Changes of synapsin I messenger RNA expression during rat brain development

Synapsin I is a synaptic phosphoprotein that is involved in the short-term regulation of neurotransmitter release. In this report we present the first extensive study of the developmental expression of its corresponding messenger ribonucleic acid (mRNA) by in situ hybridization and northern blot analysis in rat brain. Synapsin I mRNA showed pronounced differences in expression in different brain regions during postnatal development. The early expression of synapsin I mRNA in ontogenetically older regions such as the thalamus, the piriform cortex and the hippocampus coincides with the earlier maturation of these regions, in contrast to its later expression in ontogenetically younger areas such as the cerebellum and the neocortex. An intriguing expression pattern was found in the hippocampus. In all hippocampal subregions synapsin I mRNA expression increased from postnatal day (PND) 1 to 17. After PND 17, however, there was a marked dissociation between persisting high expression levels in CA3 and the dentate gyrus and a strong decline in synapsin I mRNA expression in CA1. The persistence of synapsin I in some adult rat brain regions indicates that it plays a part in synapse formation during plastic adaption in neuronal connectivities.     

Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage

The present study was designed to investigate whether or not chondrocytes in articular cartilage express type I collagen in vivo under physiological conditions. Expressions of the gene and the phenotype of type I collagen were examined in rat tibial articular cartilage in the knee joint during development. Knee joints of Wistar rats at 1, 5, and 11 weeks postnatal were fixed in 4% paraformaldehyde with or without 0.5% glutaraldehyde and decalcified in 10% EDTA. After the specimens were embedded in paraffin and serial sections made, adjacent sections were processed for immunohistochemistry and in situ hybridization for type I collagen. The epiphysis of the tibia was composed of cartilage in week-1 rats. Formation of articular cartilage was in progress in week 5 as endochondral ossification proceeded and was completed in week 11. Anti-type I collagen antibody stained only the superficial area of the epiphysis in week 1, but the immunoreactivity was expanded into the deeper region of the articular cartilage with development in weeks 5 and 11. Hybridization signals for pro-alpha 1 (I) collagen were seen in some of chondrocytes in the epiphysis of the week-1 tibia. The most intense signals were identified in chondrocytes in week 5 and the signals appeared weaker in week 11. The present study demonstrated that chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of the articular cartilage.     

Synthesis and expression of laminin during human foetal lung development

Backgrounds: The lung develops by epithelial tubes budding and branching into a flexible mesenchyme. This growth is associated with the remodelling of the epithelial basement membrane, of which laminin is a major component. Methods: Both the synthesis and expression of laminin were studied in the human lung between 10 and 31 weeks of gestation, using in sity hybridization and immunohistochemistry. Results: The synthesis of the β chain was active in the epithelial and surrounding mesenchymal cells. The mRNAs coding for the γ chain were less abundant and mainly found in the epithelium. The synthesis of these two chains continued throughout gestation, and no significant difference in the density of hybridization grains could be detected between the tips of the expanding buds and the proximal portions. Immunohistochemical localization of laminin showed important modifications of the basement membrane during gestation. In the first part of the pseudoglandular stage the epithelial basement membrane stained continuously for laminin. Later, the basement membrane was labelled in a graded fashion: at the apex of the growing buds the staining became weak with focal disruptions. Both epithelial and mesenchymal synthesis of laminin remained active, while the polypeptide was undetectable using immunohistochemistry. Conclusions: These findings suggest that the remodelling of the basement membrane during human lung morphogenesis is probably not related to a decreasing synthesis of laminin, but to either a proteolytic degradation or the assembly of an inadequate complex undetectable with the polyclonal antibody antilaminin. © 1995 Wiley-Liss, Inc.     

Transient expression of osteopontin mRNA and protein in amoeboid microglia in developing rat brain

To investigate a potential role of osteopontin (OPN) in developing rat brain, the expression of OPN mRNA and protein in the developing rat brain relative to the distribution of brain macrophages was investigated using in situ hybridization and immunohistochemistry, and the phagocytic capability of OPN-expressing cells was accessed using rhodamine isothiocyanate (RhIc) as a tracer. OPN-expressing cells appeared from embryonic day 16. During the first week of postnatal life, OPN-labeled cells increased markedly, and peaked around P7, then declined and had completely disappeared by the end of the second postnatal week. The spatiotemporal distribution pattern of OPN mRNA closely matched that of OPN protein. Their morphology and localization were compared with those of cells expressing the established microglial marker OX-42 in adjacent sections, and double-labeling studies demonstrated that OPN was localized to the amoeboid microglia which stain with the lectin GSI-B₄, another marker for microglia. Furthermore, OPN-labeled cells were confirmed to be active phagocytes emitting RhIc fluorescence indicating that the tracer into the brain tissues was engulfed by phagocytosis. Therefore, these results provide the first evidence that OPN is transiently expressed in active brain macrophages in the embryonic and early postnatal brain, and suggest that OPN may contribute to the migration and phagocytic function of brain macrophages in the developing brain.This work was supported by a Korea Research Foundation grant (KRF-2002-015-EP0106)     

