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In vivo and in vitro experiments show that betaxolol is a retinal neuroprotective agent.

The aim of the study was to determine whether betaxolol is a neuroprotective agent and can therefore slow down the changes seen in the retina following ischaemia/reperfusion. Ischaemia was induced in one rat eye by raising the intraocular pressure for 45 min. Three days later electroretinograms were recorded from both eyes and the retinas were examined immunohistochemically for the localisation of calretinin and choline acetyltransferase (ChAT) immunoreactivities. The effect of glutamate agonists, hypoxia or experimental ischaemia was examined on the GABA immunoreactivity, lactate dehydrogenase (LDH) and internal calcium levels ([Ca2+]i) of the isolated rabbit retina, rat cortical cultures and chick retinal cell cultures respectively. Betaxolol was tested to see whether it can attenuate the influence of the glutamate agonists, hypoxia or experimental ischaemia. Ischaemia for 45 min causes a change in the nature of the normal calretinin immunoreactivity, an obliteration of the ChAT immunoreactivity and a drastic reduction in the b-wave of the electroretinogram after 3 days of reperfusion. When betaxolol was injected i.p. into the rats before ischaemia and on the days of reperfusion the changes to the calretinin and ChAT immunoreactivities were reduced and the reduction of the b-wave was prevented. Rabbit retinas incubated in vitro in physiological solution lacking oxygen/glucose or containing the glutamate agonists kainate or NMDA caused a change in the nature of the GABA immunoreactivity. Inclusion of betaxolol partially prevented the changes caused by NMDA and lack of oxygen/glucose. Rat cortical cultures exposed to glutamate or hypoxia/reoxygenation resulted in a release of LDH. The release of the enzyme was almost completely attenuated when betaxolol was included in the culture medium. Kainate increased the [Ca2+]i in chick retinal cultures, as measured with Indo-1. In a medium with sodium, this kainate-induced elevation of [Ca2+]i was significantly reduced by betaxolol. The combined data show that betaxolol is a neuroprotective agent and attenuates the effects on the retina induced by raising the intraocular pressure to simulate an ischaemic insult as may occur in glaucoma.

Animals↗

Calretinin in neurochemically well-defined cell populations of rabbit retina.

In the rabbit retina, parvalbumin has been localized selectively to AII amacrine cells, while 28 kDa calbindin could be detected in horizontal cells, in one type of depolarizing cone bipolar cell and a population of wide-field amacrine cells. The distribution of the third neuronal calcium binding protein, calretinin, however, has not been studied to date in detail in the rabbit retina. Therefore in this study we aimed to describe the overall distribution of calretinin in the different retinal layers and the possible colocalization pattern with other neurochemical marker molecules. A few cone photoreceptor cells were found to be labeled, whereas the outer plexiform layer was free from immunoreactive elements. In the most proximal row of the inner nuclear layer amacrine cells were labeled, while more distally a few cells emitted beaded axon-like processes toward the outer retina. There were large (18-28 microm in diameter) cells labeled in the ganglion cell layer, of which many apparently had their axon stained. Some of the calretinin immunoreactive amacrine cells (the AII neurons) also contained parvalbumin. Colocalization of calretinin and 28 kDa calbindin could not be ascertained in the same amacrine cell populations, nor was tyrosine hydroxylase present in calretinin-containing cells. There was partial colocalization of calretinin in the gamma-aminobutyric acid-positive amacrine cell population. Parvalbumin containing ganglion cells were also positive for calretinin; however, the calretinin-positive ganglion cells were more numerous. gamma-Aminobutyric acid could be colocalized in some calretinin-positive neurons of the ganglion cell layer.

Animals↗

Laminar distribution of non-principal neurons in the rat hippocampus, with special reference to their compositional difference among layers.

