NG2 positive cells of rat spinal cord activated during experimental autoimmune encephalomyelitis are spatially associated with radially oriented astroglia and express p75 receptor: a role for nerve growth factor in oligodendrocyte progenitor migration?

Data have been provided from several studies that support the proposal that the adult oligodendrocyte progenitors migrate into the lesioned areas under conditions of experimental autoimmune encephalomyelitis (EAE). However, the routes of migration of these cells and the governing mechanisms are not clear. In the present studies, we have examined the effect of EAE upon activation of endogenous oligodendroglia progenitors and their spatial distribution in the spinal cord of Lewis rats using immunocytochemical procedures. Antibodies against the marker chondroitin sulfate proteoglycan NG2, are used for identification of oligodendroglia progenitors. We find that the activated elongated subpopulation of NG2 positive oligodendroglia progenitors of white matter is spatially associated with the radially-oriented astroglia during the acute phase of EAE. The latter re-expressed the phenotypic embryonic marker nestin while still expressing the mature astroglial marker GFAP. The elongated oligodendroglia progenitors express p75 receptor. In addition, colocalization of NG2 and p75 is observed also in ependymal neural cells of the central canal and the subventricular zone. This raises the possibility that the activated NG2+/p75+ parenchymal cell pool may also be recruited from multipotent neural cells of the germination areas. Our data suggest that, under EAE conditions, the radially oriented astroglia of juvenile phenotype may serve as scaffolding for migrating activated endogenous oligodendroglia progenitors just like radial glia provide a path for neuronal and oligodendroglia progenitor cells in embryonic stage. The expression of p75 receptor in oligodendroglia progenitors associated with radially oriented astroglia during EAE may implicate a role for NGF in the regulation of migration of oligodendroglia progenitors.

Quantitative morphological study of microglial cells in the ischemic rat brain using principal component analysis.

Pathogenic stimuli induce alterations in the morphology of microglial cells. We analysed changes in lectin-stained cells on the 1st, 3rd, 7th or 14th day after transient global ischemia. Three areas differing in the degree of microglial reaction were selected for analysis: the upper cerebral cortex, the hippocampal CA1 area, and the hilus of the dentate gyrus. Nine morphological parameters, including fractal dimension, lacunarity, self-similarity range, solidity, convexity and form factor were determined. Then the resultant data were processed using principal component analysis (PCA). We found that the two first principal components together explained more than 73% of the observed variability, and may be sufficient both to describe the morphological diversity of the cells, and to determine the dynamics and direction of the changes. In both hippocampal areas, the transformation to hypertrophied and phagocytic cells was observed, but changes in the hilus were faster than in the CA1. In contrast, in the cortex, a microglial reaction was characterised by an increase in the complexity of processes. The results presented show that the quantitative morphological analysis can be an effective tool in research on the reactive behaviour of microglia and, particularly, in the detection of small and early changes in the cells.

Morphological transformations of cells immunopositive for GFAP, TrkA or p75 in the CA1 hippocampal area following transient global ischemia in the rat. A quantitative study.

Transient global ischemia induces intensive neuronal degeneration in the hippocampal CA1 pyramidal layer, accompanied by reactive transformation of glial cells. Previously, we have shown using the double immunostaining method that the NGF receptors (NGFR) p75 and TrkA are expressed mainly on subpopulations of GFAP+ astrocytes, and this expression increases progressively after ischemia. In the presented study, we analyzed quantitatively the morphological transformations of cells immunopositive for GFAP or NGF receptors in the stratum radiatum of the CA1 hippocampal area in different survival periods after ischemia, evoked by 10-min cardiac arrest in adult rats. In control brains, NGF receptors were expressed only on small cells with poorly ramified processes. After ischemia, the NGFR+ cells increased in size and morphological complexity (measured using fractal analysis). However, even 2 weeks after ischemia these cells did not reach the size and value of the fractal dimension typical of the largest GFAP+ astrocytes. Moreover, the reaction of NGFR+ cells was significantly delayed in comparison with the total astrocyte population. The obtained results suggest that NGF receptors are expressed mainly by immature astrocytes and ischemia induces the maturation of these cells.

Concomitant up-regulation of astroglial high and low affinity nerve growth factor receptors in the CA1 hippocampal area following global transient cerebral ischemia in rat.

