Sa Sun Cho

Cerebellar alterations induced by chronic hypoxia: an immunohistochemical study using a chick embryonic model

A model of fetal aerogenic hypoxia was developed in which fertilized chicken eggs were half-painted with melted wax and incubated under normal conditions. The cerebellum of the hypoxic chick embryos at a later stage of development (E18-20) was analyzed immunochemically. Hypoxic insult resulted in considerable neurocytological deficits of the Purkinje cells and altered glial fibrillary acid protein (GFAP) immunoreactivity in the fetal cerebellum. Purkinje cells in the hypoxic embryos were marked by small cell size, poorly developed dendrites, low cell density, deletion and ectopia. On the other hand, enhanced GFAP immunoreactivity was found in astrocytes and Bergmann glia of the hypoxic embryos. Our results indicate that chronic hypoxia in the chick fetus can cause severe disorders of neuronal development as well as glial activation. We suggest that our hypoxic model of chick embryos could be an accessible animal model for further elucidating fetal hypoxia.

Glial expression of the 90-kDa heat shock protein (HSP90) and the 94-kDa glucose-regulated protein (GRP94) following an excitotoxic lesion in the mouse hippocampus

Heat shock proteins (HSPs) are immediately expressed in neuronal and glial cells under various stressful conditions and play a protective role through molecular chaperones. Although several studies have been focused on the expression of HSPs, little is known about HSP90s expression in glial cells under neuropathological conditions. In this study, we evaluated the expression pattern of the glial cell‐related HSP90 and GRP94 proteins, following the induction of an excitotoxic lesion in the mouse brain. Adult mice received an intracerebroventricular injection of kainic acid; the brain tissue was then analyzed immunohistochemically for HSPs and double labeling using glial markers. HSPs expression was quantified by Western blot analysis. Excitotoxic damage was found to cause pyramidal cell degeneration in the CA3 region of the hippocampus. In the injured hippocampus, reactive microglia/macrophages expressed HSP90 from 12 h until 7 days postlesion (PL), showing maximal levels at day 1. In parallel, hippocampal reactive astrocytes showed the expression of GRP94 from 12 h until 7 days PL. In general, HSPs expression was transient, peaked at 1–3 days PL and reached basal levels by day 7. For the first time, our data demonstrate the injury‐induced expression of HSP90 and GRP94 in glial cells, which may contribute to the mechanism of glial cell protection and adaptation in response to damage, thereby playing an important role in the evolution of the glial response and the excitotoxic lesion outcome. HSP90 may provide antioxidant protective mechanisms against microglia/macrophages, whereas GRP94 may stabilize the astroglial cytoskeleton and participate in astroglial antioxidant mechanisms.

Immunocytochemical localization of neuronal and inducible nitric oxide synthase in the retina of zebrafish, Brachydanio rerio.

Several previous studies have revealed the distribution of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in the retina of various species. However, nothing has been reported on the nNOS and iNOS expression in zebrafish retina, even though it has been used as an effective model for developmental studies. In this study on nNOS and iNOS immunoreactivity (IR) in the zebrafish retina, iNOS-IR signals were detected in the ganglion cell layer (GC), the inner plexiform layer (IP), the outer plexiform layer (OP) and the photoreceptor layer (PR). nNOS-IR signals were also intensely detected in the GC, IP, OP and PR. These signals were very similar to the iNOS-IR signals, except for some minor differences in relative intensities. This could be explained by the speculation that the synthesis of nitric oxide by iNOS may represent a compensatory mechanism in the absence of nNOS. We described for the first time the distribution of nNOS and iNOS-IR cells in zebrafish retina. This could provide the basis for further study on the nNOS and iNOS properties of zebrafish retina.

