Inhibition of Foxp4 Disrupts Cadherin-based Adhesion of Radial Glial Cells, Leading to Abnormal Differentiation and Migration of Cortical Neurons in Mice / 神经科学通报·英文版

Heterozygous loss-of-function variants of FOXP4 are associated with neurodevelopmental disorders (NDDs) that exhibit delayed speech development, intellectual disability, and congenital abnormalities. The etiology of NDDs is unclear. Here we found that FOXP4 and N-cadherin are expressed in the nuclei and apical end-feet of radial glial cells (RGCs), respectively, in the mouse neocortex during early gestation. Knockdown or dominant-negative inhibition of Foxp4 abolishes the apical condensation of N-cadherin in RGCs and the integrity of neuroepithelium in the ventricular zone (VZ). Inhibition of Foxp4 leads to impeded radial migration of cortical neurons and ectopic neurogenesis from the proliferating VZ. The ectopic differentiation and deficient migration disappear when N-cadherin is over-expressed in RGCs. The data indicate that Foxp4 is essential for N-cadherin-based adherens junctions, the loss of which leads to periventricular heterotopias. We hypothesize that FOXP4 variant-associated NDDs may be caused by disruption of the adherens junctions and malformation of the cerebral cortex.

Flow Shear Stress Enhances the Proliferative Potential of Cultured Radial Glial Cells Possibly Via an Activation of Mechanosensitive Calcium Channel

Radial glial cells (RGCs) which function as neural stem cells are known to be non-excitable and their proliferation depends on the intracellular calcium (Ca²⁺) level. It has been well established that Inositol 1,4,5-trisphosphate (IP3)-mediated Ca²⁺ release and Ca²⁺ entry through various Ca²⁺ channels are involved in the proliferation of RGCs. Furthermore, RGCs line the ventricular wall and are exposed to a shear stress due to a physical contact with the cerebrospinal fluid (CSF). However, little is known about how the Ca²⁺ entry through mechanosensitive ion channels affects the proliferation of RGCs. Hence, we hypothesized that shear stress due to a flow of CSF boosts the proliferative potential of RGCs possibly via an activation of mechanosensitive Ca²⁺ channel during the embryonic brain development. Here, we developed a new microfluidic two-dimensional culture system to establish a link between the flow shear stress and the proliferative activity of cultured RGCs. Using this microfluidic device, we successfully visualized the artificial CSF and RGCs in direct contact and found a significant enhancement of proliferative capacity of RGCs in response to increased shear stress. To determine if there are any mechanosensitive ion channels involved, a mechanical stimulation by poking was given to individual RGCs. We found that a poking on radial glial cell induced an increase in intracellular Ca²⁺ level, which disappeared under the extracellular Ca²⁺-free condition. Our results suggest that the shear stress by CSF flow possibly activates mechanosensitive Ca²⁺ channels, which gives rise to a Ca²⁺ entry which enhances the proliferative capacity of RGCs.

Effects of Histone Deacetylase Inhibitor (Valproic Acid) on the Expression of Hypoxia-inducible Factor-1 Alpha in Human Retinal Müller Cells

PURPOSE: To evaluate the effects of valproic acid (VPA), a histone deacetylase inhibitor (HDACI), on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in human retinal Müller cells under hypoxic conditions. METHODS: Chemical hypoxia was induced in human retinal Müller cells (MIO-M1) by treatment with increasing concentrations of cobalt(II) chloride (CoCl₂). Müller cells were also treated with a set concentration of CoCl₂, along with various concentrations of VPA. The expression of HIF-1α and VEGF in the treated Müller cells was determined by enzyme-linked immunosorbent assay. RESULTS: Exposure of human retinal Müller cells to increasing concentrations of CoCl₂ produced a dose-dependent increase in HIF-1α expression. The addition of increasing concentrations of VPA lead to a dose-dependent decrease in expression of HIF-1α and VEGF in Müller cells exposed to a set concentration of CoCl₂. CONCLUSIONS: HDACI VPA downregulated the expressions of HIF-1α and VEGF in human retinal Müller cells under hypoxic conditions. Using HDACI to target HIF-1α expression in Müller cells could be a new therapeutic strategy for the treatment of retinal vascular diseases.

