Distribution of ferritin in the rat hippocampus after kainate-induced neuronal injury

A gradual increase in iron occurs in the lesioned hippocampus after neuronal injury induced by the excitotoxin kainate, and the present study was carried out to investigate whether this increase in iron might be associated with changes in expression of the iron binding protein, ferritin. An increase in ferritin immunoreactivity was observed in glial cells of the hippocampus, as early as three days after intracerebroventricular injections of kainate. The number of ferritin positive cells peaked four weeks after the kainate injection, and decreased eight and twelve weeks after injection. They were found to be mostly microglia and oligodendrocytes by double immunofluorescence labeling with glial markers. A number of ferritin-labeled endothelial cells were also observed via electron microscopy. The decline in ferritin immunoreactivity four weeks after the injection of kainate is accompanied by an increase in the number of ferric and ferrous iron positive cells in the lesioned tissue. A substantial non-overlap between ferritin and iron-containing cells was observed. In particular, spherical ferric or ferrous iron-laden cells in the degenerating hippocampus were unlabeled for ferritin for long time periods after the kainate injection. An increase in iron, together with a reduced expression of iron binding proteins such as ferritin at long time intervals after kainate lesions, could result in a relative decrease in ferritin-induced ferroxidase activity and the presence of some of the iron in the ferrous form. It is postulated that this may contribute to chronic neuronal injury, following acute kainate-induced neurodegeneration.     

A light and electron microscopic study of NG2 chondroitin sulfate proteoglycan-positive oligodendrocyte precursor cells in the normal and kainate-lesioned rat hippocampus.

The adult brain contains a large population of oligodendrocyte precursor cells that can be identified using antibodies against the NG2 chondroitin sulfate proteoglycan. The functions of this newly recognized class of glial cells in the normal or pathological brain are not well understood. To begin to elucidate these functions, we have examined the morphology and distribution of oligodendrocyte precursor cells in the hippocampus and neocortex of normal and kainate-lesioned rats by anti-NG2 immunocytochemistry using light and electron microscopy. Large numbers of oligodendrocyte precursor cells were present in all layers of the neocortex and hippocampus. These cells differed in their morphology from astrocytes, oligodendrocytes and microglia. The processes of these cells often surrounded unlabeled areas of clear cytoplasm. At the electron microscopic level, some of the profiles that were enclosed by oligodendrocyte precursor cell processes contained synaptic vesicles. Other enclosed profiles were dendrites or dendritic spines. NG2-immunopositive processes were also observed to interpose between axon terminals containing round vesicles and dendrites with thick postsynaptic densities. After kainate injection, the NG2-positive oligodendrocyte precursor cells in the hippocampus displayed reactive changes characterized by swollen cell bodies, an increased number of small, filopodial-like processes, and higher levels of immunodetectable NG2. Both viable and degenerating oligodendrocyte precursor cells were observed with electron microscopy. These observations emphasize the dynamic nature of the oligodendrocyte precursor cell and suggest that, in addition to participating in the glial reactions to excitotoxic damage, oligodendrocyte precursor cells may regulate the stability, structure and function of synapses in the normal central nervous system.     

Quinacrine attenuates increases in divalent metal transporter-1 and iron levels in the rat hippocampus,after kainate-induced neuronal injury

The present investigation was carried out to elucidate the effect of the antimalarial drug quinacrine on levels of expression of the non-heme iron transporter, divalent metal transporter-1 (DMT1) and iron, in the hippocampus of rats after kainate treatment. The untreated hippocampus was lightly stained for DMT1, while an increase in DMT1 staining in astrocytes in the degenerating cornu ammonis (CA) fields, after kainate lesions. The increased DMT1 immunoreactivity was correlated with increased levels of Fe3+ and Fe2+ staining in the CA fields, as demonstrated by iron histochemistry (Perl's and Turnbull's blue stain for Fe3+ and Fe2+). The increases in DMT1 and iron staining were significantly attenuated by quinacrine. Rats injected with kainate and daily i.p. injections of quinacrine (5 mg/kg) for 7 days or 2 weeks showed significantly lower levels of DMT1 immunoreactivity and iron staining, compared with rats injected with kainate and saline. These results show that DMT1 expression is closely linked to iron levels, and provide further support for a crucial role that DMT1 plays in iron accumulation in the degenerating hippocampus.     

