M Zaremba

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.

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.

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.

GM1 ganglioside potentiates trimethyltin-induced expression of interleukin-1beta 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-lbeta (IL-1beta) and nerve growth factor (NGF) immunoreactivities in the rat hippocampus. GM1 treatment also results in an increase of the number of IL-1beta and NGF immunoreactive astrocytes. Both the intensity of gliosis and stimulation of IL-1beta 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.

The ameboid phenotype of NG2 (+) cells in the region of apoptotic dentate granule neurons in trimethyltin intoxicated mice shares antigen properties with microglia/macrophages

NG2+, stellate cells present in the adult central nervous system (CNS) have been recently recognized as a distinct glial class, identified as multipotent progenitor cells. Antigenically, they are indistinguishable from oligodendroglia progenitor cells. In response to a variety of CNS insults, these cells become rapidly activated and undergo morphological changes accompanied by increased cellular proliferation. The role they play with respect to injured neurons is not clear. In our studies, we performed immunocytochemical investigations and identified a response of NG2‐expressing cells in the model of selective neurodegeneration of murine dentate gyrus granule cells induced by systemic administration of trimethyltin. Dying neurons exhibited features of apoptotic cells. Around the region of neurodegeneration, we observed activation of NG2+ stellate cells and microglia. During the peak of apoptosis, we detected the appearance of NG2+ cells of the ameboid phenotype, intermingled with granule neurons. These cells also expressed markers of microglia/macrophages, OX42‐ and ED1‐recognized antigens, an antigen recognized by O4 antibody—a marker of more differentiated cells of the oligodendroglia lineage and, in some cases, also a protein of mature oligodendroglia adenomatus polyposis coli. They also expressed nestin. Our results suggest that the injury induces a parallel transformation of both the activated glial classes: NG2+ stellate cells and resident microglia, into ameboid cells, sharing properties of both oligodendrocyte and monocyte lineages. These cells may play a role in the phagocytosis. If this assumption is verified by electron microscopy, it would indicate a novel function of NG2 transformed cells under CNS injury conditions.

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.

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.

Trimethyltin-evoked apoptosis of murine hippocampal granule neurons is accompanied by the expression of interleukin-1beta and interleukin-1 receptor antagonist in cells of ameboid phenotype, the majority of which are NG2-positive

Interleukin-1beta (IL-1beta) has been implicated in various neuropathologies, while IL-1 receptor antagonist (IL-1ra) has been shown to reduce neuronal injury. We investigated the pattern of expression of both cytokines in murine hippocampus after trimethyltin (TMT) intoxication. Using a ribonuclease protection assay, we demonstrated induction of transcription of IL-1beta and IL-1ra 3 days following TMT treatment which correlated with the peak of neuronal apoptosis. At this time, immunocytochemical staining revealed enhanced expression of both cytokines in NG2 proteoglycan expressing ameboid cells located at the site of neurotoxic insult, some of which bound also the microglial marker, lectin. There was some overlap between NG2 and lectin staining. Our results suggest that the two cytokines are involved in apoptotic processes in dentate granule cells and indicate that the pro-apoptotic effect of IL-1beta prevails over the presumed protective action of IL-1ra. The novel finding of expression of both cytokines in NG2(+) cells of ameboid phenotype indicates that these cells, through the regulatory roles of pro- and anti-inflammatory cytokines, may be involved in control of neuronal death or survival after injury.

Anti-myelin basic protein T cells protect hippocampal neurons against trimethyltin-induced damage

We investigated the influence of administration of autoimmune T cells on trimethyltin-induced degeneration of hippocampal neurons. Female Lewis rats received 8 mg/kg trimethyltin intraperitoneally alone, or followed 24 h later by a second intravenous injection of anti-myelin basic protein T cells (green fluorescent protein-tagged). Neurodegeneration was assessed by NeuN and Nissl cell counts 21 days after trimethyltin injection. We found that neurodegeneration in the CA4 region of the hippocampus was significantly reduced in the group receiving T cells. T cells also caused an augmentation of trimethyltin-induced hippocampal astrocytic activation and astrocytic TrkA expression, which was particularly intense in the CA4 region. Our study provides the first evidence of neuroprotection evoked by transferred T cells following a neurotoxic brain insult. The data suggest that mediation of the neuroprotective effects of T-cell-released nerve growth factor occurs mainly via hippocampal astroglial TrkA receptors.

Down‐regulation of microglia and NG2‐positive cells reaction in trimethyltin‐injured hippocampus of rats treated with myelin basic protein‐reactive T cells: Possible contribution to the neuroprotective effect of T cells

In our previous investigations, we demonstrated that CD4+ antimyelin basic protein (MBP) T cells protect hippocampal neurons against trimethyltin‐induced damage. We hypothesized involvement of T cells, interacting with the various glial populations activated during the neurodegeneration process. In this study, we employ immunocytochemical methods to investigate the influence of administration of T cells on the response of microglia and of NG2+ cells to trimethyltin (TMT)‐induced damage. Female Lewis rats were treated with anti‐MBP CD4+ T cells (4 million per animal, i.v) 24 hr after TMT (8 mg/kg, i.p) intoxication. TMT caused degeneration of CA4 hipppocampal neurons and evoked an abundant reaction of microglial and NG2+ cells in the injured region. The cells changed morphology into the activated state, and the number of OX42+ and NG2+ cells increased about 4.5‐fold and 3‐fold, respectively, relative to controls as assessed on day 21 after TMT treatment. Additionally, the cells of ameboid morphology, which expressed NG2 or microglial antigens, appeared in the zone of neurodegeneration. Furthermore, certain cells of ameboid phenotype shared both antigens. In rats treated with T cells, down‐regulation of the activation of both glial classes and reduction of formation of their ameboid forms was observed. The number of the total OX42+ and NG2+ cells decreased by 21% and 54%, respectively, and the number of their ameboid forms decreased by 46% and 73%, respectively. Our data suggest that the diminished activation of microglia and NG2+ cells, particularly the reduced number of their ameboid forms, may contribute to the neuroprotective effect of T cells.