Expression of c-maf and mafB genes in the skin during rat embryonic development

The maf oncogene (v-maf) was initially identified in an avian oncogenic retrovirus, AS42, which induces musculoaponeurotic fibrosarcoma in vivo and transforms chicken embryo fibroblasts in vitro. Genes of the maf family have important roles in embryonic development and cellular differentiation. Both genes are expressed in a wide variety of tissues including spleen, kidney, lens and liver. The present study was performed to analyze expression of c-maf-1 and mafB genes in skin of embryonic stages from 15 days onwards using in situ hybridization. Expression of c-maf mRNA was first detected on embryonic day (ED) 16 in the nuclei of cells in the basal layer in developing epidermis. On ED 19, high expression was detected in the nucleus of basal keratinocytes and developing hair germs. On postnatal day (PD) 3, expression of c-maf had disappeared in epidermis and hair follicles. MafB showed similar expression patterns as c-maf. Our findings indicate that c-maf and mafB are involved in embryonic development of epidermis and hair follicles.     

Regional and temporal expression of sodium channel messenger RNAs in the rat brain during development

Summary The distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.Supported by Deutsche Forschungsgemeinschaft grant no.: Cr 30/16     

Growth of postnatal rat retina in vitro. Development of neurotransmitter systems

In this study, we demonstrate that explanted neonatal rat retina can be maintained in culture for periods up to 3 weeks. The cultured retinas displayed a distinct layering that was almost identical to litter-matched retinas of the same age, but the majority of the ganglion cells did not survive and photoreceptor outer segments did not develop properly. Distinct synaptophysin immunoreactivity was expressed in both the inner and outer plexiform layers of cultured retina and the pattern mimicked that one observed in vivo. After 2-3 weeks in vitro, the inner retina expressed immunoreactivities to various components of the cholinergic and nitrergic transmitter systems, including nitric oxide activated cyclic GMP immunoreactivity. The investigated cell populations displayed similar distribution patterns as in situ, but morphological differences appeared in vitro. Such differences were mainly observed as irregularities in the arborization patterns in the inner part of the inner plexiform layer. We suggest that these discrepancies may arise as a result of reduced ganglion cell survival. Our observations demonstrate that some neurotransmitter systems develop in vitro and their neural circuitry appears similar to the in vivo situation. The presence of synapses, receptor proteins and transmitter substances implies that neural communication can occur in cultured retinas.     

视网膜特异基因（PcR─18）产物的形态定位研究

为了解ＰｃＲ－１８基因在大鼠视网膜上的细胞定位，用原位分子杂交组织化学方法（ＩＳＨＨ），观察了代号ＰｃＲ－１８的视网膜特异基因表达细胞在大鼠视网膜内的分布。结果显示，ＰｃＲ－１８基因主要在视网膜节细胞层内的直径较大、甲苯胺蓝染色较深的细胞中表达，该种细胞多分布于黄斑以外区域及视网膜周边部，双极细胞层及视网膜其他各层未见特异ＰｃＲ－１８基因表达细胞。结合视网膜细胞形态－功能关系的有关研究基础，分析了该基因产物在视觉传导中可能的功能意义，认为该基因产物可能与低、中频率刺激传导乃至运动信息的处理及启动固视反射有关。本文还对ＩＳＨＨ方法在研究异源群体中，某一特异基因表达变化时所显示的优势进行了讨论     

Expression of preproenkephalin mRNA in rat superior cervical ganglion during postnatal development

The number of principal neurons in the rat superior cervical ganglion (SCG) exhibiting enkephalin-peptide immunoreactivity is reported to be limited. To better determine the degree of enkephalinergic phenotype in sympathetic neurons, sections of SCGs from rats aged newborn to adult were processed for in situ hybridization histochemistry, using a [³⁵S]cRNA probe directed against preproenkephalin (PPENK). > 50% of principal ganglion neurons express mRNA for PPENK in adults. Striking variability in labeling intensity is observed. PPENK mRNA is detected in developing ganglia beginning at postnatal days 4–7. Both the number of cells and intensity of labeling increases with postnatal development. These results indicate that expression of PPENK mRNA is more widespread than expression of enkephalin peptides and develops postnatally.     