Heme oxgenase-1 is expressed in viable astrocytes and microglia but in degenerating pyramidal neurons in the kainate-lesioned rat hippocampus

The present study aimed to elucidate the distribution of heme oxygenase-1 (HO-1) in the hippocampus after intracerebroventricular injections of kainate. Very little or no staining of HO-1 was observed in the normal CA1, whilst moderate staining of dentate hilar neurons was observed in the dentate gyrus, in the normal hippocampus. At postinjection day 1, a slight increase in immunoreactivity in the neuropil of the lesioned CA fields and a marked increase in HO-1 immunoreactivity in glial cells of the stratum lacunosum moleculare of CA fields and the stratum moleculare of the dentate gyrus was observed. Electron microscopy showed that the glial cells had features of viable astrocytes. At postinjection day 3, glial cells in the dentate gyrus continued to express HO-1, whilst pyramidal neurons in the degenerating CA fields started to express intense HO-1 immunoreactivity in their cell bodies. At postinjection weeks 1–3, HO-1 was observed in glial cells in the center of the lesion, but also in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, whilst the neurons contained discontinuous cell membranes and nuclear outlines, and had features of degenerating neurons. Intense immunoreactivity was observed in the cytoplasm of the degenerating neurons. The density of staining was greater than that observed in astrocytes or microglia. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than 15-fold) were associated with significant oxygen toxicity. These and the present observations suggest a destructive effect of increased expression of HO-1 in neurons, and possible novel therapeutic approaches involving overexpression of HO-1 must therefore be approached with caution. Electronic Publication     

An immunocytochemical study of glutamate receptors and glutamine synthetase in the hippocampus of rats injected with kainate

Immunocytochemistry was used to study the distribution of the kainate receptors GluR1, GluR2/3 and GluR4 and of the N-methyl-d-aspartate (NMDA) receptor NMDAR1 as well as the astrocyte markers glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) in the hippocampus of normal and kainate-lesioned rats. Hippocampal pyramidal neurons and dentate granule neurons were labelled heavily for GluR1 and GluR2/3, but only lightly for GluR4. Dense GluR4 immunopositivity was, however, observed in oligodendrocyte-like glial cells. Hippocampal pyramidal neurons and dentate granule neurons were moderately labelled for NMDAR1. Intravenous kainate injections resulted in a decrease in GluR1 and GluR2/3 immunoreactivity on the apical dendrites of pyramidal neurons as early as 7 h postinjection. At 18 h, there was a marked reduction in GluR1 and GluR2/3 receptors in the terminal tuft of dendrites of most hippocampal pyramidal neurons in the affected area, although some cells showed labelling in other portions of the apical dendrites and in basal dendrites. Immunostaining for GluR4 and NMDAR1 was also reduced at this time. At postinjection day 3, only the cell bodies and the basal dendrites of a few scattered pyramidal cells were labelled. Taken together, these results indicate a progressive loss of glutamate receptors, which affects the apical dendritic tree before the basal dendritic tree. The decrease in receptor immunoreactivity could be due to a downregulation of the receptors, since it occurred as early as 7 h postlesion, before cell death was evident in Nissl-stained sections. At long intervals after kainate injection, all pyramidal cells at the centre of the lesion showed a lack of glutamate receptor staining, and no partially labelled pyramidal cells were observed. The periphery of the lesion, however, contained many partially labelled pyramidal neurons among the unlabelled cells and had features of early lesions. The present study also showed an early decrease in GS immunoreactivity in the affected CA fields of the hippocampus (18 h to 3 days postinjection), followed by a medium-term increase (5–68 days) and a late decrease in GS immunoreactivity (81 days). The decrease in GS immunoreactivity at 81 days is not due to an absence of astrocytes, since GFAP staining showed many densely labelled astrocytes in the affected CA field.     

Changes in glutathione in the hippocampus of rats injected with kainate: depletion in neurons and upregulation in glia

The aim of the present study was to elucidate the distribution of glutathione immunoreactivity in the normal hippocampus and after kainate-induced neuronal injury. A specific antibody was used that recognizes both the reduced (GSH) and oxidized (GSSG) forms of glutathione. Immunoreactivity to glutathione was observed in neurons, but few immunolabeled glial cells were observed in the normal hippocampus. After kainate injection, a decrease in glutathione immunoreactivity was observed in pyramidal neurons from as early as 1 day after injection. In contrast, dense staining to glutathione was observed in large numbers of reactive astrocytes at 3 days to 6 weeks after kainate injection. This suggests upregulation of glutathione synthesis in these cells. One possibility is that the high content of glutathione is protective to reactive astrocytes. Another possibility is that the high glutathione concentration in reactive astrocytes may be protective to neurons around the glial scar.     