In the present study we examined the laminar distributions of four types of chemically defined subpopulations of non-principal neurons, that is, those immunoreactive for parvalbumin (PV), calretinin (CR), nitric oxide synthase (NOS) and somatostatin (SS), in the rat hippocampus, by estimating their approximate numerical densities (NDs) and percentages in specific layers according to the 'disector' principle. CR-immunoreactive (CR-IR) neurons and NOS-IR neurons were scattered throughout layers, but among layers in each subdivision their NDs were largest in the principal cell layers, where 30-45% of CR-IR and NOS-IR somata in each subdivision were located. In addition, CR-IR and NOS-IR somata were also concentrated at the border between the stratum radiatum (SR) and stratum lacunosum moleculare (SLM) in the CA1 region, where the NDs of these neurons were far larger than those in the SR/SLM as a whole and close to those in the stratum pyramidale (SP) (CR-IR somata at the ventral level and NOS-IR somata at the dorsal level) or larger (NOS-IR neurons at the ventral level). The NDs of CR-IR somata were dorsoventrally different in all layers of the CA3 region, the SR/SLM in the CA1 region and the hilus and the granule cell layer (GCL) of the dentate gyrus (DG), whereas the NDs of NOS-IR somata were dorsoventrally different in all layers of the CA3 region and the SP in the CA1 region. In contrast, approx. 90% of somatostatin-like immunoreactive (SS-LIR) neurons were located in the stratum oriens/alveus (SO/SA) in the CA1 region and in the hilus of the DG, where they were the most predominant cell type among the four types of non-principal cells. In contrast, in the CA3 region, SS-LIR somata were scattered in various layers. The majority (50-70%) of PV-IR neurons were located in the principal cell layers, whereas one-fourth to one-third of them were located in the SO/SA and hilus. The NDs in the SP of the CA1 and CA3 regions showed a significant dorsoventral difference. Although PV-IR somata were most numerous among the four non-principal cell groups in the SP of the dorsal CA1 region, they were not necessarily predominant in the principal layers in other regions, that is, in the ventral CA1 region, CA3 region and DG, where the NDs of CR-IR and/or NOS-IR somata were nearly equal to or larger than that of PV-IR somata. The present study not only reveals the laminar distribution patterns of four types of non-principal neurons in each subdivision quantitatively, but also illustrates the prominent differences in the compositions of four types of non-principal cells in each layer of each subdivision.

Animals↗

Coexpression of calretinin and parvalbumin in Ruffini-like endings in the rat incisor periodontal ligament.

The coexpression of calretinin- (CR) and parvalbumin-immunoreactivities (irs) was examined in oro-facial tissues of the rat. Nerve fibers coexpressing these calcium-binding proteins (CaBPs) were observed in the lingual periodontal ligament of incisors but not other tissues. In the part of periodontal ligament adjacent to the alveolar bone, such nerve fibers left nerve bundles and formed bush-like endings, i.e., they ramified repeatedly and terminated with one to four twigs. An immunoelectron microscopic method indicated that these endings were identical to Ruffini-like endings, 4% of trigeminal neurons retrogradely labeled from the inferior alveolar nerve coexpressed CR- and parvalbumin-irs. The present observations suggest that the coexpression of these CaBPs may be a specific marker for low-threshold mechanoreceptors in the trigeminal ganglion.

Animals↗

Calretinin-like immunoreactivity in the Ruffini endings, slowly adapting mechanoreceptors, of the periodontal ligament of the rat incisor.