Relative sparing of calretinin containing neurons in the substantia nigra of 6-OHDA treated rat Parkinsonian model

A certain calcium binding protein (CaBP) has been known to exert a neuroprotective effect in various neurodegenerative diseases. Using the 6-OHDA induced rat Parkinsonian model, we examined if calretinin (CR), one of CaBP family, could play the similar role in the Parkinsons disease because CR is profusely localized in dopaminergic neurons of the substantia nigra pars compacta (SNPC) of the rat. Employing immunohistochemical analyses, we found that the survival rate of CR neurons was significantly higher than that of tyrosine hydroxylase (TH) neurons in the SNPC of the Parkinsonian rat. Furthermore double-labeled fluorescent microscopy revealed that almost all surviving TH neurons were also positive to CR. Our data suggest that CR-positive neurons are less vulnerable to 6-OHDA and CR in the dopaminergic neurons may have a protective function for survival of these neurons in the experimentally induced Parkinsonian rat.

Constitutive expression of c-myb mRNA in the adult rat brain

In this study, we demonstrated the c-myb mRNA expression in the adult rat brain using an in situ hybridization technique. We found c-myb mRNA signals in the various regions of the forebrain and midbrain including the cerebral cortex, thalamus, hippocampus, hypothalamus, superior and inferior colliculi and central gray. In the cerebellum, a diffuse signal was found in the granular layer while some positive cells were detected in the molecular layer as well. In addition, a number of cells showed intense signals in many nuclei of the medulla oblongata. The constitutive expression of c-myb mRNA in the different kinds of neural cells suggests that this gene might be involved in the normal function of these neurons.

Distribution of transferrin binding protein immunoreactivity in the chicken central and peripheral nervous systems

Transferrin binding protein (TfBP) is a glycoprotein originally purified from chicken oviduct that exhibits transferrin binding activity. Recent work has shown that TfBP is a post‐translationally modified form of the heat shock protein (HSP108), the avian homologue of a glucose regulated protein, GRP94. The function of this protein, however, has not yet been clearly defined. Antiserum to TfBP was found to selectively stain oligodendrocytes of the avian brain. In this study, we further describe these oligodendrocytes and other cell types positive to anti‐TfBP in the chick nervous system. In accordance with previous studies, the most prominent cell type that labels with antiserum to TfBP is the oligodendrocyte. At the electron microscopic level, the immunoreactive product is confined to the perinuclear cytoplasm and fine processes of the oligodendrocytes, whereas myelin and axoplasm are devoid of staining. The immunoreactive product is found both in the cytoplasmic matrix and bound to the endoplasmic reticulum and plasma membrane, suggesting that TfBP may have properties of both a soluble and an integral membrane protein. There is great variability in the number of TfBP‐oligodendrocytes in different areas of the central nervous system (CNS); large numbers of cells are associated with the white matter regions and are found in the myelinated tracts, whereas few cells are present in the gray matter regions. In the retina, TfBP is localized specifically in the cells that are morphologically oligodendrocytic and is present in the optic nerve fiber layer and the ganglion cell layer. Obvious staining is also seen in the Bergmann glial cells of the cerebellum and in the Schwann cells of the sciatic nerve. Furthermore, the choroid plexus cells similarly exhibit a strong reaction. The association of TfBP in these specific cell types responsible for myelination and sequestering iron and transferrin implies that TfBP may be involved in myelination and iron metabolism of the chick nervous system, perhaps through a role in transferrin concentration in these cells. A putative role of TfBP, as HSP108, is considered. J. Comp. Neurol. 260‐271, 1997.

Chronological changes of N-methyl-D-aspartate receptors and excitatory amino acid carrier 1 immunoreactivities in CA1 area and subiculum after transient forebrain ischemia