Análise imunocitoquímica e de expressão gênica de efeitos do bevacizumabe em explantes de retina de ratos lister e em linhagem celular de glia de Müller humana

INTRODUÇÃO: As doenças retinianas associadas à neovascularização, tais como a degeneração macular relacionada à idade e as retinopatias diabética e da prematuridade são as principais e mais importantes causas da cegueira em todo o mundo. Nos últimos anos, injeções intravítreas de fármacos com ação antiangiogênica, como o bevacizumabe (BVZ), têm sido de grande valia tanto em pacientes na fase adulta quanto nos recém-natos. Todavia, estudos experimentais in vitro e in vivo sugerem que essas drogas promovam efeitos adversos sobre alguns processos celulares, interferindo diretamente em mecanismos fisiológicos que mantém a homeostase do tecido retiniano, incluindo os mecanismos de proliferação, diferenciação e morte celular. OBJETIVO: investigar o efeito do BVZ nos processos de transcrição e tradução de marcadores da gliose: GFAP e vimentina, de morte celular, caspase-3 e beclina-1, e dos proteoglicanos relacionados à manutenção e desenvolvimento de tecido retiniano: neurocam, fosfacam e sindecam-3. MÉTODOS: Dois modelos experimentais foram usados nesse estudo: 1) linhagem celular de Müller de Glia humana adulta (MIO-M1), cultivada em meio de cultura D-MEM na presença e ausência de BVZ por 12 e 24 horas nas concentrações de 0,25 mg/mL e 0,50 mg/mL e 2) explantes de retinas de ratos 2 dias pós-nascidos submetidos à 0,50 mg/mL da droga por 48 horas. Durante este período foram mantidos a 5% de dióxido de carbono à temperatura de 37°C. A análise de proteínas foi realizada por imunocitohistoquímica e Western Blotting e a expressão de RNAm, pela reação em cadeia da polimerase em tempo real (PCR Real Time). Foi utilizado o Teste de ANOVA - fator único para a comparação entre os grupos controle e tratados com BVZ de um mesmo período (12h ou 24h) e o teste t de Student para a comparação entre as mesmas concentrações de 12h e 24h, e para a comparação entre os grupos controle e tratado com BVZ dos explantes (p < 0,05). RESULTADOS...

Backgraound: Vasoproliferative retinal disorders such as age-related macular, degeneration, diabetic retinopathy and retinopathy of prematurity are major causes of blindness in the world. In recent years, intravitreal injections of drugs with antiangiogenic action, as bevacizumab, have been very useful for both patients in adulthood and in newborns. However, experimental studies, in vivo and in vitro, suggest that antiangiogenic drugs may promote side effects in cellular proceedings, interfering directly in physiological mechanisms of cellular proliferation, differentiation and death. POURPOSE: Investigate the bevacizumab effects in transcription and translation processes of gliosis, GFAP and vimentin, cellular death markers, caspase-3 and beclin-1, and proteoglycans involved in retinal tissue maintenance and development, neurocan, phosphacan and syndecan-3. METHODS: Two experimental models were used on this research: cellular lineage of adult and human Müller glial cell(MIO-M1) were cultivated on D-MEM medium with 0,25 and 0,50 mg/mL bevacizumab for 12 and 24 hours, and two days old rat retinal explants submitted to 0,50 mg/mL for 48 hours. During this period were stored in laboratory ovens at 5% carbon dioxide pressure and 37 °C average temperature. Molecular techniques were used to evaluate gene expression and protein content. Protein assessments were performed by immunocytochemistry and western blotting analysis, while Real Time PCR was used to measure mRNA content. ANOVA tests one factor were applied to compare the control and BVZ groups of the same period (12h or 24h) and t test from Student to compare the same conditions of 12h and 24h, and to compare the control and BVZ retinal explants groups (p<0.05). RESULTS...