The distribution and ultrastructural localization of calretinin (CR)-like immunoreactivity (-LI) were investigated in the lingual periodontal ligament of rat incisors. Some thick nerve fibers within the nerve bundle displayed CR-LI; these CR-like immunoreactive (-IR) nerve fibers entered the alveolar half of the lingual periodontal ligament of the incisor where dendritic terminal arborization was exhibited. Thin and beaded CR-IR nerve fibers were rarely observed in the periodontal ligament. Observations of adjacent sections immunostained with protein gene-product 9.5 (PGP 9.5) revealed that most, if not all, PGP 9.5-IR nerve terminals showing a dendritic arborization expressed CR-LI. Immunoelectron microscopic observations showed that electron-opaque immunoreaction products were localized in the axoplasm of the axon terminals, except for the mitochondria, which were surrounded by Schwann sheaths and multiple-layered basal lamina. Neither cell bodies, the cytoplasmic extension of terminal Schwann cells, nor other cellular elements such as periodontal fibroblasts exhibited CR-LI. The present findings suggest that Ruffini endings, an essential mechanoreceptor in the periodontal ligament and categorized as a slowly adapting mechanoreceptor, express CR-LI, and that CR may participate in the Ca2+ homeostasis against external stimuli in the periodontal Ruffini endings.

Animals↗

Calretinin-like immunoreactivity in the regenerating periodontal ruffini endings of the rat incisor following injury to the inferior alveolar nerve.

Regeneration of calretinin (CR)-like immunoreactive (IR) nerve fibers was investigated in the periodontal ligament of the rat lower incisor following resection of the inferior alveolar nerve (IAN). In addition, the degeneration and regeneration processes of periodontal nerve fibers were examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker. In normal animals, the periodontal nerve fibers showing PGP 9.5-like immunoreactivity (LI) formed either periodontal Ruffini endings with expanded arborization and thin free nerve endings in the alveolar half of the ligament. Thick CR-IR nerve fibers also appeared in a dendritic fashion in the same region, but thin CR-IR nerve fibers were rarely observed. Five days following resection of the IAN, a major population of PGP 9.5-IR and all CR-IR nerve fibers disappeared except for some thin PGP 9.5-IR nerves in the periodontal ligament. Regenerated PGP 9.5-IR nerve fibers appeared around 7 days following resection, in contrast to a very small number of regenerated CR-IR nerve fibers. Around 14-21 days following resection, the number and terminal morphology of regenerated PGP 9.5-IR nerve fibers were comparable to those observed in normal animals, but the number of regenerated CR-IR nerve fibers was still smaller than that of normal animals. The number of regenerated CR-IR nerve fibers increased to return to normal by 56 days following injury. The delay of expression of CR-LI in the regenerated periodontal Ruffini endings suggests that functional recovery of periodontal Ruffini endings occurred after the completion of the regeneration of periodontal nerve fibers.

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Calretinin is not a marker for subdivisions within the suprachiasmatic nucleus.

In this study, we report the immunocytochemical localization of the calcium-binding protein calretinin (CAL) in the suprachiasmatic nuclei (SCN) of male and female rodents including rats, mice, golden hamsters, and Arvicanthis niloticus. The results revealed that CAL is present in different subdivisions of the SCN in the different species studied and CAL can, therefore, not be considered a marker for particular subdivisions within the SCN. No differences were found between males and females.

Animals↗

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 Parkinson's 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.

Animals↗

Cellular distribution of vanilloid VR1 receptor immunoreactivity in the guinea-pig myenteric plexus.

Recent investigations suggest that vanilloid receptor-1 (VR1) immunoreactivity occurs in the intestine. We have determined and quantified this immunoreactivity in the myenteric plexus with respect to cholinergic and neurofilament protein-positive neurones. Guinea-pig and rat preparations were dual-labelled with specific antibodies raised in rabbit or goat against vanilloid receptor-1 and against other neurochemical markers. In the rat ileum, both vanilloid receptor antibodies were co-distributed, whereas in the guinea-pig ileum and colon, tertiary fibres were also detected with the goat antibody. In the guinea-pig, all vanilloid receptor-1-immunoreactive cell bodies were choline acetyltransferase-immunopositive (100%) and showed some immunoreactivity to neurofilament proteins (NFP-200 kDa (79%) or triplet (10.8%)) or calretinin. Immunoreactive fibres in the secondary plexus co-localised with calcitonin gene-related peptide (CGRP) and with substance P, calretinin and synapsin I in the tertiary plexus. Subpopulations of cholinergic neurones including sensory, interneuronal and secretory neurones express vanilloid receptor-1. Co-localisation with substance P and calretinin in fibres suggests that vanilloid receptor-1 may be expressed by excitatory motor neurones. The association of vanilloid receptors with calcitonin gene-related peptide and synaptic protein in fibres implies a role for vanilloid receptors in neurotransmitter/neuropeptide release. Although it is likely that at least some of the vanilloid receptor-bearing fibres originate in immunopositive myenteric soma, the origin of all these fibres cannot be identified in the present study.