Spatial and temporal expression of UDP-galactose: ceramide galactosyl transferase mRNA during rat brain development

 We studied the expression of UDP-galactose: ceramide galactosyl transferase (CGT) mRNA in postnatal rat brains using an in situ hybridization technique. From P0 to P16, there was a defined temporal and spatial pattern to the earliest acquisition of CGT mRNA expression. In the forebrain, CGT mRNA-expressing (CGT⁺) cells were first detected in regions outside the subventricular zone around the lateral ventricle at P2. Cells in the external capsule, internal capsule and corpus callosum were later found to be CGT-positive. At P8 to P16, CGT⁺ cells were found in the thalamus, striatum, occipital and frontal cortex. In the case of midbrain and hindbrain, the first CGT⁺ signals were detected in the medullary raphe of the medulla oblongata at P0. CGT⁺ cells were subsequently located in the cerebellum, midbrain and pons from P4 to P16. That is, in regions closer to the areas in which CGT⁺ cells were first found, CGT mRNA expression was observed much earlier. These findings support the notion that there are at least two discrete waves of CGT mRNA signal expression in the forebrain and hindbrain. Accepted: 3 February 1999     

The immunocytochemical localization of neuronal nitric oxide synthase in the developing rat retina

The localization of nitric oxide synthase (NOS) was investigated in the developing rat retina by immunocytochemistry and western blot analysis, using an antiserum directed against neuronal NOS. NOS-labeled cells were first detected at postnatal day 5 (P5) in the inner row of the neuroblastic layer. These cells were considered to correspond to the type 1 cell of the adult rat retina. Type 2 cells, characterized by a small soma and weak immunoreactivity, and a class of displaced amacrine cells were detected at P9 and P7, respectively. By P14 or P15, the time of eye opening, NOS immunoreactivity appeared in some bipolar cells. NOS was first expressed at the protein level at P9. Thereafter, quantitative evaluation by immunoblotting confirmed that the intensity of the immunoreactive bands increased abruptly, reaching the same value as is found in the adult retina at P21. Our results demonstrate that differentiation of NOS-labeled cells follows a discrete developmental pattern and is most active during the 2nd postnatal period in the rat retina.     

Expression of managanese superoxide dismutase in rat submandibular gland demonstrated by in situ hybridization and immunohistochemistry

Superoxide, an active oxygen species, plays an important role in protecting against bacterial infection. However, it also has adverse effects on health. Manganese superoxide dismutase (Mn-SOD) is a scavenger of superoxide. Antioxygen enzymes such as Mn-SOD exist in various tissues, and provide protections against oxidative injury. We investigated both the expression of Mn-SOD mRNA and the localization of Mn-SOD in adult rat submandibular glands using in situ hybridization and immunohistochemistry. Both Mn-SOD mRNA and Mn-SOD were detected in striated duct cells, and in some granular duct cells and excretory duct cells. With immunoelectron microscopy, many immunolabelings were observed on the mitochondria. These findings suggest that the expression of Mn-SOD mRNA and the localization of Mn-SOD in submandibular glands correlate with the number of mitochondria in cells.     

Plasmablastic lymphoma of the oral cavity in an HIV-negative patient

Plasmablastic lymphoma (PBL) is a rare, highly aggressive lymphoma typified by immunoblast-like cells with abundant basophilic cytoplasm and paranuclear hof. It shows absent expression of CD45 and CD20. In contrast, it displays a constant reaction with CD138 and VS38c. It may be easily misinterpreted as some other lymphoma. An exhaustive integration of clinical, morphologic, phenotypic, and molecular features is important to exclude misdiagnosis and inappropriate treatment. We report a case of HIV-negative PBL arising on the left areas of posterior teeth mucosa of a 58-year-old man. Immunohistochemically, the tumor cell was immunoreactive for CD138, VS38c, VEGF, and vimentin; Ki-67 showed a high proliferation rate. Epstein-Barr virus (in situ hybridization) was nonreactive, and IgH gene rearrangement was identified by polymerase chain reaction amplification products. A diagnosis of PBL was rendered.     

Cellular hybridization for BDNF. trkB, and NGF mRNAs and BDNF-immunoreactivity in rat forebrain after pilocarpine-induced status epilepticus

The messenger RNAs (mRNAs) for the neurotrophins, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), are upregulated during epileptic seizure activity, as visualized by in situ hybridization techniques. Neurotrophins might be protective against excitotoxic cell stress, and the upregulation during seizures might provide such cell protection. In this study, a high dose of pilocarpine (300 mg/kg) was used to induce long-lasting, limbic motor status epilepticus and a selective pattern of brain damage. The regulation of BDNF, trkB, and NGF mRNA was studied by in situ hybridization at 1, 3, 6, and 24 h after induction of limbic motor status epilepticus. BDNF immunoreactivity was examined with an anti-peptide antibody and the neuropathological process studied in parallel. BDNF mRNA increased in hippocampus, neocortex, piriform cortex, striatum, and thalamus with a maximum at 3–6 h. Hybridization levels increased earlier in the resistant granule and CA1 cells as compared to the vulnerable CA3 neurons. BDNF immunoreactivity was elevated in dentate gyrus at 3–6 h. trKB mRNA increased in the entire hippocampus. NGF mRNA in hippocampus appeared in dentate gyrus at 3–6 h and declined in hilar neurons at 6–24 h. Cell damage was found in the CA3 area, entire basal cortex, and layers II/III of neocortex. Endogenous neurotrophins are upregulated during status epilepticus caused by pilocarpine, which is related to the coupling between neuronal excitation and trophic factor expression. This upregulation of neurotrophic factors may serve endogenous protective effects; however, the excessive levels of neuronal hyperexcitation resulting from pilocarpine seizures lead to cell damage which cannot be prevented by endogenous neurotrophins.     