We have examined the effect of global transient cerebral ischemia, evoked in rat by 10 min of cardiac arrest, upon the changes in the cellular expression of two nerve growth factor (NGF) receptors (TrkA and p75) in the hippocampus. We have used immunocytochemical procedures, including a quantitative analysis of staining, along with some quantitative morphological analyses. We have found, under ischemic conditions, a decrease of TrkA immunoreactivity in degenerating CA1 pyramidal neurons and in neuropil. On the other hand, a strong, ischemia-induced up-regulation of TrkA and p75 immunoreactivity was observed in the majority of reactive astroglia population in the adjacent CA1 hippocampal region. The colocalization of the two receptors in the same reactive astroglial cells was evidenced by double immunostaining and further supported by quantitative morphological analysis of TrkA and p75 immunoreactive glial cells. Our data implicate the involvement of NGF receptors in the postischemic regulation of astrocytic function; however, the lack of NGF receptor expression on some astrocytes suggests heterogeneity of astroglia population. Our results also indicate that the lack of neuroprotective action of astroglial NGF induced in the ischemic hippocampus [J Neurosci Res 41 (1995) 684; Acta Neurobiol Exp 57 (1997) 31; Neuroscience 91 (1999) 1027] is not caused by a paucity of NGF receptors but may rather be due to the counteraction of some proinflammatory substances, released simultaneously by glia cells. On the other hand, the up-regulated astroglial TrkA receptor may be an important target for exogenous NGF, which, as previously described [J Neurosci 11 (1991) 2914; Neurosci Lett 141 (1992) 161], exerts a neuroprotective effect in ischemia.

High-affinity NGF receptor in the rat spinal cord during acute and chronic phases of experimental autoimmune encephalomyelitis: a possible functional significance.

The biological effects of Nerve Growth Factor (NGF) are primarily mediated via its high affinity receptor-TrkA. In the present study, we examined the effect of experimental autoimmune encephalomyelitis (EAE) upon the expression of TrkA in neuronal and non-neuronal cells of the spinal cord of Lewis rats during the acute (14 days postimmunization) and chronic (12 months postimmunization) phases of the disease. In the normal spinal cord, both of mature and aged rats, we found TrkA immunoreaction (TrkA-IR) in the motoneurons of the Rexed lamina IX and in both oligo- and astroglia cells. In the acute phase of the disease, we found a reduction of TrkA immunoreactivity in motoneurons and its up-regulation in oligodendroglia, mainly in the white matter. We also confirmed our previous findings concerning the up-regulation of TrkA-IR in astroglia. Both neuronal and non-neuronal changes of TrkA immunoreactivity had a transient character: they were not seen in the chronic phase of the disease. Our results suggest that both neuronal and glial TrkA expression changes depend on inflammation. Moreover, our data indicate that, during the acute phase of EAE, the glial cells become more receptive to NGF, pointing to glia as an important target for pharmacological manipulations, particularly for exogenously administered NGF.

Dentate granule neuron apoptosis and glia activation in murine hippocampus induced by trimethyltin exposure.

We investigated the effect of trimethyltin (TMT), a well-known neurotoxicant, on murine hippocampal neurons and glial cells. Three days following intraperitoneal (i.p.) injection of TMT into 1-month-old Balb/c mice at a dose of 2.5 mg/kg body weight we detected damage of the dentate gyrus granular neurons. The dying cells displayed chromatin condensation and internucleosomal DNA fragmentation, which are the most characteristic features of apoptosis. To study, if prolyl oligopeptidase is engaged in neuronal apoptosis following TMT administration, we pretreated mice with the specific inhibitor--Fmoc-Pro-ProCN in doses of 5 and 10 mg/kg body weight (i.p. injection). Three days following injection we did not observe any attenuation of neurotoxic damage, regardless of inhibitor dose, indicating the lack of prolyl oligopeptidase contribution to neuronal injury caused by TMT. The neurodegeneration was associated with reactive astrogliosis in whole hippocampus, but particularly in injured dentate gyrus. The reactive astrocytes showed an increased nerve growth factor (NGF) expression in ventral as well as dorsal hippocampal parts. NGF immunoreactivity was also augmented in neurons of CA3/CA4 areas, which were almost totally spared after TMT intoxication. It suggested a role for this neurotrophin in protection of pyramidal cells from loss of connection between CA3/CA4 and dentate gyrus fields. The granule neurons' death was accompanied by increased histochemical staining with isolectin B4, a marker of microglia, in the region of neurodegeneration. The microglial cells displayed ramified and ameboid morphology, characteristic of their reactive forms. Activated microglia were the main source of interleukin 1beta (IL-1beta). It is possible that this cytokine may participate in neurodegeneration of granule cells. Alternatively, IL-1beta elaborated by microglia could play a role in increasing NGF expression, both in astroglia and in CA3/CA4 neurons.