An immunocytochemical study of calpain II in the hippocampus of rats injected with kainate

The distributions of the kainate/dl-alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (KA/AMPA) receptors GluR1 and calcium-activated neutral protease II (calpain II) in the hippocampus of normal and kainate-lesioned rats were studied by immunocytochemistry. There was a reduction in GluR1 immunoreactivity and a slight increase in calpain II immunoreactivity on the dendrites of pyramidal neurons in CA fields affected by the kainate at 18 h postinjection. Calpain II immunoreactivity was associated with amyloid fibrils at electron microscopy. These fibrils were most often intracellular, in membrane-bound profiles, some of which were contacted by axon terminals and were identified as degenerating dendrites. There was extensive destruction of mitochondrial membranes in degenerating profiles, and accumulations of amyloid fibrils were often localised in mitochondria in a calpain-positive profile. This was unlike other, calpain-negative degenerating profiles, that contained tubulovesicular profiles or multilamellar bodies, where mitochondrial membranes were preserved. Many more calpain-positive profiles were observed at electron microscopy 6 days after kainate injection. The enzyme was present in macrophages and astrocytes in lesioned areas.     

Delayed apoptotic pyramidal cell death in CA4 and CA1 hippocampal subfields after a single intraseptal injection of kainate

We have performed a detailed time-course analysis of cell death in the hippocampal formation, basal forebrain and amygdala following a single intraseptal injection of kainate in adult rats. Acetylcholinesterase histochemistry revealed a profound loss of staining in the medial septum but not in the diagonal band, and cholinergic fiber density was highly reduced in the hippocampus and amygdala at 10 days postinjection. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphatebiotin nick end labeling (TUNEL) histochemistry was performed for precise location of apoptotic cells. Both the medial septum and amygdala exhibited numerous TUNEL-positive nuclei after the intraseptal injection of kainate, while the lateral septum exhibited a lower but significant incidence in terms of apoptotic cells. In the medial septum, the presence of apoptotic cells was at a location displaying acetylcholinesterase staining. TUNEL histochemistry revealed a time-dependent sequential apoptotic cell death in hippocampal pyramidal cells. During the first two days postinjection, apoptosis in the hippocampus was only evident in the CA3 region. At five days postinjection, the entire CA4 region became apoptotic. At 10 days postinjection, the whole extent of the CA1 pyramidal cell layer exhibited numerous TUNEL-positive nuclei. The time-course of kainate-induced apoptosis in Ammons's horn correlated with the disappearance of hippocampal pyramidal neurons as detected by Nissl staining, which is suggestive of a prominent apoptotic death for these cells. The temporal delayed distant damage to CA4 and CA1 hippocampal subfields after a single intraseptal kainate injection is not seen in other models employing kainate and may be a valuable tool for exploring the cellular mechanisms leading to cell death in conditions of status epilepticus.     

Kainate-induced neuronal injury leads to persistent phosphorylation of cAMP response element-binding protein in glial and endothelial cells in the hippocampus

Intracerebroventricular kainate treatment in rats induces neuronal cell death, followed by proliferation and hypertrophy of glial cells in the lesioned area. To further understand the activated signal transduction pathways and to get insights into potential target gene activation, the present study aims to elucidate long-term effects on the phosphorylation state of cAMP response element-binding protein (CREB) in the hippocampal formation. One to four weeks after kainate injection, we found high levels of phosphorylated and hence activated CREB (pCREB) in glial cells of the degenerating CA fields. As shown by electron microscopy, pCREB immunoreactivity was present in reactive astrocytes, oligodendrocyte precursor cells and endothelial cells of blood vessels. It is postulated that pCREB could drive the expression of downstream genes in these cells to promote cell proliferation and survival.     

Mechanism of Theiler's virus-induced demyelination in nude mice

In its natural murine host, infection with Theiler's murine encephalomyelitis virus (TMEV) produces a chronic, progressive demyelinating disease. To help elucidate the role of host immune mechanisms involved in demyelination, we studied TMEV infection in Nude mice. These animals demonstrated rising titers of infectious virus within the central nervous system and failed to produce anti-TMEV antibody. Neurologic signs including the development of severe hind limb paralysis were evident approximately 2 weeks postinfection with most animals succumbing within the first month. Immunoperoxidase studies demonstrated viral antigen in the cytoplasm of neurons and glial cells for the entire period of observation. Plaques of demyelination associated with scanty inflammatory infiltrates were present in the spinal cord by 14 days postinfection. Electron microscopic studies of the involved white matter revealed numerous degenerating glial cells, many of which contained paracrystalline arrays of picornavirus within their cytoplasm. Some of the infected glial cells were identified as oligodendrocytes by demonstrating their myelin-plasma membrane connections. The studies indicate that in Nude mice TMEV causes a lytic infection of oligodendrocytes producing demyelination independent of the T lymphocyte immune system.     