Effect of Bushen Huoxue Compound on Retinal Müller Cells in High Glucose or AGEs Conditions / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of Bushen Huoxue Compound (BHC) on lactate dehydrogenase (LDH) leakage, expressions of vascular endothelial growth factor (VEGF) and VEGF mRNA in retinal Muller cells under high glucose condition or advanced glycosylation end products (AGEs) condition by using serum pharmacological method.</p><p><b>METHODS</b>The retinal Müller cells of 5-7 days post-natal Sprague Dawley (SD) rats were cultured with modified enzyme-digestion method. Purified retinal Muller cells were cultured in normal conditions, high glucose condition (50 mmol/L) or AGEs (50 mg/L and 100 mg/L) conditions, and BHC-containing serum was added to culture medium. The LDH leakage and VEGF expressions were measured by enzyme-linked immunosorbent assay (ELISA). In addition, the relative expression of VEGF mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Compared with the normal control group, expressions of VEGF and VEGF mRNA were significantly increased in the high glucose group, the low dose AGEs group and the high dose AGEs group (all P < 0.01). The LDH leakage was obviously increased in the high dose AGEs group, when compared with the normal control group and the high glucose group (P < 0.01). The LDH leakage, expressions of VEGF and VEGF mRNA were obviously decreased by BHC-containing serum both in high glucose and AGEs conditions (P < 0.05, P < 0.01). BHC-containing serum had no significant effect on the LDH leakage and expressions of VEGF and VEGF mRNA in normal conditions (P > 0.05).</p><p><b>CONCLUSIONS</b>AGEs intervention could obviously lower the stability of Müller cell membrane. Up-regulated expressions of VEGF and VEGF mRNA in cultured Müller cells could be induced by AGEs or high glucose. BHC-containing serum could stabilize the stability of Müller cell membrane, inhibit the transcription of VEGF mRNA and decrease the protein expression of VEGF, which might be one of important mechanisms for preventing and treating diabetic retinopathy.</p>

Roles of retinal Müller cells in health and glaucoma / 生理学报

In the vertebrate retina, Müller cells are principal glial cells which stretch across the whole thickness of the retina and contact with the somata and processes of all retinal neurons, thus forming an anatomical and functional link between glial cells and retinal neurons. Numerous studies have shown that Müller cells express various neurotransmitter receptors, transporters, ion channels and enzymes that are relative to cellular activities. In addition, the cells also release factors, such as D-serine and glutamate etc., to regulate the neuron excitability. Therefore, retinal Müller cells may play more curious roles in addition to supporting the retinal neurons. The information exchange and interaction between Müller cells and neurons may regulate and maintain retinal neuronal functions. In the glaucomatous retina, Müller cells are reactivated (gliosis). Reactivated Müller cells undergo a variety of changes in cellular physiology, biochemistry and morphological features. Meanwhile, the reactivated Müller cells may produce and release cytotoxic factors, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), reactive oxygen species (ROS) and prostaglandin E2 (PGE2), thus involving in the induction of retinal ganglion cell apoptosis and death. Here, we reviewed the physiological properties of retinal Müller cells, and the functional changes of Müller cells in the glaucomatous retina.

Effects of avastin on expression of AQP4 in Müller cells under hypoxia / 华中科技大学学报（医学）（英德文版）

The aim of this study was to investigate the effects of Avastin on aquaporin4 (AQP4) expression in human retinal Müller cells in vitro under hypoxia, so as to explore the mechanism of Avastin treating retinal edema. The human Müller cells were cultured using the enzymatic digestion method. Müller cells were identified under the transmission electron microscopy and by using immunofluorescence staining. By using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the expression of AQP4 mRNA and VEGF mRNA in Müller cells cultured with 500 μmol/L CoCl(2) for 0, 3, 6, 12 and 24 h, and with 0, 100, 300, 500 and 700 μmol/L CoCl(2) for 24 h was detected. The expression of AQP4 mRNA in Müller cells cultured with 50 ng/mL exogenous vascular endothelial growth factor (VEGF) for 0, 0.5, 1, 2 and 4 h, and with 0, 25, 50 and 75 ng/mL VEGF for 24 h was detected. Amplified cDNA products of AQP4 mRNA in Müller cells cultured with 500 μmol/L CoCl(2) and 200 μg/mL Avastin for 24 h were detected. The results showed that more than 95% cells displayed positive immunofluorescence reaction. Characteristic 8-10 nm intracellular filaments could be seen in the cytoplasm under the transmission electron microscopy. In the CoCl(2) experimental groups, the expression of AQP4 mRNA and VEGF mRNA in Müller cells was increased as compared with the control group. Alteration of AQP4 mRNA and VEGF mRNA levels showed a significantly positive correlation (r (2)=0.822, P<0.05). The expression of AQP4 mRNA in Müller cells was increased by VEGF. The expression of AQP4 mRNA was significantly decreased by Avastin as compared with the control group. It is suggested that Avastin can decrease the expression of AQP4 mRNA in human Müller cells under chemical hypoxic conditions partially via VEGF path, which may be one of the mechanisms of Avastin treating retinal edema.