The upregulation of nerve growth factor receptors in reactive astrocytes of rat spinal cord during experimental autoimmune encephalomyelitis.

Using immunocytochemistry, we have examined the effect of experimental autoimmune encephalomyelitis (EAE) upon the expression of nerve growth factor (NGF) and its TrkA and p75 receptors in astroglia cells of the spinal cord of Lewis rats. We have found that, in normal spinal cord, astroglia of white matter expressed both NGF receptors while those in gray matter expressed only TrkA and no astroglia expressed NGF. During EAE, strong upregulation of TrkA in the astroglia of gray and white matter was found, particularly in a population of radially oriented astrocytes. An upregulation of p75 was noted in radial astroglia and, to some extent, also in the stellate astrocytes of white matter. In general, the upregulation of NGF receptor immunoreactivities in astroglia correlated with the strong intensification of glial fibrillary acidic protein immunocytochemistry, a prominent feature of EAE. No NGF immunoreactivity appeared in any astroglia cells during EAE. Our results suggest that, during EAE, astroglia of the spinal cord become particularly receptive to NGF, possibly as part of a mechanism enabling astroglial cells to respond to localized release of neurotrophins. Moreover, our data suggest that spinal cord astroglia cells may be a potential target for pharmacological manipulations in EAE.

Long-term microglial and astroglial activation in the hippocampus of trimethyltin-intoxicated rat: stimulation of NGF and TrkA immunoreactivities in astroglia but not in microglia.

In the present study we investigated the microglial and astroglial response after trimethyltin (TMT) exposure over a prolonged period of time. Male Wistar rats were given a single dose of TMT (8 mg/kg, i.p.) and survived 4, 7, 21, 60 and 180 days after the administration of the toxin. Histochemistry (Griffonia simplicifolia lectin staining) and immunocytochemistry for GFAP were applied to identify micro- and astroglial cells, respectively. To assess the trophic response of glial cells (NGF and TrkA expression), single or double staining experiments were performed. In addition, the biochemical evaluation of GFAP and NGF were carried out at chosen timepoints using immunoblotting technique and ELISA, respectively. The main findings of our study were as follows. (1) A protracted activation of microglia (at least up to 2 months posttreatment). (2) A long-lasting expression of GFAP immunoreactivity (at least up to 6 months posttreatment) and a steady increase in GFAP content (at least up to 2 months posttreatment). (3) The appearance of enormously enlarged, round-shape astrocytes exclusively localized to CA1 and observed 2 months posttreatment. (4) The stimulation of NGF and TrkA expression in reactive astrocytes. (5) The strongest activation of micro- and astroglia coincided with the most prominent neurodegeneration in the hippocampus, i.e., in CA4/CA3c and CA1. It is tempting to assume that the activation of glial cells in the hippocampal areas particularly vulnerable to TMT may affect neuronal fate after neurotoxic insult.

Prolonged and concomitant induction of astroglial immunoreactivity of interleukin-1beta and interleukin-6 in the rat hippocampus after transient global ischemia.

We have investigated the pattern of expression of IL-1beta and IL-6 immunoreactivities in rat hippocampus after transient complete brain ischemia evoked by a 10 min cardiac arrest, at survival times ranging from 1 day to 28 days. To identify the cell types expressing the two immunoreactivities we used specific cell markers and combined staining procedures. In the intact brain IL-1beta and IL-6 were mainly localized in neurons particularly in pyramidal and granular cell layers. Ischemic insult resulted in a concomitant induction of IL-1beta and IL-6 immunoreactivities in multiple astroglia especially in the CA1 area which is the most vulnerable to ischemic insult as manifested by a massive delayed neuronal death accompanied by an intense gliosis. The number of astroglia expressing both immunoreactivities and the intensity of staining was maximal at the 14th day and remained at the same level at the 28th day. Our data suggest that the astroglial IL-1beta and IL-6 may affect the neurodegeneration of CA1 neurons in the ischemic hippocampus and that the prolonged proinflammatory effects of IL-1beta prevail over the presumed protective action of IL-6.

GM1 ganglioside potentiates trimethyltin-induced expression of interleukin-1 beta and the nerve growth factor in reactive astrocytes in the rat hippocampus: an immunocytochemical study.