Demyelination, and remyelination by Schwann cells and oligodendrocytes after kainate-induced neuronal depletion in the central nervous system.

Excitotoxins are thought to kill neurons while sparing afferent fibers and axons of passage. The validity of this classical conclusion has recently been questioned by the demonstration of axonal demyelination. In addition, axons are submitted to a profound alteration of their glial environment. This work was, therefore, undertaken to reassess axonoglial interactions over time after an excitotoxic lesion in the rat. Ultrastructural studies were carried out in the ventrobasal thalamus two days to 18 months after neuronal depletion by in situ injections of kainic acid. In some cases, lemniscal afferents were identified by using anterograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei. Two and four days after kainate injection, numerous dying axons displaying typical signs of Wallerian degeneration were observed in a neuropile characterized by the loss of neuronal somata and dendrites, an increase in number of microglia/macrophages and the disappearance of astrocytes. Ten and 12 days after kainate injection, degenerating axons were no longer observed although myelin degeneration of otherwise unaltered axons was ongoing with an accumulation of myelin remnants in the neuropile. At 16 and 20 days, the demyelination process was apparently complete and axons of different diameters were sometimes packed together. One and two months after kainate injection, the axonal environment changed again: remyelination of large-caliber axons occurred at the same time as reactive astrocytes, oligodendrocytes and numerous Schwann cells appeared in the tissue. Schwann cell processes surrounded aggregates of axons of diverse calibers, ensheathed small ones and myelinated larger ones. Axons were also remyelinated by oligodendrocytes. Horseradish peroxidase-labeled lemniscal afferents could be myelinated by either of the two cell types. After three months, the neuropile exhibited an increase in number of hypertrophied astrocytes and the progressive loss of any other cellular or axonal element. At this stage, remaining Schwann cells were surrounded by a glia limitans formed by astrocytic processes. These data indicate that although excitotoxins are sparing the axons, they are having a profound and complex effect on the axonal environment. Demyelination occurs over the first weeks, accompanying the loss of astrocytes and oligodendrocytes. Axonal ensheathment and remyelination takes place in a second period, associated with the reappearance of oligodendrocytes and recruitment of numerous Schwann cells, while reactive astrocytes appear in the tissue at a slightly later time. Over the following months, astrocytes occupy a greater proportion of the neuron-depleted territory and other elements decrease in number.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quinacrine abolishes increases in cytoplasmic phospholipase A<Subscript>2</Subscript> mRNA levels in the rat hippocampus after kainate-induced neuronal injury

The present investigation was carried out to study the possible effects of quinacrine in modulating cytoplasmic phospholipase A₂ (cPLA₂) mRNA levels in rat hippocampus after kainate treatment. Injections of kainate into the right lateral ventricle resulted in significant increases in cPLA₂ mRNA levels in the hippocampus, at 3 days and 7 days after injection. The elevation in cPLA₂ mRNA levels is consistent with previous observations of increased cPLA₂ immunoreactivity in degenerating neurons and astrocytes at these times. Rats that received once daily intraperitoneal injections of quinacrine (5 mg/kg) after the intracerebroventricular kainate injections showed almost complete attenuation of increased cPLA₂ expression, at both 3 and 7 days after kainate injection. These results show that in addition to its well-known effect of inhibition of PLA₂ activity, quinacrine could also inhibit cPLA₂ expression, and further supports a role for PLA₂ in kainate-induced neuronal injury. Electronic Publication     

Discrimination between different types of neuroglial cells in rat central nervous system using combined immuno- and enzyme-histochemical methods