Animals↗

AII amacrine cells express the MT1 melatonin receptor in human and macaque retina.

AII amacrine cells are critical interneurons in the rod pathway of mammalian retina, active primarily in dim lighting conditions. Melatonin, a neuromodulator produced at night in the retina, is believed to induce retinal adaptation to dim lighting conditions in most vertebrate species examined to date, including humans. We hypothesized that melatonin may influence retinal light adaptation by acting on AII cells directly and thus investigated whether melatonin receptors were expressed in AII neurons. Postmortem nonpathological eyes from four human donors as well as two eyes from two Macaque Fasicularis monkeys were analyzed. Double immunocytochemistry was performed using an anti-MT(1) antibody and an antibody to calretinin, an AII marker. Analysis utilized confocal microscopy. A polyclonal anti-calretinin antibody labelled amacrine cells exhibiting the distinct AII morphology, in both human and macaque retina. MT(1) immunoreactivity in macaque retina was similar to human staining, in that horizontal, amacrine and ganglion cell bodies were stained, as were inner segments of photoreceptors. In human retina 86% of calretinin positive cells expressed the MT(1) receptor peripherally, whereas centrally, 78% colocalization was observed. In the macaque retina, 100% of AII amacrine cells expressed MT(1) immunoreactivity both centrally and peripherally. That virtually all AII neurons express the MT(1) receptor in both human and macaque retina, may provide the first evidence demonstrating a role for melatonin in AII regulation, furthering the hypothesis of melatonin function in retinal light adaptation.

Amacrine Cells↗

Colchicine causes excessive ocular growth and myopia in chicks.

Colchicine has been reported to destroy ganglion cells (GCs) in the retina of hatchling chicks. We tested whether colchicine influences normal ocular growth and form-deprivation myopia, and whether it affects cells other than GCs. Colchicine greatly increased axial length, equatorial diameter, eye weight, and myopic refractive error, while reducing corneal curvature. Colchicine caused DNA fragmentation in many GCs and some amacrine cells and photoreceptors, ultimately leading to the destruction of most GCs and particular sub-sets of amacrine cells. Colchicine-induced ocular growth may result from the destruction of amacrine cells that normally suppress ocular growth, and corneal flattening may result from the destruction of GCs whose central pathway normally plays a role in shaping the cornea.

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The immunohistochemical diagnosis of epithelial mesothelioma.

Although it is generally accepted that immunohistochemistry can assist in distinguishing between epithelial mesotheliomas and metastatic adenocarcinomas to the serosal membranes, a great deal of controversy exists regarding not only the practical value of some of the markers currently used, but also which should be included as part of the routine diagnostic panel. Until recently, these panels consisted primarily of antibodies that stained adenocarcinomas but not mesotheliomas. Over the last few years, however, markers that are commonly expressed in mesotheliomas but not in adenocarcinomas have been recognized. The focus of this review is on those markers for which there is an indication that they have a practical use in the diagnosis of mesothelioma. Special emphasis is placed on those which have been most recently recognized.

Adenocarcinoma↗

Colocalization of calretinin and glucagon in rat pancreatic A cells.