The Morphological Study of Muller Cells with Enzyme Histochemical Staining in Retina of Induced Diabetic Rats

PURPOSE: To evaluate the morphological changes in Muller cells of an induced diabetic rat model with carbonic anhydrase histochemical staining. METHODS: Retinae of three normal rats and four streptozotocin-induced diabetic rats were used. The morphological changes in the Muller cells of these retinae were observed using enzyme histochemical staining. RESULTS: The numbers of positive staining Muller cells in diabetic rats retinae were significantly lower than those of the normal rats. In addition, the shape of the Muller cell bodies in the streptozotocin-induced diabetic rat model's retina changed from polygonal to abnormally flat. Furthermore, the staining of the Muller cells' segment in the outer nuclear layer of the diabetic rat's retinae were weaker, and some Muller cell segments were not stained at all. CONCLUSIONS: The numbers of Muller cells in diabetic rats' retinae were significantly lower than those of the normal rats. In addition, the features of Muller cell bodies of the diabetic rats were changed morphologically.

Effects of Hyperhomocysteinemia on the Immunohistochemical Reactivity for Vimentin in the Retinal Glial Cell

It has been suggested that the elevated plasma homocysteine may lead to retinal dysfunction. We investigated the effects of plasma levels of homocysteine and folate on the retinal glial cells' injuries. Male Sprague-Dawley rats were raised either on a control diet or on an experimental diet containing 3.0 g/kg homocystine without folic acid for 10 weeks. Plasma homocysteine concentrations were measured by a HPLC-fluorescence detection method. Plasma folate and vitamin B12 levels were analyzed by a radioimmunoassay. The response of Muller cells which are the principal glial cells of the retina was immunohistochemically examined using an antibody for vimentin, a cytoskeletal protein belonging to the family of intermediate filament. At 2 weeks, the homocystine diet induced a twofold increase in plasma homocysteine, and a concomitant increase in the expression of vimentin in the Muller cells' processes spanning from the inner to outer membranes of the retina indicating arterial degeneration. At 10 weeks, the homocystine diet induced a fourfold increase in plasma homocystine, but vimentin immunoreactivity in the retinas was similar in both groups. In conclusion, increased plasma homocysteine levels have influence on morphological and functional changes of Muller cells in the retina.

Enzyme Histochemical Study of Retinoblastoma

PURPOSE: The histiogenesis of retinoblastoma, the most common intraocular malignancy of childhood, has been investigated from the early times. But in spite of this effort, its origin has been controversial. This study was performed to investigated the cell of origin for retinoblastoma using enzyme histomchemistry for carbonic anhydrase. METHODS: We obtained enucleated eye that was diagnosed as retinoblastoma and its section was stained for hematoxylin-eosin for diagnosis of retinoblastoma. We used enzyme histomchemistry for carbonic anhydrase distinguishing Muller's cells, red-and green-sensistive cones from neuro-retinal cells. RESULTS: They were disagnosed as relatively well-differentiated retinoblastoma by hematoxylin-eosin staining and composed of tumor cells with numerous rosette. Neither numeric nor morphologic changes of Muller cells that are suspected of malignant features in enzyme histochemistry for carbonic anhydrase was found. CONCLUSIONS: The cells of retinoblastoma were originated from the two layers, inner nuclear and ganglion cell layer. The enzyme histochemistry for carbonic anhydrase is the one of the useful methods to investigate the origin of retinoblastoma although more cases is needed to assess.

The Effects of Growth Factors on the Contractility of Muller Cells

PURPOSE: To assess the ability of retinal Muller cells that are to generate tractional forces during culture and to evaluate their responsiveness to contraction-stimulating growth factors. METHODS: After being dissociated from porcine retina, Muller cells were cultured, and identified by immunocytochemistry. The cells were applied to the collagen gel, and changes in the thickness of the collagen layer over time were measured. Then these values were used to estimate Muller cell's contractility indirectly. Each of the applications was classified by an initial cell population and added IGF-I and PDGF concentrations. RESULTS: The contraction rate of collagen at 24 hours into incubation differed significantly between the cell groups, with group 1 having a ratio of 5.08 +/- 0.81, group 2; 7.96 +/- 0.44, group 3; 21.46 +/- 0.86, and group 4; 28.36 +/- 1.64% (p=0.000). The contraction rate of the IGF-treated groups and the PDGF-treated groups are increased by their concentrations (P<0.05), and the contraction rate of the IGF-treated groups was higher than the PDGF-treated groups at all concentration (P<0.05). CONCLUSIONS: As the cell number and concentration of growth factors were increased, the contractility of Muller cells was elevated. The development of neutralizing antibody to IGF-I and PDGF can be one of the ways for prevention of proliferative vitreoretinopathy clinically.

