weeks, some amacrine cells and a few horizontal cells showed necrotic features. Three to seven cellular layers in the outer nuclear layer and nerve terminals, rolled by the fine processes of the Müller cells near the somata of the degenerated ganglion cells, were apparent at 24 weeks. Apoptosis appeared in a few photoreceptor cells at 4 weeks, and the number of apoptotic photoreceptors increased thereafter.Conclusion/interpretationThese findings suggest that the visual loss associated with diabetic retinopathy could be attributed to an early phase of substantial photoreceptor loss, in addition to later microangiopathy.

Reaction of Müller cells after increased intraocular pressure in the rat retina

Abstract Using light microscopy and immunocytochemistry, we investigated the morphological changes of retinal tissues and the reaction of Müller cells in the ischemic rat retina induced by increasing intraocular pressure. At early stages (from 1 h to 24 h after reperfusion), cells in the ganglion cell layer and in the inner nuclear layer showed some degenerative changes, but at later stages (from 72 h to 4 weeks) marked degenerative changes occurred in the outer nuclear layer (ONL). At 4 weeks after reperfusion, the ONL was reduced to 1 or 2 cell layers. Immunoreactivity for glial fibrillary acidic protein (GFAP) appeared in the endfeet and distal processes of Müller cells as of 1 h after reperfusion. GFAP immunoreactivity in Müller cells increased up to 2 weeks and then decreased at 4 weeks after reperfusion. Our findings suggest that Müller cells are involved in the pathophysiology of retinal ischemia through the expression of GFAP. The degree of GFAP expression in Müller cells closely correlated with that of the degeneration of retinal neurons.

The Synaptic Complex of Cones in the Fovea and in the Periphery of the Macaque Monkey Retina

Parallel pathways for visual information processing start at the first synapse of the retina, at the cone pedicle. At least eight different types of bipolar cells receive direct synaptic input from an individual cone. The present study explores whether enough synaptic sites are available at the cone pedicle to supply all these bipolar cells. Monkey retinae were optimally fixed for electron microscopy. Serial horizontal sections were cut through the cone pedicle layer in a piece close to the fovea (eccentricity: 0.75 mm) and in a peripheral piece (eccentricity: 5-6 mm). The ribbon synapses (triads) at the cone pedicle base were analysed. The average number of synaptic ribbons per cone pedicle increased from 21.4 +/- 1.6 (n = 26) in central retina to 41.8 +/- 3 (n = 14) in peripheral retina. Five central and five peripheral pedicles were reconstructed and the invaginating bipolar cell dendrites forming the central elements of the triads were characterized. Close to the fovea an average of 18 invaginating bipolar cell dendrites was found, in peripheral retina the average was 90. Pedicles of foveal cones have one invaginating central process per ribbon, pedicles of peripheral cones have two. It is possible that midget bipolar cell dendrites occupy the majority of triads in the fovea, while in peripheral retina both midget and diffuse bipolar cell dendrites share the triads.

Changes in retinal neuronal populations in the DBA/2J mouse

DBA/2J (D2) mice develop a form of progressive pigmentary glaucoma with increasing age. We have compared retinal cell populations of D2 mice with those in control C57BL/6J mice to provide information on retinal histopathology in the D2 mouse. The D2 mouse retina is characterized by a reduction in retinal thickness caused mainly by a thinning of the inner retinal layers. Immunocytochemical staining for specific inner retinal neuronal markers, viz., calbindin for horizontal cells; protein kinase C (PKC) and recoverin for bipolar cells, glycine, γ-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and nitric oxide synthase (NOS) for amacrine cells, and osteopontin (OPN) for ganglion cells, was performed to detect preferentially affected neurons in the D2 mouse retina. Calbindin, PKC, and recoverin immunoreactivities were not significantly altered. Amacrine cells immunoreactive for GABA, ChAT, and OPN were markedly decreased in number, whereas NOS-immunoreactive amacrine cells increased in number. However, no changes were observed in the population of glycine-immunoreactive amacrine cells. These findings indicate a significant loss of retinal ganglion and some amacrine cells, whereas glycinergic amacrine cells, horizontal, and bipolar cells are almost unaffected in the D2 mouse. The reduction in amacrine cells appears to be attributable to a loss of GABAergic and particularly cholinergic amacrine cells. The increase in nitrergic neurons with the consequent increase in NOS and NO may be important in the changes in the retinal organization that lead to glaucomain D2 mice. Thus, the D2 mouse retina represents a useful model for studying the pathogenesis of glaucoma and mechanisms of retinal neuronal death and for evaluating neuroprotection strategies.

