Fig 4 - uploaded by Elena Vecino
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A and B are sections of the retina (R) and optic nerve head (ONH) taken in the longitudinal axis of the optic nerve. Bv, blood vessel; V, vitreous. A: Toluidin blue-stained section. Scale bar 5 40 µm. B: Adjacent section of A labeled for S100. The arrowhead points to the limit between optic nerve head and the retina where the growth cone-like processes of astrocytes form a boundary between both structures. The arrow points to one astrocyte cell body. Scale bar 5 40 µm. C: Higher magnification picture of the area arrowed in B. Scale bar 5 20 µm.
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The distribution of S100 immunoreactivity within the normal and regenerating retina and optic nerve head of the teleost Tinca tinca L. has been investigated using the avidin-biotin complex (ABC) method and a polyclonal antibody against S100. Astrocytes and Müller cells were labeled with this antibody. This represents the first description of astroc...
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Context 1
... as those in the optic nerve fiber layer were clustered tightly in the region of the optic nerve head (Fig. 3A,B). One of the processes of these cells was directed to the optic nerve and the other to the retina. The processes directed to the retina ended with a thick growthconelike profile, forming a meshwork just at the entrance to the retina (Fig. 4A). These cells were mainly located around the central retinal artery (Figs. 1A,D, ...
Citations
... Among other functions, the S100 protein plays a role in cytoskeleton dynamics and regulates cytosolic Calcium levels by modulating several cellular processes such as the cell cycle, cell growth, and differentiation, transcription, and secretion (Marenholz et al., 2004). In teleosts immunoreactivity to S100 protein was reported in hair cells of the saccule and saccular nerve (Foster et al., 1993), Muller cells and astrocytes of the retina (Vecino et al., 1997), kidney juxtaglomerular cells (de Girolamo et al., 2000), hair cells of the lateral line (Germana et al., 2004a;Montalbano et al., 2018), crypt olfactory neurons (Germana et al., 2004b), and glial cells of the central nervous system (Manso et al., 1997), and in macrophages in the gut . In addition, the considerable number of teleost species evaluated in a study by Vigliano et al. (Vigliano et al., 2009), that cross-reacted with the antibody demonstrated that the S100 protein is highly conserved through the process of evolution, although some variation in species sequences could lead to no cross-reaction. ...
Rodlet cells (RCs) have always been an enigma for scientists. RCs have been given a variety of activities over the years, including ion transport, osmoregulation, and sensory function. These cells, presumably as members of the granulocyte line, are present only in teleosts and play a role in the innate immune response. RCs are migratory cells found in a variety of organs, including skin, vascular, digestive, uropoietic, reproductive, and respiratory systems, and present distinct physical properties that make them easily recognizable in tissues and organs. The development of RCs can be divided into four stages: granular, transitional, mature, and ruptured, having different morphological characteristics. Our study aims to characterize the different stages of these cells by histomorphological and histochemical techniques. Furthermore, we characterized these cells at all stages with peroxidase and fluorescence immunohistochemical techniques using different antibodies: S100, tubulin, α-SMA, piscidin, and for the first time TLR-2. From our results, the immunoreactivity of these cells to the antibodies performed may confirm that RCs play a role in fish defense mechanisms, helping to expand the state of the art on immunology and immune cells of teleosts.
... Morphological Staining and IHC. The S100 protein immunoreaction (S100-IR) can be used as a specific marker of the glia, because this protein family is secreted mainly by the glia in the brain [18,19]. Glutamate receptors are located primarily on the membranes of neuronal cells [20]; therefore, glutamate-immunoreaction (Glu-IR) is used as a marker of neurons in this paper. ...
The aim of this research was to assess the degree of injury in the cerebellums of loaches (Misgurnus anguillicaudatus) exposed to a subchronic concentration of mercury chloride (HgCl2), based on the glia-neuron ratio (GNR). Under exposure to subchronic HgCl2, immunohistochemistry showed that the density of S100-IR glial cells increased continuously and the density of Glu-IR neurons showed a slight increase during the first 9 days in the loach cerebellum. Subsequently, the S100-IR glia density was enhanced, while the density of the Glu-IR neurons began to decrease. There was a significant time-related increase (P<0.05) in the GNR value during the whole course. These results confirmed that immunohistochemistry can be an effectual tool to detect the neurovirulence of mercury contamination, and more importantly, that GNR can be used to assess the neurotoxicity of pollution..