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.

The Morphological Changes of Muller Cell in Human Diabetic Retinopathy

PURPOSE: Vascular cells may not be the only cells affected by diabetes in the retina. In particular, b-wave abnormalities of the electroretinogram in diabetic patients with absentor minimal microangiopathy have suggested to possible dysfunction of Muller cells. METHODS: This study was performed to investigate the morphological changes of Muller cell in human diabetic retinopathy. Thirteen human retinas were obtained from donor eyes. These eyes were enucleated immediately after death. Five eyes were used as normal controls without specific medical history. Eight eyes were obtained from diabetes patients and four eyes of them had diabetic retinopathy in gross finding. RESULTS: In normal control group, Muller cells were observed in nerve fiber layer and inner nuclear layer of the retina. The Muller cells were found to have stained strong positive reaction and polygonal pattern. In the group of diabetic history without diabetic retinopathy, Muller cells had similar pattern with control group. But, in diabetic retinopathy, Muller cells had lightly positive pattern in inner nuclear layer and nuclei were oval, compared with polygonal shape in normal retina. CONCLUSIONS: These findings suggested that Muller cells might have functional and morphological changes in diabetic retinopathy, and these changes can induce the diabetic microvascular abnormalities.

The Morphological Changes of Muller Cell after Argon Laser Photocoagulation in Pigmented Rabbit

PURPOSE: Blood vessels within the retina are surrounded by Muller cells, and it is known that Muller cells may be related with the pathogenesis of diabetic retinopathy based on this histologic structure. Argon laser photocoagulation is routinely performed in the treatment of diabetic retinopathy by inhibiting neovascularization and edema, but its mechanism remains unclear. Muller cell changes were demonstrated utilizing carbonic anhydrase immunohistochemical staining to know a relation between argon laser photocoagulation and the effect of Muller cells in the rabbit retina. METHODS: Author used 16 rabbit retinas which were obtained from 8 rabbits. Exposure time and spot size were kept 0.15 second and 500 microgram. 150~350 mW of power intensity was needed to produce moderate degree coagulation in rabbit retina. RESULTS: We observed retina and its histological changes at 1 week, 2 weeks, 3 weeks and 4 weeks after photocoagulation by using carbonic anhydrase staining. The differences in the morphological changes in Muller cells and retina layers were observed between moderate and severe degree coagulation. With severe degree coagulation, the loss of all the retinal layers was observed. On the other hand, with moderate degree coagulation, proliferated pigment epithelial cells and chorioretinal adhesion were observed with loss of photoreceptor and outer nuclear layer. Muller cells were observed by carbonic anhydrase staining with proliferated Muller cells with increased nuclei and proliferated process. CONCLUSIONS: These findings suggest that Muller cells might be important in the scar formation by argon laser photocoagulation and that the proliferaration of Muller cells play a certain role in the therapeutic mechanism.

Morphological Changes of Astrocytes and Muller Cells in the Neonatal Rat Model of Retinopathy of Prematurity / 대한해부학회지