We investigated changes of immunoreactivities of N-methyl-D-aspartate receptor (NR) and of excitatory amino acid carrier 1 (EAAC-1), the neuronal glutamate transporter, in the vulnerable CA1 area and the less vulnerable subiculum of the gerbil hippocampus at various times following transient forebrain ischemia. At 30 min after ischemia-reperfusion, the intensity of NR immunoreactivity increased markedly in neurons of CA1 and subiculum, particularly NR2A/B, while EAAC-1 immunoreactivity was reduced in CA1. At 3 hr after reperfusion, the density of NR1 immunoreactivity markedly decreased in CA1. In contrast EAAC-1 immunoreactivity increased in CA1 and in the subiculum. At 12 hr after reperfusion, the decrease of NR1 immunoreactivity was not detected whereas EAAC-1 immunoreactivities in the CA1 area were intensified. In the subiculum, both NR subunits immunoreactivities decreased significantly, in contrast to the maintenance of EAAC-1 immunoreactivity. At 24 hr after reperfusion, both NR2A/B and EAAC-1 immunoreactivities decreased markedly in CA1 and subiculum. We tentatively suggest that the increase of NR immunoreactivity in CA1 at early times after ischemia-reperfusion may increase the delayed neuronal death, and that the increase or maintenance of EAAC-1 immunoreactivity at early times after ischemia-reperfusion may be an important factor in survival of neurons.

Glial cells in the bird retina: immunochemical detection.

The avian retina is remarkably different from its mammalian counterpart in macroglial cell appearance. First, it is completely devoid of astrocytes. Thus, Müller cells constitute the only astrocytic‐like cell population in avian retinae, whereas mammalian retinae also contain astrocytes in close association with blood vessels. Second, axons in the optic nerve layer of the retina of birds are myelinated, unlike those found in most mammalian species, with the exception of the rabbit, in which the medullary rays of the retina are myelinated by oligodendrocytes. Recent studies have revealed evidence that bird retinae contain a large number of oligodendrocytes, but which glial cell type myelinates axons intraretinally is still controversial. Apart from macroglial appearance, microglia in the bird retina show a very similar pattern of distribution to that of mammalian counterparts. This article reviews the existing data, including our new observations, and discusses the issues that remain to be resolved. Microsc. Res. Tech. 50:151–160, 2000.

Fas ligand mRNA expression in the mouse central nervous system

Fas ligand (FasL) expressing cells delete Fas bearing T cells, thereby enabling privileged immune status in the brain. Although the presence of FasL immunoreactivity has been shown in various cell types in the central nervous system, the precise in vivo distribution of FasL mRNA in mammals is not known. Accordingly, we localized intense FasL mRNA signals in neuroglial cells mainly within the white matter regions. Using a combined labeling technique of immunocytochemistry and in situ hybridization, we confirmed that FasL signals were due to neuroglial cells rather than neurons. This study shows that FasL mRNA is constitutively expressed in the normal mouse brain, and suggests that the Fas/FasL system protects the CNS from immunological damage.

Immunocytochemical study with an anti-transferrin binding protein serum : a marker for avian oligodendrocytes

We have investigated immunocytochemically the localization of a transferrin binding protein (TfBP) in adult CNS of avian and mammalian species using a polyclonal antibody raised against the protein purified from hen oviduct membranes (alpha OV-TfBP). TfBP has recently been shown to be HSP108. An overall strong immunoreactivity was revealed in most parts of the avian brains, especially in the white matter. The main immunoreactivity originated in small, intensely reacting cells interpreted as oligodendrocytes. The density of TfBP-labeled oligodendrocytes of the avian brains was generally proportional to the degree of myelination. There were no marked differences in TfBP-immunostaining pattern between avian species (chick, pigeon and lovebird). On the other hand, in rat, rabbit and cat brains we could not find any TfBP-immunoreactivity. Immunoelectron microscopy has further revealed that TfBP is present in the light and medium types of oligodendrocytes which are known to have high metabolic activities. TfBP reaction product was homogeneously dispersed throughout the perinuclear cytoplasm and fine processes of oligodendrocytes. The intracytoplasmic organelles such as mitochondria and Golgi apparatus were devoid of reaction product. The presence of TfBP in oligodendrocytes implies that this protein may play an important role in transferrin-mediated iron metabolism in the CNS. The complete lack of cross-reactivity between alpha OV-TfBP and mammalian tissues suggests that there is species variability in TfBP structure. We conclude that this chick TfBP antiserum will prove useful in studies of oligodendrocytes and myelination in the avian CNS.