显色原位杂交检测乳腺癌HER2基因扩增的应用价值

目的评估显色原位杂交（CISH）技术在检测乳腺癌标本中HER2基因扩增的应用价值。方法分别采用免疫组织化学（IHC）EnVision和CISH两种方法,检测165例乳腺癌中HER2蛋白表达及HER2基因扩增的情况。结果（1）IHC检测HER2蛋白表达阴性者107例,表达阳性1＋者24例,CISH均未检测到HER2基因扩增;（2）IHC检测HER2表达3＋者22例,CISH检测中21例呈现HER2基因的高倍扩增,仅1例呈低倍扩增,HER2基因高倍扩增及其蛋白表达之间的符合率为95．5％;（3）IHC检测HER2表达2＋者12例,CISH检测有3例HER2基因呈高倍扩增,6例呈低倍扩增,3例无扩增。结论CISH在检测HER2基因扩增与IHC检测的结果具有较高的一致性和敏感性,可以作为一种检测乳腺癌HER2基因状况的方法。     

The birthdates of GABA-immunoreactive amacrine cells in the rat retina

The birthdates of GABAergic amacrine cells in the rat retina were investigated by immunocytochemistry using anti-GABA and anti-bromodeoxyuridine (BrdU) antisera. The ratio of co-localization of GABA to BrdU increased gradually from embryonic-day 13 (E13) and showed a peak value on E18 in the central retina and on E20 in the periphery. After birth, until postnatal-day 3 (P3), a few co-localized cells were observed in the inner nuclear layer (INL). However, in the peripheral retina, co-localized cells were observed in the INL and ganglion cell layer until P5. Our results suggest that the birthdates of GABA-immunoreactive cells vary, depending on cell-type and that there is a temporal lag in the GABA-immunoreactive cell production in the peripheral retina relative to the central retina. Received: 11 January 1999 / Accepted: 20 April 1999     

NR2B反义寡核苷酸对大鼠海马CA1区NR2B mRNA表达的影响

目的：观察NMDA受体2B亚单位（NR2B）反义寡核苷酸（ANR2B）对海马CA1区NR2B mRNA表达的影响,筛选有效的ANR2B,探讨改变NR2B mRNA表达的新方法.方法：正常SD大鼠,海马CA1区立体定位注射ANR2B,灌注取脑,连续冰冻切片,原位杂交组织化学方法染色,光镜下观察NR2B mRNA在ANB2B作用下的表达变化.结果：正常大鼠海马CA1区立体定位注射ANR2B后,注射点及其周围NR2B mRNA原位杂交染色强度明显下降,仅有少量锥体细胞和颗粒细胞散在分布;而在注射NR2B正义寡核苷酸（SNR2B）、生理盐水（NS）及NS插针不注射（NSNO）的海马切片上,海马CA1区的染色特征与注射ANR2B有明显差别,其作用区锥体细胞、颗粒细胞及顶树突的NR2B原位杂交染色强度无明显变化.结论：ANR2B能够降低NR2B mRNA在正常大鼠海马CA1区的基础性高表达.     

Immunohistochemical localization of protein kinase C-alpha in the retina of pigs during postnatal development

The cellular localization and protein expression level of protein kinase C (PKC)-alpha was examined in pig retina at different ages. Western blot analysis detected PKC-alpha in the retinas of 3-day-old piglets and indicated significantly increased expression in 6-month-old young adult and 2-year-old adult pigs. Immunohistochemistry of 3-day-old retinas revealed intense PKC-alpha reactivity in the inner plexiform and inner nuclear cell layers, weak reactivity in the ganglion cell layer, and few positive cells in the outer nuclear cell layer. The cellular localization of PKC-alpha in the adult retina was similar, with staining more intense than that in neonates. PKC-alpha was co-localized in some glial fibrillary acidic protein-positive cells and glutamine synthetase-positive cells in the retina. This study demonstrates that the protein level of retinal PKC-alpha is increased with maturation and suggests that PKC-alpha plays a role in signal transduction pathways for postnatal development in porcine retina.