OBJECTIVES: Retinopathy of prematurity (ROP) is one of the major cause of vision loss among children. Recently, the prevalence of ROP is markedly increasing as the survival rate of very-low-birth-weight premature infants has been improved. It is widely accepted that retinal hypoxia results in the release of factors influencing new blood vessel growth. But, it is little known about the morphological changes of retinal astrocytes and Muller cells in the ROP model. So, we planned to investigate the morphological changes of those retinal glial cells induced by alternating hyperoxic and hypoxic injury in ROP. METHODS: Newborn rats (postnatal day 6) were exposed to two different oxygen concentrations alternating every 24 hours until postnatal day 14. Used oxygen concentrations were 10~15% for hypoxic episode and 55~80% for hyperoxic episode. Afterthen, they were returned to room air. A group of animal served as a room air control. Retinal vascularity was assessed by ADPase reaction and morphology of retinal glial cells was observed using transmisson electron microscope. RESULTS: Preretinal neovascular tufts were observed in 2 out of 12 animals of group III (75/10%) and 4 out of 12 animals of group IV (80/10%), respectively. There was no remarkable structural change of astrocytes. But we could observe some morphological changes of Muller cells. Retraction of the radial processes of Muller cells and breaking of basal lamina were noted at the site of preretinal neovascularization. Decrease in the space occupied by the cytoplasmic processes of Muller cells was observed in the inner nuclear layer of group IV retinae. Infiltration of microglia or macrophage into the vitreo-retinal interface and the site of extravasation was noted. Findings suggestive of neuronal cell death were also observed especially in the inner nuclear layer. CONCLUSIONS: Morphological change of Muller cells and resultant loss of integrity of internal limiting membrane seemed to be the most important step for preretinal neovascularization. But, no structural changes of astrocytes were noted.

Morphological Study of GFAP Immunoreactive Cells on Fetuses and Neonate Telencephalon of Korean Native Goat / 대한해부학회지

GFAP (Glial Fibrillary Acidic Protein) was one of the intermediate filament group and used as an astrocyte marker. The numerous studies about GFAP immunoreactive cell's distribution were investigated for fetus, neonate and aged brains. There are several reports about that GFAP immunoreactive cells were appeared at early fetus or after birth. In cases of mammalian fetus radial glia cells migrated toward pial surface at early stage and revealed GFAP immunoreactivity by the immunostain. But in cases of rodents, they migrated last gestation or after birth. This study, the GFAP immunoreactive cells' localizations and distribution in the fetuses (the 30th, 45th, 60th, 90th, 95th, 105th 120th of gestation) and neonate telencephalon of Korean native goat were investigated by immunohisto-chemistry (ABC method). The results obtained in this study were summarized as followings; 1. Multipolar astrocytes of 60 days of gestation were found cerebral cortex, in 95 days of gestation were found cerebral medulla, in 105 days of gestation were found lateral ventricle. 2. Radial glial cell presented 45 days of gestation and process of GFAP immunoreactive was to stretch out from ventricular to pia mater. And the nonpolar immunoreactive cells were transformed bipolar immunoreactive cells and they were transformed to monopolar and multipolar immunoreactive cell. 3. The number of GFAP immunoreactive cells of a field were gradually increased from 45 days of gestation till 90 days of gestation and decreased from 90 days of gestation till 105 days of gestation. But in 120 days of gestation and newborn were slightly increased. 4. Immunoreactivity of GFAP immunoreactive cells were gradually decreased from 95 days of gestation till 120 days of gestatioin. However, most pia mater areas and ventricles had high immunoreactivity and medulla part had low immunoreactivity. These results were suggested that radial glial cell of cerebral cortex and cerebral medulla were developed faster than lateral ventricle.

Morphological Study of GFAP Immunoreactive Cells on Fetuses and Neonate Mesencephalon of Korean Native Goat / 대한해부학회지

GFAP (Glial Fibrillary Acidic Protein) was one of the intermediate filament group and used as an astrocyte marker. The numerous studies about GFAP immunoreactive cell's distribution were investigated for fetus, neonate and aged brains. There are several reports about that GFAP immunoreactive cells were appeared at early fetus and after birth. In cases of mammalian fetus radial glia cells migrated toward pial surface at early stage and revealed GFAP immunoreactivity by the immunostain. But in cases of rodents, they migrated at late gestation or after birth. This study, the GFAP immunoreactive cells' localizations and distribution in the fetuses (the 30 th, 45 th, 60 th, 90 th, 105 th, 120 th of gestation) and neonate mesencephalon of korean native goat were investigated by immunohistoche-mistry (ABC method). The results obtained in this study were summarized as followings; 1. Multipolar astrocytes at 60 days of gestation were found in midbrain, in 90 days of gestation were found in cerebral aqueduct. 2. Radial glial cell presented 60 days of gestation and process of GFAP immunoreaction was to stretch out from ventricular to pia mater and nonpolar immunoreactive cell was transformed to bipolar, monopolar and multipolar immunoreactive cell. 3. The number of GFAP immunoreactive cells of field were gradually decreased from 90 days of gestation till 105 days of gestation. But in 120 days of gestation and newborn were slightly increased. 4. Immunoreactivity of GFAP immunoreactive cells were gradually decreased from 95 days of gestation till 120 days of gestatioin. These results were suggested that radial glial cell of midbrain developed very earlier than that of cerebral aqueduct. However, cerebral aqueduct developed lately than that of midbrain, but faster developing than other.