Expression of vascular endothelial growth factor mRNA following transient forebrain ischemia in rats

Using a reproducible two-vessel occlusion model for forebrain ischemia in rats, we investigated the temporal and spatial changes of vascular endothelial growth factor (VEGF) expression after transient forebrain ischemia with Northern blot analysis and in situ hybridization. Northern blot analysis revealed that VEGF mRNA of the hippocampus was increased from 12 h after reperfusion, with a peak at 1 day. In situ hybridization and double labeling for VEGF mRNA and glial fibrillary acidic protein showed a transient induction of VEGF mRNA in the neurons of the hippocampus from 12 h of reperfusion with a peak at 1 day, and in the astrocytes of the hippocampus, caudoputamen, thalamus and cortical regions at 1 day. After 3 days, no more VEGF signal was detected. Our results demonstrate that astrocytes and neurons each upregulate VEGF mRNA in different temporal and spatial patterns after transient forebrain ischemia in the rat, and these patterns appear to be different from those in transient focal cerebral ischemia.

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

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.

Upregulation of ciliary neurotrophic factor in reactive Müller cells in the rat retina following optic nerve transection.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following optic nerve transection. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following optic nerve transection, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between three and seven days post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression increased continuously up to 7 days after optic nerve transection (to 430% of control levels), but decreased again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells may play a protective role for axotomized ganglion cells in the rat retina.

Upregulation of gp130 and STAT3 activation in the rat hippocampus following transient forebrain ischemia

To determine whether the pathophysiological processes after transient forebrain ischemia are mediated via a signal pathway involving gp130 (a signal transducer for the interleukin‐6 family), we analyzed changes in the expression of gp130 and its downstream transcription factor, signal transducer and activator of transcription factor 3 (STAT3), in the rat hippocampus of a four‐vessel occlusive ischemia model. Expression of gp130 mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Four hours after ischemic injury, astrocytes expressed gp130 mRNA. Expression of gp130 increased preferentially in the CA1 and dentate hilar regions, and was maintained for at least 2 weeks. Increase in gp130 expression was accompanied by the activation of STAT3 following ischemic injury. Four hours after injury, STAT3 and phosphorylated STAT3 (pSTAT3) were observed in the nuclei of the dentate hilar region, and sequentially in the CA1 region at day 1. By day 3, STAT3 immunoreactivity markedly increased in these areas, where small cells with the morphology of astrocytes showed nuclear and cytoplasmic STAT3 and nuclear pSTAT3 immunoreactivities. These patterns were especially maintained in the CA1 area until 14 days of reperfusion. Double‐labeling experiments revealed that the cells expressing STAT3 and pSTAT3 were glial fibrillary acidic protein‐expressing reactive astrocytes. These results show a coordinated and long‐lasting upregulation of gp130 mRNA and STAT3 activation in reactive astrocytes of the postischemic hippocampus, indicating that they may be involved in the astrocytic response to an ischemic insult. GLIA 41:237–246, 2003.

Expression of CNTF in Müller cells of the rat retina after pressure-induced ischemia.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following ischemia induced by transiently increasing the intraocular pressure. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following ischemia and reperfusion, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between 1 day and 2 weeks post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression continuously increased up to 2 weeks after ischemic injury (to 600% of control levels), but had declined again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells has a protective function for lesioned neurons following transient ischemia.

Upregulation of phospholipase D in astrocytes in response to transient forebrain ischemia

Previous in vitro studies using cell cultures or brain slices have demonstrated that phospholipase D (PLD) in the nervous system is involved in the signaling mechanism in response to a variety of agonists. However, little is known about the pathophysiological role of PLD‐mediated signaling in the adult brain. We examined the changes in the expression of a PLD isozyme, PLD1, in the adult rat hippocampus, using immunological approaches and an assay for PLD activity after transient forebrain ischemia (four‐vessel occlusion model) that results in the selective delayed death of CA1 pyramidal cells and induces reactive astrocytes in the CA1 subfield. In the control hippocampus, PLD1 the level of immunoreactivity was very low. After ischemia, in parallel with the results of Western blot analysis and the PLD activity assay, immunohistochemical analysis of PLD1 demonstrated that the immunoreactive proteins peaked at 7–14 days and were most prominent in the CA1 and the dentate hilar region. The temporal and spatial patterns of immunoreactivity of both PLD1 and glial fibrillary acidic protein (GFAP) were very similar, indicating that reactive astrocytes express PLD1, confirmed by double staining for PLD1 and GFAP. These results demonstrate that reactive astrocytes upregulate PLD in vivo after injury in the adult rat hippocampus. GLIA 30:311–317, 2000.