Calretinin is a calcium-binding protein whose major functions are assumed to be buffering, transport of Ca2+, regulation of various enzyme systems and cellular protection. Moreover, calretinin does not seem to be a specific marker for any particular cell since it has been discovered in various mammalian and avian organs. In order to give clue to its function(s), we investigate its distribution in the rat pancreas using immunocytochemistry and transmission electron microscopy. We have found calretinin immunoreactive cells in rat pancreas only in the islets of Langerhans. These cells correspond to A cells producing glucagon. The colocalization of calretinin and glucagon was confirmed with double-labelling immunofluorescence. Nevertheless, the role of calretinin and its relationship with the production and/or the exocytosis of glucagon granules remains to be elucidated.

Animals↗

A sequential protocol combining dual neuroanatomical tract-tracing with the visualization of local circuit neurons within the striatum.

We describe here an experimental approach designed to aid in the identification of complex brain circuits within the rat corpus striatum. Our aim was to characterize in a single section (i) striatal thalamic afferents, (ii) striatopallidal projection neurons and (iii) striatal local circuit interneurons. To this end, we have combined anterograde tracing using biotinylated dextran amine and retrograde neuroanatomical tracing with Fluoro-Gold. This dual tracing protocol was further implemented with the visualization of different subpopulations of striatal interneurons. The subsequent use of three different peroxidase substrates enabled us to unequivocally detect structures that were labeled within a three-color paradigm.

3,3'-Diaminobenzidine↗

Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain.

The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral 'tiers' with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei. We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway. Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively.

Animals↗

A polyclonal goat antiserum against the calcium-binding protein calretinin is a versatile tool for various immunochemical techniques.

Specific antibodies are useful tools to label particular neurons and at times to delineate neuronal circuits--a task not easily achieved by other techniques. Human recombinant calretinin, a protein belonging to the EF-hand family of Ca2+-binding proteins, was used to produce an antiserum in goat. The specificity of the antiserum to recognize calretinin was demonstrated in brain extracts from mouse, rat, and chick and in extracts from human tumor cell lines known to express this protein. Immunohistochemically, the antiserum-stained specific neurons in human, rhesus monkey, mouse, and rat brain. The goat anti-calretinin antiserum is an appropriate tool for double- or triple-immunolabeling studies along with previously-established rabbit and mouse antibodies. Thus, it allows for the concomitant staining with antibodies directed against other EF-hand calcium-binding proteins including calbindin-D28k and parvalbumin. The antiserum can further be used for the quantification of calretinin in different tissues or cell lines in a sandwich ELISA. Additionally, it is well suited for the detection of calretinin in certain cell lines or malignant pleural mesotheliomas. Immunostaining of these samples is comparable to that with the well-characterized calretinin-specific polyclonal rabbit antiserum 7696.

Animals↗

Calretinin and FMRFamide immunoreactivity in the nervus terminalis of prenatal tree shrews (Tupaia belangeri).

The distribution and development of FMRFamide- and calretinin-immunoreactive neurons were investigated in the nervus terminalis of prenatal tree shrews from gestation day 19 onwards. The first FMRFamide-immunoreactive cells were observed medially in the olfactory epithelium on gestation day 20. From gestation day 23 onwards, the migrating nervus terminalis ganglion cells showed FMRFamide calretinin immunoreactivity. The distribution pattern of FMRFamide- and calretinin-immunoreactive cells was similar along the migratory route and in the ganglion of the terminal nerve. However, most probably calretinin and FMRFamide were expressed in separate neuronal populations. For the first time in a mammal, FMRFamide and calretinin are reported to occur in the migrating perikarya and neuronal processes of the nervus terminalis during prenatal development. The results suggest (i) an early activation of the rostral FMRFamide-immunoreactive migratory stream comparable to that described for the GnRH-immunoreactive part of the terminal nerve in other mammals and possibly (ii) an involvement of calretinin in mechanisms of cell migration and outgrowth of neuronal processes in the terminal nerve during the studied period.

Animals↗