... To further characterize Pax2 þ astrocytes in the ONH during regeneration, we have performed ON crush and analyzed both the modifications in the pax2 gene expression level and in the Pax2 þ astrocyte population. To study the Pax2 þ astrocytes we used several astrocyte markers whose antibody specificity has been widely demonstrated in our laboratory and others: Pax2 transcription factor (Macdonald et al., 1997;Parrilla et al., 2009), GFAP and Ck intermediate filaments (Lillo et al., 2002;Parrilla et al., 2009), glutamine synthetase (GS) an enzyme which converts the glutamate into glutamine (Clemente et al., 2008;Parrilla et al., 2009), zonula ocludens1 (ZO1) located in the tight junctions (Mack and Wolburg, 2006) and S100 calcium-modulated protein (Jimeno et al., 1999;Vecino et al., 1997;Velasco et al., 1997). Moreover, we analyzed cell division in the ONH after ON crush and identified a Pax2 þ /PCNA þ astroblast pool in the ONH which is activated after an injury (Parrilla et al., 2009). ...
... Our results in control animals suggests that Pax2 þ and S100 þ astrocytes form different subpopulations in fish ONH (Lillo et al., 2002;Parrilla et al., 2009). Our results and others show that the ONH S100 þ astrocyte population does not undergo modifications after ON crush (Vecino et al., 1997), while the Pax2 þ astrocytes react strongly, supporting the idea that they are two astrocyte populations. This S100 þ astrocyte behavior in the ONH after ON crush differs from that following PGZ cryolesion, when there is a strong reaction after the injury (Jimeno et al., 1999;Parrilla et al., 2012). ...
The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and the Pax2(+) astrocyte population in the ONH strongly reacted during the regeneration of the retina after a lesion in goldfish. In the present work we have analyzed Pax2 expression in the goldfish ONH after optic nerve (ON) crush. At one week post-injury, when the regenerating axons arrive at the ONH, the pax2 gene expression level increases as well as the number of Pax2(+) astrocytes in this region. These Pax2(+) astrocytes show a higher number of Cytokeratin (Ck)(+)/GFAP(+) processes compared with control animals. In contrast, a different S100(+) astrocyte population is not modified and persists similar to that of controls. Furthermore, we find a ring that surrounds the posterior ONH that is formed by highly reactive astrocytes, positive to Pax2, GFAP, Ck, S100, GS and ZO1. In this region we also find a source of new astrocytes Pax2(+)/PCNA(+) that is activated after the injury. We conclude that Pax2(+) astrocytes constitute a subpopulation of ONH astrocytes that strongly reacts after ON crush and supports our previous results obtained after retina regeneration. Altogether, this suggests that pax2 gene expression and Pax2(+) astrocytes are probably directly involved in the process of axonal regeneration.
... During both development and, in fish, also during continuous growth, Müller cells establish contacts with the growing axons and release different adhesion and guidance molecules, such as laminins, ephrins or immunoglobulins to the basal lamina participating in the guidance of axons through the retina [4,5,6]. Moreover, astrocytes in the nerve fiber layer (NFL) pack the RGC axons and seem to participate in guiding young RGC axons coming from the PGZ [7,8]. In the ONH, both types of glial cells are involved in the formation of a favorable environment, expressing guidance molecules such as R-Cadherin, Netrin1 or Slit2, which promote the axon growth and their incorporation into the optic nerve [4,5,6]. ...
... Using the goldfish (Carassius auratus) as an animal model of central nervous system regeneration, we find that the Pax2 + astrocyte population and pax2a gene expression are affected in the absence of young RGC axons after PGZ elimination and react strongly when the regenerating axons reach the ONH again. We also compare Pax2 + astrocytes [19] with the S100 + astrocyte population in the retina and ONH, previously described [7,8,17], and we find that they belong to different astrocyte subpopulations. Moreover, we discover that S100 + astrocytes are not only involved in ONH reorganization after an injury, but also in PGZ regeneration. ...