Carbonic Anhyd rase Activity in Muller Cell

Carbonic anhydrase, an enzyme catalysing the reversible hydration of carbon dioxide, is present in the Muller cells.Because the enzyme is not present in other uroretinal cells in the retina, it can be used as a marker for Muller cells.Carbonic anhydrase activity was demonstrated bnzymehistochemically in human and rabbit Muller cells to know a relation of metabolic functions and carbonic anhydrase activity.Human retinas were obtained from donor eyes.The eyes were enucleated immediately after death forenzymatic activity. In human retina, heavy staining was found in the inner nuclear layer and nerve fiber layer, moderate staining in the outer nuclear layer and weak or no staining in the plexiform layers.In rabbit retina, heavy staining was found in the nerve fiber layer and nuclear layers and weak reaction in the two plexiform layers. These findings suggest that Muller cells may participate in CO2 homeostasis mechanism of carbonic anhydrase in the retina.

Expression of GABA Transporters in the Rat's Olfactory Bulb during Development

Developmental changes in the expression of two GABA (gamma-aminobutyric acid) transporter proteins, GAT-1 and GAT-3, in the olfactory bulb of embryonic and postnatal rats were examined with immunocytochemistry using antisera against GAT-1 and GAT-3. The expression and localization of GAT-1 and GAT-3 showed distinct temporal patterns during olfactory bulb development. GAT-1 immunoreactivity appeared weakly in most likely growing axons of the presumptive glomerular layer from embryonic day 18 and increased during the first postnatal week. In contrast, GAT-3 immunoreactivity, first detected at E16, was found in radial glial cell fascicles and was replaced by what were likely astroglial cells postnatally. At P7, GAT-1 and GAT-3 immunoreactivities reached the adult pattern i.e., GAT-1 immunoreactivity was observed in the labeled punctate structures in all layers of the olfactory bulb except the nerve fiber layer, while GAT-3 immunoreactivity was observed in the astroglial processes of all layers of the olfactory bulb. Our results suggest that GABA transporters, especially GAT-3, play important roles in regulating the GABA levels of developing olfactory bulbs.

Apoptosis of Retinal Ganglion Cell after Ischemia-reperfusion Injury of Optic Nerve in Rabbits

To develop a new animal model for ischemia-reperfusion infury of the optic nerve in rabbits and to investigate the pattern of retinal ganglion cell apoptosis and bcl-2 staining in the model, occlusion of the posterior ciliary arteries for 2 hours and reperfusion were performed in one eye of thirty rabbits. In Group I(15 eyes), the optic nerve and the posterior ciliary arteries were tied while in Group II(15 eyes), the posterior ciliary arteries only were tied. The contralateral eyes received sham operation without occlusion. Following reperfusion of 24 hours(5 eyes), 48 hours(5 eyes), and 1 week(5 eyes), in each group respectively, both eyes were enucleated. TUNEL(TdT-mediated dUTP nick end labeling) staining for DNA fragmentation and bcl-2 immunohistochemical staining were done. The numbers of TUNEL-postitive ganglion cells were significantly increased at 24 and 48 hours in Group I and II compared to the control eyes(P<0.05). The numbers of TUNEL-positive ganglion cells in Group I were significantly larger than in Group II at 48 hours(p=0.01). Though the numbers of TUNEL-positive ganglion cells decreased progressively until 1 week, those in Group II at 1 week were still significantly larger than in control eyes(P=0.04), Which suggested the ischemia-reperfusion induced apoptosis of ganglion cell occurred at least until 1 week. Bcl-2 was stained strongly positive at the TUNEL-positive area and in Muller cells compared to the control eyes. The ischemia-reperfusion injury of the optic nerve induced apoptosis of retinal ganglion cell in the new animal model. The overexpressionof bcl-2 at the TUNEL-positive area and in Muller cells can be assumed to be a defense mechanism against retinal ganglion cell death by apoptosis under the ischemic condition. The results of this study will provide baseline data ganglion cell death.