The central nervous system (CNS) contains several types of neuroglial cells. In the present study, we characterized different types of glial cells in rat CNS by using single and combined immuno- and enzyme-histochemical methods, and immunofluorescence techniques. Two recently developed monoclonal antibodies (mAbs) against rat macrophages-associated antigens appeared to recognize a subpopulation of glial cells in the CNS of normal adult rats. These ED4- and ED8-positive glial cells were predominantly located in the white matter of adult rat CNS and shared morphological features with microglia. ED4 and ED8 were applied in a double staining combined with mAbs and an antiserum raised against galactocerebroside (GalC) to identify oligodendrocytes, or with anti-glial fibrillary acidic protein antiserum (GFA) to identify astrocytes. We also used a mAb against myelin basic protein (MBP) to identify oligodendrocytes. It appeared that ED4 and ED8 recognized a subpopulation of oligodendrocytes. MAbs against GalC and MBP recognized cells in an immunoperoxidase staining with a morphology identical to that of the ED8-positive cells and part of the ED4-positive cells. Frozen sections of Lewis rats CNS with acute experimental allergic encephalomyelitis (EAE) were investigated, where infiltrating brain macrophages could be found which stained positively with ED4 and ED8 as well as with the monocyte/macrophage mAbs ED1 and ED2. These brain macrophages did not stain when GalC, MBP and GFA markers were applied. Furthermore, ED4+GalC+ and ED8+GalC+ oligodendrocytes were present in the CNS white matter of EAE animals with similar appearance as in normal adult rats. With the currently used markers, we could not detect a third type of neuroglial cell, besides the astrocytes and oligodendrocytes. Thus, none of our anti-macrophage monoclonals recognized the presumptive microglia. Only under pathological conditions, e.g., in inflammatory infiltrates in the course of EAE, could brain macrophages be detected in the CNS parenchyma and only in the direct vicinity of blood vessels, indicating their hematogenous origin.     

Induction of P-glycoprotein expression in astrocytes following intracerebroventricular kainate injections

 The expression of P-glycoprotein (PGP) was studied by immunocytochemistry and light and electron microscopy, in normal rats and after intracerebroventricular kainate injections. Two antibodies to PGP, mdr (Ab-1) and c-219, were used. As in previous studies (Thiebault et al. and Jetté et al.), labelled capillaries were observed in normal rats. Kainate injections resulted in death of pyramidal neurons in the hippocampus, and a proliferation of glial cells in the affected cornu ammonis fields. An increase in PGP expression was observed in reactive astrocytes as early as 1 day postinjection. Immunoreactivity peaked at 2 weeks postinjection, but was still visible as late as 10 weeks postinjection. Similar results were observed using the two antibodies. Double immunolabelling and confocal microscopy also showed that PGP was colocalised with GFAP, a marker for astrocytes. The expression of PGP in astrocytes was confirmed by electron microscopy, which showed immunoreaction product in cells containing dense bundles of glial filaments and features of reactive astrocytes. The increased PGP expression in reactive astrocytes could be part of a cellular stress response program in these cells. Received: 17 August 1998 / Accepted: 26 January 1999     

A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus

We have studied by immunocytochemistry, the distribution of DMT-1, a cellular iron transporter responsible for transport of metal irons from the plasma membrane to endosomes, in the normal monkey cerebral neocortex and hippocampus. Light to moderate DMT-1 staining was observed in glial cell bodies in the neocortex, the subcortical white matter, and the hippocampus. Despite light labeling of cell bodies, glial end feet around cortical and subcortical blood vessels were heavily labeled. In the neocortex, the glial cell bodies displayed the morphological features of protoplasmic astrocytes. Labeled glial cells in the subcortical white matter contained dense bundles of glial filaments and were identified as fibrous astrocytes. The observation that DMT-1 was present on astrocytic endfeet suggests that these cells are involved in uptake of iron from endothelial cells. It is possible that the iron could then be redistributed into the extracellular space in the brain parenchyma.     

Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation

We investigated the effects of erythropoietin (Epo) in glial cell development, especially the maturation of late stage immature oligodendrocytes and the proliferation of astrocytes. Epo mRNA level in oligodendrocytes was much more prominent than those in neurons or astrocytes, which were the same as those in the young adult kidney, while Epo receptor (Epo-R) mRNA level were almost the same among neural cells, kidney and liver tissues. On immunohistochemical examination, Epo-R expression was also detected in O4-positive immature oligodendrocytes and glial fibrillary acidic protein positive astrocytes. These results suggested that types of both glial cells are responsive to Epo. The numbers of mature oligodendrocytes, which are characterized by myelin basic protein and process development, were increased by treatment with recombinant human Epo (rhEpo) (0.001-0.1 U/ml). The maturation of oligodendrocytes was also enhanced by coculture with astrocytes in vitro. However, when mixed cultured cells (oligodendrocytes+astrocytes) were treated with anti-Epo antibody and/or soluble Epo-R, the differentiation of oligodendrocytes was partially inhibited. Interestingly, high dose rhEpo (1, 3, 10 U/ml) markedly enhanced the proliferation of astrocytes. These results suggested that Epo not only promotes the differentiation and/or maturation in oligodendrocytes, but also enhances the proliferation of astrocytes. It is generally accepted that astrocytes produce Epo, and therefore Epo might act on astrocytes in an autocrine manner. The astrocytes stimulated with Epo may further accelerate the maturation of oligodendrocytes. These comprehensive effects of Epo might also affect the ability of oligodendrocyte lineage cells to promote myelin repair in the normal and damaged adult central nervous system.     