... 6D–E). Both S100 + /GFAP 2 (Fig. 6D) cells and S100 + / GFAP + cells (Fig. 6E) showed the same elongated morphology and location in the NFL, suggesting that both of them are astrocytes, as previously proposed for tench [8]. ...
The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2(+) astrocytes located in the ONH and S100(+) astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2(+) and S100(+) astrocytes have different responses. At 7 days after injury the number of Pax2(+) cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100(+) astrocytes in the retina surrounding the ONH and S100(+) processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100(+) astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2(+) cells increase. At the same time, S100(+)/GFAP(+)/GS(+) astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2(+)/PCNA(+) astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2(+) astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process.
... In teleosts, immunoreactivity to S100 protein was reported in hair cells of the saccule and saccular nerve [24], Müller cells and astrocytes of the retina [25], kidney juxtaglomerular cells [26], hair cells of the lateral line [27], crypt olfactory neurons [28], and glial cells of central nervous system [29], but never in any cellular type with contractile capability. With respect to the S100 protein immunosignal in rodlets of mature stages, it also could be explained on the basis of granule composition. ...
... Sato and Hitomi [14] reported that differentiated human oesophageal epithelial cells express S100 protein, whereas S100 immunoreaction in immature proliferating cells located in basal strata of the epithelium is absent, thus demonstrating that S100 protein expression is closely associated with cell differentiation. In teleosts, immunoreactivity to S100 protein was reported in hair cells of the saccule and saccular nerve [24], Müller cells and astrocytes of the retina [25], kidney juxtaglomerular cells [26], hair cells of the lateral line [27], crypt olfactory neurons [28], and glial cells of central nervous system [29], but never in any cellular type with contractile capability. With respect to the S100 protein immunosignal in rodlets of mature stages, it also could be explained on the basis of granule composition. ...
Rodlet cells are an enigmatic cell type described in tissues of both marine and freshwater teleosts. Although their structure is well established, up to date their function remains subject of debate. However, there is consensus among the majority of researchers that rodlet cells play an important role within immune system, and this function is probably related with the release of rodlets due to contractile capability of their fibrous layer. Regulation of the contraction mechanism would require proteins that modulate Ca(++) intracellular concentration to be expressed in rodlet cells. We performed a morphological and immunohistochemical study at light and electron microscopy levels to assess S100 protein immunoreactivity in developing rodlet cells. Immature stages did not exhibit immunoreactive signal; however, immunoreactivity was observed in the fibrous layer of both transitional and mature rodlet cells. The latter stage also showed immunosignal within the rodlets. These findings suggest a clear association between S100 protein expression and rodlet cell development that could be linked to the regulation of rodlet activity and contractile property of their fibrous layer. Furthermore, S100 protein antibody constitutes a novel marker for rodlet cells that could be used in future studies of this particular cell type.
... The primary antibodies and their concentrations are listed in Table 1. Specificities of the antibodies have been described previously (Dahl et al., 1985;Mastronardi et al., 1993;Vecino et al., 1997;Mack et al., 1998;Wang, 2001;Sueiro et al., 2003;Raymond et al., 2006). Anti-keratin was used to identify epithelial cells and antibodies against GFAP, Cx43, GS, and S100 to specify astroglial cells. ...
Astroglial cell lines have many applications for advancing neural developmental and functional studies. However, few astroglial cell lines have been reported from fish. In this study, we report the characterization of the immortal cell line TB2 isolated from adult tilapia brain tissue. The cell line was established at 25 degrees C in L15 medium supplemented with 15% fetal bovine serum. Most of the cells displayed a fibrous morphology and were immunoreactive for A2B5 antigen, glial fibrillary acidic protein (GFAP), keratin, vimentin, and the gap junction protein connexin 43 (Cx43). They weakly expressed glutamine synthetase (GS), S100 protein, and the neural stem cell markers Sox2 and brain lipid binding protein (BLBP). In contrast to astroglia in vivo, most TB2 cells also expressed galactocerebroside (GalC), substance P (SP), and tyrosine hydroxylase (TH). By immunoblot and RT-PCR, the cells also expressed myelin basic protein (MBP), proteolipid protein (PLP), and Cx35. On a poly-L-lysine-coated substrate in vitro, TB2 cells showed increases in neuronal dopamine decarboxylase (DDC) and microtubule-associated protein 2 (MAP2), indicating that they can initiate differentiation into neurons. Taken together, the results suggest that TB2 cells are astroglial progenitor cells (neural stem cells) and may develop into oligodendrocytes and neurons in a suitable environment. The present study advances our knowledge of fish astroglia. However, the factors that affect neural development in fish remain unknown, as do the characteristics of the intermediate differentiation stages between stem cells and mature nerve cells. The TB2 cell line will promote these investigations.
