Pro-inflammatory Cytokine-induced Activation Of Glial Cells And Bcl-2 Protein Expression In The Mouse Brain And The Relationship Are Related With Age

Objective This project was designed to investigate the mice brain response to an inflammatory challenge and whether parameters of this response vary in adulthood during lifetime. In particular, the project examined the features of activation of neuroglial cells in mice upon exposure to a mixture of proinflammatory cytokines (PICs), represented by interferon-γand tumor necrosis factor-α, and analyzed whether this activation varies with age. Furthermore, the project investigated the effect of PICs on neuronal survival in the periventricular parenchyma and its interaction with the activation of glial cells.Methods C57BL/6J mice of different ages were divided into three age groups, which consisted of young (2-3.5 months), middle-aged (10-11 months) and old (18-20 months) animals. A mixture of the PICs of 2μl recombinant murine interferon-γand of 2μl recombinant murine tumor necrosis factor-α(the biological activity of either cytokine was 500 units/perμl) was injected stereotaxically into the lateral cerebral ventricle of mice; vehicle (phosphate-buffered saline, PBS) was injected as control. After a survival of 1, 2 (all age groups) and 4 days (young and middle-aged animals) respectively, we examined the response of glial cells using glial fibrillary acidic protein (GFAP) as astrocyte marker, and the antibodies F4/80 and CD11b, which recognize microglial antigens, as markers of microglia. Expression of the anti-apoptotic protein Bcl-2 in the brain of mice of different ages was also investigated. Astrocytes and microglia were labeled with immunohistochemistry, and their number and immunosignal intensity were evaluated with quantitative image analysis. Neuronal cell death was examined in different brain structures using methods suited to detect necrotic or apoptotic phenomena, namely Fluoro-Jade B (FJB) histochemistry and the TUNEL technique, which were performed in adjacent sections. Furthermore, Bcl-2 expression was investigated with immunohistochemistry and Western blot in different brain regions after the intracerebroventricular (icv) injection of PICs. Double labeling with immunofluorescence was used to investigate whether Bcl-2 was expressed not only by neurons but also by glial cells.Results In both the PBS-injected control cases and the PICs-treated ones, GFAP-positive astrocytes in the brain of mice of the three age groups were observed. They were located mainly in the piriform and entorhinal cortex, the septum, throughout the hippocampal formation, amygdaloid complex, and in the white matter (subcortical white matter, as well as in the main fiber tracts). Qualitative differences in cell morphology were assessed between the PIC-injected and PBS-injected experimental groups. In the brain of PBS-injected control cases, glial cells did not display overt features of activation, consisting of smaller cell bodies and thinner processes. At 2 days following PIC icv treatment, in all age groups astrocytes were markedly activated in the periventricular regions, and especially in the hippocampus, exhibiting hypertrophy of cell bodies and processes and enhanced GFAP immunostaining. Such features of astrocytic activation persisted through the analyzed time course, i.e. up to 4 days following PIC treatment.Interestingly, PICs-induced astrocyte activation was significantly more marked in the brain of the aged animals, both in terms of cell hypertrophy and increased GFAP expression, than of younger age groups. This was documented by the quantitative analysis of the number and immunostaining intensity of GFAP positive astrocytes, by means of cell counts and densitometric evaluation, respectively. These analyses and their statistical evaluation showed a highly significant increase (P＜0.001) of these parameters in the old mice with respect to younger age groups in the CA1 field of the hippocampus and in the striatum at 2 days after PIC icv injections.Concerning microglia, in control brains of the three age groups most of the F4/80-positive elements exhibited the phenotype of resting microglia, with relatively small cell bodies and thin, long and ramified cell processes. After PIC icv injections, F4/80-positive cells within the brain parenchyma exhibited phenotypic changes indicative of microglial activation, which were well evident already at 24 h. These immunostained cells were characterized by hypertrophic cell bodies with thicker and stouter processes, as well as an amoeboid or rod shape, and intense immunostaining. The most prominent changes in F4/80-positive cell reactivity were detected in periventricular regions as well as in deeper brain areas, including the hippocampal formation, septum, striatum, cerebral cortex, periventricular thalamic regions, and anterior hypothalamus. As observed for astrocytes, the features of activation of F4/80-immunopositive cells were more marked in the brain of the old mice than in those of the young and middle-aged ones. Among other features, in the brain of PIC-treated old mice numerous ameboid microglial