Minisegments of newborn rat optic nerves in vitro: gliogenesis and myelination

Summary The question of whether the development of CNS glial cells requires the presence of axons or not can be studied with in vitro systems. In order to compare the differentiation of glial cells during development in vitro with that in situ, we have selected the optic nerve, which is anatomically as well as histotypically a well defined structure. For the in vitro investigations, small explants, called minisegments, of newborn rat optic nerves were cultivated taking four major conditions into account: (1) the regular size of the minisegments should guarantee a permanent exchange of the culture medium in order to avoid cell death, (2) neither mechanical nor enzymatic dissociation of the tissue were applied, (3) the minisegments were explanted into flasks without substrate for cell adhesion and (4) the minisegments were under constant gyratory agitation. The following in situ results were obtained: optic nerves of newborn rats are morphologically characterized by the presence of naked axons, astrocytes, glial precursors, and the absence of both differentiated oligodendrocytes and myelin. At postnatal day 5 myelin sheaths are still absent. Two weeks after birth, differentiated oligodendrocytes and microglial cells are present and numerous axons are surrounded by compact myelin. The in vitro experiments show the following main results, which were obtained after 14 h, 2 d, 5 d and 14 d in culture: during time in culture, the shape of minisegment of newborn rat optic nerves undergoes drastic changes, which indicate high cellular dynamics. After 14 h in vitro, axonal profiles, cells with pyknotic nuclei as well as clusters of astrocytes and glial precursors are present. After 2 days in culture the axonal profiles disappeared and the number of degenerating cells decreased drastically. Many large cells, probably phagocytes containing inclusions and more cells are differentiated. At the stage of 5 d in vitro 4 major types of cells can be distinguished: differentiated oligodendrocytes, which form compact and loose myelin, astrocytes, large and small glioblasts and phagocytes. Immunoprecipitates for myelin basic protein and/or myelin associated glycoprotein were found in oligodendrocytes, in their processes and associated to the myelin. Processes of some astrocytes showed immunoreactive products of glial fibrillary acidic protein. After two weeks in culture, the minisegments were mostly composed of astrocytes, whereas oligodendrocytes became rare and phagocytes disappeared. It can be concluded that CNS glial cells can attain their structural and immunocytochemical characteristics in the total absence of neuronal cell bodies and axons. However, it can be speculated that neurons (or neuronal factors) could regulate the number of astrocytes and oligodendrocytes and keep these glial cells in a physiological equilibrium.     

In vivo Tracking of Mesenchymal Stem Cells Labeled with a Novel Chitosan-coated Superparamagnetic Iron Oxide Nanoparticles using 3.0T MRI

This study aimed to characterize and MRI track the mesenchymal stem cells labeled with chitosan-coated superparamagnetic iron oxide (Chitosan-SPIO). Chitosan-SPIO was synthesized from a mixture of FeCl₂ and FeCl₃. The human bone marrow derived mesenchymal stem cells (hBM-MSC) were labeled with 50 µg Fe/mL chitosan-SPIO and Resovist. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity, phenotype and differentiation potential. Electron microscopic observations and Prussian blue staining revealed 100% of cells were labeled with iron particles. MR imaging was able to detect the labeled MSC successfully. Chitosan-SPIO did not show any cytotoxicity up to 200 µg Fe/mL concentration. The labeled stem cells did not exhibit any significant alterations in the surface markers expression or adipo/osteo/chondrogenic differentiation potential when compared to unlabeled control cells. After contralateral injection into rabbit ischemic brain, the iron labeled stem cells were tracked by periodical in vivo MR images. The migration of cells was also confirmed by histological studies. The novel chitosan-SPIO enables to label and track MSC for in vivo MRI without cellular alteration.