... As far as we known the presence of S100 protein in the central nervous system of the zebrafish (Danio rerio) has been never reported. Nevertheless, its presence has been confirmed in different sensory organs of the teleosts including the retina (Vecino et al., 1997;Velasco et al., 1997), neuromasts of the lateral line system (Abbate et al., 2002;Germanà et al., 2002), inner ear and olfactory epithelium (Catania et al., 2007;Germanà et al., 2004aGermanà et al., ,b, 2007. ...
... On the other hand, the immunohistochemical study we have conducted the complete map of the distribution of S100 protein in the zebrafish, since this protein has been localized previously only in some mechanosensory (Abbate et al., 2002;Germanà et al., 2004aGermanà et al., , 2007 and chemosensory (Germanà et al., 2004b) cells. The information so far available about the occurrence and distribution of S100 protein in teleosts other than zebrafish is also primarily focused on some sensory organs (Vecino et al., 1997;Velasco et al., 1997), the mechanosensory cells (Foster et al., 1993;Germanà et al., 2002;Saidel et al., 1990), or the kidney (de Girolamo et al., 2000(de Girolamo et al., , 2003. Only Manso et al. (1997) have reported the distribution of S100 protein-like in the trout brain. ...
S100 proteins are EF-hand calcium-binding protein highly preserved during evolution present in both neuronal and non-neuronal tissues of the higher vertebrates. Data about the expression of S100 protein in fishes are scarce, and no data are available on zebrafish, a common model used in biology to study development but also human diseases. In this study, we have investigated the expression of S100 protein in the central nervous system of adult zebrafish using PCR, Western blot, and immunohistochemistry. The central nervous system of the adult zebrafish express S100 protein mRNA, and contain a protein of approximately 10 kDa identified as S100 protein. S100 protein immunoreactivity was detected widespread distributed in the central nervous system, labeling the cytoplasm of both neuronal and non-neuronal cells. In fact, S100 protein immunoreactivity was primarily found in glial and ependymal cells, whereas the only neurons displaying S100 immunoreactivity were the Purkinje's neurons of the cerebellar cortex and those forming the deep cerebellar nuclei. Outside the central nervous system, S100 protein immunoreactivity was observed in a subpopulation of sensory and sympathetic neurons, and it was absent from the enteric nervous system. The functional role of S100 protein in both neurons and non-neuronal cells of the zebrafish central nervous system remains to be elucidated, but present results might serve as baseline for future experimental studies using this teleost as a model.
... Four different antibodies were used to examine the glial population of the ONH and retina. Rabbit anti-S100 (Dako; Carpinteria, CA), previously used in teleosts to label mature astrocytes, labeled part of the Müller cell population and horizontal cells (Vecino et al. 1997; Velasco et al. 1997). The mouse anti-GS antibody (Chemicon; Temecula, CA), previously used in fish (Mack et al. 1998; Peterson et al. 2001) labeled the entire population of Müller cells. ...
... (D) Magnification of the square (2) in B, showing two kinds of junctions among the astrocytes' processes that form the glia limitans of the optic nerve, desmosomes (arrows) and tight junctions (arrowheads ). Bars 0.15 m.Easter et al. 1984; Bastmeyer et al. 1995; Vecino et al. 1997; Ott et al. 1998; Petrausch et al. 2000), but none of them describes the particular distribution of the vitreal processes of the Müller cells that we have shown in the optic disc of tench. This area appears to be exposed to several mechanical forces because it is a transition zone between the neural retina and the optic nerve. ...