cells were observed. Quantitative analysis and statistical evaluation implicating pairwise post hoc comparison documented in the analyzed structures in each age group highly significant increases (P＜0.0001, in all groups) of the number of cells and their immunostaining intensity after PIC treatment with respect to the matched control experiments. Quantitative analysis of microglia activation also revealed significant age-by-treatment interactions in both cell counts and densitometric evaluation. In particular, PIC-dependent increases in the number of activated microglial cells were selectively larger in old mice than in young or middle-aged mice. Likewise, the immunostaining intensity of microglial cells (F4/80 optical density, OD; values) exhibited a significantly higher increase in the old mice than in the younger ones in all the analyzed structures. The results observed with CD11b immunohistochemistry were similar to those obtained with F4/80 antibodies. Neither FJB-stained neurons nor TUNEL-positive cell nuclei were found in neurons in the brain of the young and old mice after PIC icv injection, indicating that the observed features of glial activation were non secondary to neurodegenerative phenomena.Bcl-2-immunopositive cells were distributed widely in the brain of mice, in the cortex, hypothalamic parenchyma surrounding the third ventricle, in the diagonal band, in the hippocampus, septum and in the red nucleus of midbrain. Bcl-2 staining consisted of brownish reaction products mostly localized in the cytoplasm and processes of the neurons. In control brains of the three age groups, the density of Bcl-2-immunopositive elements was higher in the old mice than in the younger animals, especially in the hippocampus and cortex. At 2 days after PIC injections, the density of Bcl-2-positive neurons increased in the cortex and hippocampus with the matched control groups (P＜0.01, in all groups), and this persisted for 4 days. Thus, quantitative analysis of the number and immunostaining intensity of Bcl-2 positive cells were highly significantly increased with respect to controls in the cortex in three age groups at 2 days after PIC icv injections. The semiquantitative findings were similar in the hippocampus. Bcl-2 induction in response to PICs, IFN-γ+TNF-α, varied with age, being stronger in old mice than in the younger and middle-aged at 2 days after PIC icv injections. A significant age×treatment interaction and a significant age effect were found for cell number and density of Bcl-2 positive cells at 2 days in the analyzed structures. Expression of the antiapoptotic protein Bcl-2 was found in glial cells in the cortex, septum, hippocampus and corpus callosum at 4 days after PIC icv administration (middle-aged animals, of 10-11 months of age, were used for this experiment). Double staining for Bcl-2 and GFAP showed that these glial cells were indeed astrocytes, and the processes of activated astrocytes were found to contact and surround Bcl-2 positive neurons at 4 days after icv PIC administration.Western blot analysis detecting Bcl-2 protein in the cortex and hippocampus of young, middle-aged and old mice at 4 days following treatment with PICs and PBS, respectively. Bcl-2 is a single protein band which weight is 25-26 KDa. Densitometric evaluation of 25-26 kDa band (Bcl-2) in the brain of young, middle-aged and old mice revealed that there was a significant increase in Bcl-2 protein levels in the aged mice hippocampus and cortex at 4 days after PBS and PIC (P＜0.01)icv injections as compared to levels observed in young and middle-aged mice.Conclusion By the icv injection of a mixture of interferon-γand tumor necrosis factor-α, an acute neuroinflammatory challenge was applied to the mouse brain, and an array of parameters of the response and age-dependence of this phenomenon was investigated. The exposure to PICs induced activation of astrocytes and microglia, documented by the upregulation of glial antigens (GFAP, and the antigens recognized by F4/80 and CD11b) in the mouse brain tissue. Activation of microglia preceded that of astrocytes. The brain inflammatory reaction to PICs showed a strong age-dependence, being much more intense at an advanced age than at younger ones. This indicates the old brain is more sensitive to inflammatory stimulation than that of younger subjects, and further supports the concept that the activation of glial cells participates in the central nervous system immune-regulated response.Exposure to PICs through icv injections was also found to upregulate the expression of the Bcl-2 protein, which is well known to exert an anti-apoptotic role. This may have participated in the neuroprotective response to the inflammatory challenge. Thus, the old brain is more susceptible to inflammatory stimuli than that of younger subjects. The activation of astrocytes which express Bcl-2 also indicated that this glial cell type is implicated in a self-protective response to an inflammatory insult. Finally, the finding that processes of activated astrocytes contact and surround Bcl-2 positive neurons further points out a close relationship between astrocytes and neurons.