Won-Kyu Ju

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.

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.

Immunocytochemical localization of nitric oxide synthase in the mammalian retina

The localization of nitric oxide synthase (NOS) was investigated by immunocytochemistry and immunoblotting using an antiserum against neuronal NOS in the rat, mouse, guinea pig, rabbit and cat retinae. Western blot analysis of retinal tissue extracts showed that the NOS-immunoreactive band of 155 kDa was present in all species. In the rat, mouse, guinea pig and rabbit retinae, two types of amacrine cells and a class of displaced amacrine cells were consistently NOS-labeled. In the cat retina, unlike other mammals, one type of amacrine cells and two types of displaced amacrine cells showed NOS immunoreactivity. NOS immunoreactivity was further found in some bipolar cells of the rat and guinea pig, some interplexiform cells of the mouse, some photoreceptor cells of the rabbit and some Müller cells of the cat.

Nitric oxide is involved in sustained and delayed cell death of rat retina following transient ischemia

We have investigated the role of nitric oxide (NO) in the rat retina following ischemic injury induced by transient increase of intraocular pressure. The thickness of both the inner plexiform layer and inner nuclear layer decreased during early postischemic stages (up to 1 week). In late postischemic stages (2-4 weeks), the thickness of the outer nuclear layer (ONL) decreased markedly. Thus, mechanisms other than excitotoxic ones may contribute to postischemic retinal cell death. Treatment of rats with N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, significantly reduced ischemic damage. Our findings suggest that NO is involved in the mechanism of ischemic injury, and plays a key role in the delayed and sustained cell death in the ONL following transient retinal ischemia.

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus following transient global ischemia

To investigate a potential role of ciliary neurotrophic factor (CNTF) in transient global ischemia, we have studied the postischemic regulatory changes in the expression of CNTF and its receptor, the ligand-binding alpha-subunit (CNTFRalpha). Immunoblot analysis demonstrated CNTF levels were slightly upregulated already during the first day after ischemia and then increased markedly by more than 10-fold until 2 weeks postischemia. Immunoreactivity for CNTF became detectable 1 day after ischemia and was localized in reactive astrocytes. The intensity of the immunolabeling was maximal in CA1 during the phase of neuronal cell death (days 3-7 postischemia) and in the deafferented inner molecular layer of the dentate gyrus. Upregulation of CNTF expression was less pronounced in CA3 and absent in the stratum lacunosum moleculare and the outer molecular layer of the dentate gyrus and thus did not simply correlate with astroliosis as represented by upregulation of glial fibrillary acidic protein (GFAP). As shown by in situ hybridization, expression of CNTFRalpha mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Following ischemia, reactive astrocytes, identified by double labeling with antibodies to GFAP, transiently expressed CNTFRalpha mRNA with a maximum around postischemic day 3. This astrocytic response was most pronounced in CA1 and in the hilar part of CA3. These results show that CNTF and its receptor are differentially regulated in activated astrocytes of the postischemic hippocampus, indicating that they are involved in the regulation of astrocytic responses and the neuronal reorganizations occurring after an ischemic insult.

Ganglion cells of the rat retina show osteopontin-like immunoreactivity

Osteopontin (OPN) is a negatively charged, highly acidic glycosylated phosphoprotein that contains an GRGDS amino acid sequence, characteristic of proteins that bind to integrin receptors, thereby playing crucial roles in a number of physiological processes. This study was conducted to examine the expression of OPN in the rat retina by Northern blot analysis, Western blot analysis and immunocytochemistry. The expression of OPN was identified in the retina and OPN-like immunoreactivity was present in a number of ganglion cells. Thus, OPN appears to be important in the retinal homeostasis.

Increased expression of ciliary neurotrophic factor receptor α mRNA in the ischemic rat retina

Abstract Using in situ hybridization, we investigated the expression of ciliary neurotrophic factor receptor ((CNTFRα) mRNA in the rat retina rendered ischemic by elevation of the intraocular pressure (IOP). The IOP was increased to 120 mmHg and maintained for 60 min. The rats were sacrificed on the day of reperfusion (DRP) 1, 3, 7, 14, and 28. In the normal retina, the signal for CNTFRα mRNA was present in retinal cells in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). On DRP 1, numerous cells in the INL and GCL showed a CNTFRα mRNA signal. From DRP 3 onwards, CNTFRα mRNA appeared in photoreceptor cells located in the outer part of the outer nuclear layer. The signal in these cells increased up to DRP 14 and then decreased at DRP 28. Our findings suggest that cells expressing CNTFRα mRNA may resist the degenerative processes induced by ischemic insult in the rat retina.

Expression of brain/kidney protein in Müller cells of rat retina following transient ischemia

We have investigated the expression and cellular localization of brain/kidney (B/K) protein in the rat retina following transient ischemia. In the normal retina, strong B/K immunoreactivity was localized to some ganglion cells. In addition, a few radial Muller cell processes showed B/K immunoreactivity. Following ischemia and reperfusion, most B/K-labeled ganglion cells were lost, whereas between 1 day and 2 weeks post-lesion B/K immunoreactivity appeared in many more Muller cell processes with increasing intensity. Quantitative evaluation by immunoblotting confirmed that B/K expression then decreased progressively, to 35% of control values at four weeks post-lesion. Our findings suggest that Muller cells are involved in the pathophysiology of retinal ischemia through the expression of B/K following transient ischemia.

The expression and cellular localization of brain/kidney protein in the rat retina.

Brain/kidney (B/K) protein is a new protein of 474 amino acids, which contains two C2 domains structurally homologous to those present in synaptotagmins. The expression of B/K protein was identified in the rat retina and B/K protein immunoreactivity was localized to a number of ganglion cells, a few amacrine cells and the radial processes of Müller cells. Thus, B/K protein appears to be important in the homeostasis in these cells of the rat retina.