... In the intraocular portion of the ONH of animals that lack or present a poorly developed lamina cribrosa , as in mouse (Fujita et al. 2000), rat (Wolburg and Buerle 1993) and turtle (Dávila et al. 1987), this area is occupied by several thick astrocytic processes. In a previous IHC study of the tench ONH, the authors described many cells around the optic disc that are S100 (Vecino et al. 1997) and suggested that these cells could form an interface between the retina and the optic nerve. Moreover, in a previous study of the GFAP labeling pattern in the visual pathway of the goldfish (Levine 1989 ), it was shown that both the astrocytes of the ONH and those of the optic tract are GFAP , although the astrocytes of the rest of the optic nerve are GFAP-negative (GFAP ). ...
This study demonstrates the peculiarities of the glial organization of the optic nerve head (ONH) of a fish, the tench (Tinca tinca), by using immunohistochemistry and electron microscopy. We employed antibodies specific for the macroglial cells: glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), and S100. We also used the N518 antibody to label the new ganglion cells' axons, which are continuously added to the fish retina, and the anti-proliferating cell nuclear antigen (PCNA) antibody to specifically locate dividing cells. We demonstrate a specific regional adaptation of the GS-S100-positive Müller cells' vitreal processes around the optic disc, strongly labeled with the anti-GFAP antibody. In direct contact with these Müller cells' vitreal processes, there are S100-positive astrocytes and S100-negative cells ultrastructurally identified as microglial cells. Moreover, a population of PCNA-positive cells, characterized as glioblasts, forms the limit between the retina and the optic nerve in a region homologous to the Kuhnt intermediary tissue of mammals. Finally, in the intraocular portion of the optic nerve there are differentiating oligodendrocytes arranged in rows. Both the glioblasts and the rows of developing cells could serve as a pool of glial elements for the continuous growth of the visual system.
... In the present study we have observed, for the first time, a high density of glial cells in the visual pathways of tench using immunocytochemical methods. In a recent article [58] we classified S-100-positive cells of the tench optic nerve as astrocytes. However, S-100-stained glial cells in the remaining portions of the tench visual pathway were found to be more similar to the oligodendrocytes described in previous studies [16,32], although S-100 could have labeled more than one type of glial cell. ...
Glial cells in the normal and regenerating visual pathways of Tinca tinca (Cyprinid, Teleost) were studied by labelling with anti-S-100 antibody. In normal fish, S-100-positive bipolar cells were found in the optic nerve, optic tract, and in the diencephalic visual pathways. After crushing the left optic nerve, the distribution and the number of S-100-immunoreactive cells were modified. In the injured nerve, 7 to 15 days after crushing no immunoreactive cell bodies were found in the crushed area, but a great number of S-100-positive cells were found on both sides of the injured area. Sixty days after crushing, positive cells penetrating the crushed area were observed; the normal pattern was almost restored 200 days after crushing. In the diencephalon, 25 days after crushing, the number of S-100-positive cells increased remarkably and the most intense immunostaining of glial processes was observed 60 days after crushing. The density of S-100-labelled cells decreased after 4 months postcrushing. However, in the optic tectum no changes were observed. The increase of glial cells in the lesioned visual pathway suggests that they could play an important role in axonal regeneration after crushing.
The purpose of this chapter is to review some of our studies on the localisation of neurotrophins and their receptors in glial cells of the optic nerve and retina of the fish (tench) and rat. The fish optic nerve has the capacity to regenerate after damage and the retina grows throughout life. These characteristics are not associated with the same tissues of the rat. Neurotrophins are thought to be involved in development and regeneration so a difference in the distribution of neurotrophins and their receptors in retina/optic nerve in rat and fish may relate to such functions. At least five different-types of neurotrophin molecules exist of which nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) have been best studied. Neurotrophin receptors of which there are two kinds, the low affinity receptor (p75) and the high affinity tyrosine kinase receptors (Trk) exists at varying states and have varying affinities for the different type of neurotrophins. The functional role of the neurotrophins and their receptors and ways of studying these molecules in the retina are also discussed in this overview.