This study demonstrates potentiation by GM1 ganglioside treatment of trimethyltin (TMT) induced reactivity of astrocytes, and the expression of astroglial interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF) immunoreactivities in the rat hippocampus. GM1 treatment also results in an increase of the number of IL-1 beta and NGF immunoreactive astrocytes. Both the intensity of gliosis and stimulation of IL-1 beta and NGF expression in astrocytes mostly occurs in the regions of heaviest neurodegeneration in the hippocampus (CA4/CA3c and CA1). It is tempting to assume that enhancement of astroglial NGF expression by GM1 ganglioside may play a role in the protective action of GM1 against neurotoxic insult.

Differential susceptibility of senile and lesion-induced astrogliosis to phosphatidylserine.

The susceptibility of age- and lesion-induced astrogliosis to the treatment with phosphatidylserine was investigated with the use of GFAP immunoblotting. The existence of age-induced upregulation of GFAP content was confirmed in the hippocampus, septum, and corpus callosum of the rat. The Ptd-Ser treatment of the aged rats further increased the GFAP content in the hippocampus and corpus callosum. The GFAP content increase in the corpus callosum was additionally illustrated by the upregulation in GFAP immunostaining. In the septum no further elevation of GFAP was observed after Ptd-Ser treatment, and in the striatum the compound elicited significant GFAP content increase, absent in the untreated aged rat brain striatum. In the intact adult rat brain no effect of Ptd-Ser on GFAP content was observed; neither did the compound elicit any modulation of the astrogliosis related to the mechanical lesion of the brain in the septum, hippocampus, cortex, and striatum. In the corpus callosum, Ptd-Ser potentiated the GFAP content increase related to the mechanical lesion, pointing to the structure-related heterogeneity of astrocytic population. Because it has been previously found that Ptd-Ser partly reverses one of the aspects of rodent brain aging, the aging-induced decrease of the acetylcholine release, the possibility exists that the effects of Ptd-Ser administration on glia and neurons in the aged brain may be related.

Axonal accumulation of p75NTR and TrkA in the septum following lesion of septo-hippocampal pathways.

Septal cholinergic neurones depend on trophic support by nerve growth factor (NGF) which can rescue them from injury-induced degeneration. Since NGF exerts its effects via p75NTR and TrkA receptors coexpressed in vast majority of these neurones and down-regulated without NGF treatment after injury, in this study we aimed to examine how does the lesion to the cholinergic tracts affect distribution of both types of receptor proteins in damaged fibres. Early changes (two and seven days) were examined immunocytochemically within the septum and supracallosal stria after unilateral lesion to the supracallosal area and cingulum transecting some septal cholinergic efferents. We found accumulation of p75NTR and TrkA immunoreactive material (so-called "pile-up") within axonal segments of distended appearance proximal to the transection at two days postlesion and its translocation towards cell bodies seven days postsurgery. We observed p75NTR pile-up to be more intense than TrkA, which may indicate different cellular concentrations of both receptors. Receptor pile-up resembled acetylcholinesterase pile-up reported previously, suggesting a common response mechanism involving axonal transport disturbances.

Difluoromethylornithine counteracts lesion-induced astrogliosis in rat hippocampus: enhancement of inhibitory effect by combined treatment with GM1 ganglioside.

The effect of difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the rate limiting enzyme of polyamine biosynthesis, and its combined action with GM1 ganglioside, was studied on the GFAP content in a model of remote astrogliosis evoked in the hippocampus by lateral fimbria transection. DFMO markedly suppressed hippocampal gliosis as measured by GFAP immunoblotting seven days postsurgery. Combined treatment with DFMO and GM1 ganglioside--a substance which alone also counteracts hippocampal gliosis, produced a stronger suppressive effect than DFMO. The results support the hypothesis of a causal link between lesion induced events: polyamine biosynthesis and astroglial reaction. Potentiation of the inhibitory effect of DFMO by GM1 ganglioside suggests that the latter does not act through the mechanism involving ODC suppression.

Trimethyltin-induced plastic neuronal changes in rat hippocampus are accompanied by astrocytic trophic activity.

Partial deafferentation of the hippocampus due to trimethyltin (TMT) intoxication has been reported to induce plastic rearrangements of neuronal elements but the factors that direct these responses are unknown. To assess the possible involvement of nerve growth factor (NGF) in the phenomenon we evaluated the presumable changes in the expression pattern of NGF immunoreactivity (NGF-IR) in rat hippocampus 21 days after administration of TMT (8 mg/kg, i.p.) when reactive changes are fully developed. Immunolabelling for TrkA known to mediate biological effects of NGF and for GFAP to identify astroglial cells as a one of presumed source of postinjury produced factors was carried out on adjacent sections to establish the relation between expression of these proteins. In control hippocampus NGF-IR and TrkA-IR were localized in neurons and/or neuropil. After exposure to TMT remarkable non-neuronal expression of both proteins was observed. The distribution pattern of NGF, TrkA and GFAP overlapped suggesting that reactive astrocytes may not only produce NGF but also may become responsive to this neurotrophin. Zones of extensive NGF and TrkA astroglial expression corresponded to areas of axonal-dendritic rearrangements reported earlier. The data suggest that astroglia-derived trophic activity may be involved in neuronal plastic events associated with treatment with TMT.

Bilateral gliosis in unilaterally lesioned septohippocampal system: changes in GFAP immunoreactivity and content.

Unilateral damage to the lateral fimbria led to a bilateral gliosis in the septum and hippocampus. The gliosis was manifested by an increase in GFAP staining, accompanied by an increased number of glial fibrillary acidic protein (GFAP)(+) cells and GFAP content; the latter however was not visible in the contralateral septum. In general, the contralateral reaction appeared weaker than the ipsilateral one. The pattern of contralateral increase in GFAP-immunoreactivity (IR) matched almost exactly that observed on the ipsilateral side in the hippocampus (the most evident increase was seen in the oriens and pyramidal layers of cornu Ammonis 3 and in polymorphic area of gyrus dentatus). In the septum the bilateral increase in GFAP-IR was mainly visible in the dorsolateral quadrant of the structure; however in the ipsilateral side it spread over the whole half of the structure. The astrocytic responses in the septum and hippocampus were not equivalent: they differed mainly with regard to the increase of GFAP(+) cells (over 300% of control in the anterior part of the septum and only about 120% in the dorsal hippocampus). The differences between the percentage increases of other gliotic indices: GFAP-IR and GFAP content. Various possibilities that may account for the occurrence of contralateral gliosis are discussed, the most plausible being the contribution of interhemispheric and intraseptal links and the action of some diffusible agents. We suggest that bilateral gliosis may have an impact on compensatory postlesion processes, possibly by providing trophic support to impaired neurons.

Expression of astroglial nerve growth factor in damaged brain.

This article provides an overview of different brain insults leading to induction of astroglial NGF expression. It discusses mechanisms of induction of astroglial NGF in a damaged brain and a possible role of its expression in pathophysiology of Alzheimer's disease. It emphasizes that the pharmacological manipulation of astroglial production of NGF may serve as a possible therapeutic strategy to ameliorate the pathological changes occurring after brain damage.

Differential effects of GM1 ganglioside treatment on glial fibrillary acidic protein content in the rat septum and hippocampus after partial interruption of their connections.

The glial fibrillary acidic protein (GFAP) content was investigated using immunoblotting techniques in the septum and hippocampus of the rat after bilateral lateral fimbria transection. Seven days after surgery GFAP content increased significantly both in the septum (140% of control) and hippocampus (120% in dorsal, the less denervated, and 145% in the most denervated ventral part), indicating the occurrence of reactive gliosis. The GM1 treatment caused statistically significant attenuation of GFAP increment in all hippocampal parts. In contrast, GM1 treatment has no influence on the increase of GFAP content in the septum. Results suggest a differential effect of GM1 on the two gliotic reactions formed as a consequence of the lesion at the level of the source of innervation (septum) and the target (hippocampus).

Effects of partial lesion of dorsal hippocampal afferent and GM1 ganglioside treatment on conditioned emotional response and hippocampal afferent markers in rats.

Acquisition of the conditioned emotional response (CER) in 32 male hooded rats previously learned to press a bar for food and divided into four groups was studied. Two groups received electrolytic lesions of the dorsal hippocampal afferent and were thereafter injected either with GM1 ganglioside (30 mg/kg daily) or with buffer. Two remaining groups were sham operated and similarly injected. The partial hippocampal deafferentation evoked immediate enhancement of bar presses rate which persisted during the 2-week period of testing. CER training undertaken 2 days after surgical procedures appeared unsuccessful, whereas similar training with a cue of different modality initiated a week later resulted in acquisition of conditioned suppression of bar presses in all groups. Toward the end of training the conditioned suppression was more pronounced in lesioned than in control rats. The GM1 injections attenuated the conditioned suppression in control rats, presumably due to an antinociceptive role of ganglioside treatment. Behavioural training did not change the normal distribution pattern in cholinergic and serotonergic hippocampal afferent markers showing dorso-ventral gradient along longitudinal axis. The lesion-induced decrease pattern was also not affected. However, in contrast to previous findings in non-trained animals, the GM1 treatment was not effective in protecting against degenerative changes in the hippocampus of trained rats.