Cytokine-induced activation of glial cells in the mouse brain is enhanced at an advanced age

Neuroscience 141 (2006) 645– 661

CYTOKINE-INDUCED ACTIVATION OF GLIAL CELLS IN THE MOUSE BRAIN IS ENHANCED AT AN ADVANCED AGE X.-H. DENG,a,b G. BERTINI,a Y.-Z. XU,a,b Z. YANa1 AND M. BENTIVOGLIOa*

Numerous neurological diseases exhibit an age-related prevalence. For example, multiple sclerosis prevails in young adult and middle-aged individuals, whereas aging is a well-ascertained risk factor for neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases. This indicates that the response to neuroinflammatory and neuronal death-inducing processes varies in the CNS during the lifetime. Inflammatory responses within the CNS have been the focus of increasing interest because they have been observed in a variety of pathological conditions such as stroke, infection, neurotrauma, demyelinating and neurodegenerative diseases (see, for example, McGeer and McGeer, 1998; Williams, 2002; Blasko et al., 2004). It has been suggested that neuronal loss associated with the progression of Alzheimer’s disease may be partly due to the chronic presence of inflammatory proteins produced by activated glial cells (McGeer et al., 1995; McGeer and McGeer, 1998). It has also been proposed that neurodegeneration in Alzheimer’s disease could be driven by a cytokine-mediated vicious circle of self-sustained immunological processes following the activation of glial cells (Mrak and Griffin, 2005). On the other hand, injury-induced glial activation can exert both harmful and protective effects on nervous tissue components via the synthesis of a variety of molecules, including cytokines (Raivich et al., 1999; Allan and Rothwell, 2001; Raivich and Banati, 2004). There is evidence that chronic neuroinflammation consequent to glial activation can affect per se neuronal function and survival. For example, chronic infusion of the endotoxin lipopolysaccharide (LPS) in the fourth ventricle elicits in the hippocampus of young rats glial activation, gene expression changes including cytokine induction, as well as neurodegenerative phenomena and cognitive impairment, which may mimic some of the changes found in Alzheimer’s disease in humans (Hauss-Wegrzyniak et al., 1998, 2002). In the same paradigm, behaviorally induced gene expression in the hippocampus is altered only in regions showing microglia activation (Rosi et al., 2005). A wealth of evidence indicates that normal aging is characterized by low-grade chronic inflammatory activity, with increased production of cytokines in the blood and in the brain, and an imbalance between pro- and anti-inflammatory cytokines (Saurwein-Teissl et al., 2000; Bruunsgaard et al., 2001; Bodles and Barger, 2004), whose functional and cellular correlates are not yet fully understood (Bodles and Barger, 2004). Cytokines can be produced both by cells that can be recruited into the CNS, such as activated T cells and natural killer cells, and cells resident in the CNS, such as activated glial cells and

a Department of Morphological and Biomedical Sciences, University of Verona, Faculty of Medicine, Strada Le Grazie 8, 37134 Verona, Italy b Department of Anatomy and Neurobiology, Xiangya Medical College, Central South University, Changsha, Hunan, PR China

Abstract—Numerous neurological diseases which include neuroinflammatory components exhibit an age-related prevalence. The aging process is characterized by an increase of inflammatory mediators both systemically and in the brain, which may prime glial cells. However, little information is available on age-related changes in the glial response of the healthy aging brain to an inflammatory challenge. This problem was here examined using a mixture of the proinflammatory cytokines interferon-␥ and tumor necrosis factor-␣, which was injected intracerebroventricularly in young (2–3.5 months), middle-aged (10 –11 months) and aged (18 –21 months) mice. Vehicle (phosphate-buffered saline) was used as control. After a survival of 1 or 2 days (all age groups) or 4 days (young and middle-aged animals), immunohistochemically labeled astrocytes and microglia were investigated both qualitatively and quantitatively. In all age groups, astrocytes were markedly activated in periventricular as well as in deeper brain regions 2 days following cytokine treatment, whereas microglia activation was already evident at 24 h. Interestingly, cytokine-induced activation of both astrocytes and microglia was significantly more marked in the brain of aged animals, in which it included numerous ameboid microglia, than of younger age groups. Moderate astrocytic activation was also seen in the hippocampal CA1 field of vehicle-treated aged mice. FluoroJade B histochemistry and the terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling technique, performed at 2 days after cytokine administration, did not reveal ongoing cell death phenomena in young or aged animals. This indicated that glial cell changes were not secondary to neuronal death. Altogether, the findings demonstrate for the first time enhanced activation of glial cells in the old brain, compared with young and middle-aged subjects, in response to cytokine exposure. Interestingly, the results also suggest that such enhancement does not develop gradually since youth, but appears characterized by relatively late onset. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: neuroinflammation, aging, interferon-␥, tumor necrosis factor-␣, microglia, astrocytes. 1

Present address: Beijing Institute of Neuroscience, Capital University of Medical Science, Beijing, PR China. *Corresponding author. Tel: ⫹39-045-8027158; fax: ⫹39-045-8027163. E-mail address: [email protected] (M. Bentivoglio). Abbreviations: ANOVA, analysis of variance; DG, dentate gyrus; FJB, FluoroJade B; GFAP, glial fibrillary acidic protein; IFN-␥, interferon-␥; LPS, lipopolysaccharide; OD, optical density; PBS, phosphate-buffered saline; TNF-␣, tumor necrosis factor-␣; TUNEL, terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.04.016

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macrophages (see, for example, Aloisi, 2001; Neumann, 2001; Ladeby et al., 2005). Gene expression profiling in the aging brain of rodents has revealed changes of genes related to inflammatory responses, including transcriptional alterations of genes involved in microglial activation, increase of markers indicative of primed microglia, and overexpression of astrocytic proteins (Griffin et al., 1998; Lee et al., 2000; Sly et al., 2001; Weindruch et al., 2002; Godbout et al., 2005; Wu et al., 2005). Basal levels of proinflammatory cytokines were found to be increased in cultured glial cells isolated from aged rat brains, which were unable to rescue neurons from amyloid ␤ toxicity (Yu et al., 2002), and released more nitric oxide in response to stimulation than glia derived from young donors (Yu et al., 2002; Xie et al., 2003). On the other hand, immunohistochemical studies of the CNS of normal aged rodents have repeatedly demonstrated changes in non-neuronal cells, such as the presence of macrophages in the neuronal parenchyma (Ogura et al., 1994) and features of microglial activation in the white matter (Perry et al., 1993; Ogura et al., 1994) that were also documented in nonhuman primates (Sheffield and Berman, 1998; Hinman et al., 2004). Since microglia represent the resident immune cells in the CNS (Raivich et al., 1999; Aloisi, 2001; Ladeby et al., 2005), these findings indicate that the immune-related state of alert of the CNS varies during aging. Age-related changes also involve astrocytes, which in the senescent brain are characterized by an early stage of reactivity, whose functional consequences on the glia-neuronal crosstalk remain to be determined (Cotrina and Nedergaard, 2002; Finch, 2003). Despite the wealth of data pointing out a switch to a proinflammatory condition during aging and the involvement of brain glia in this process, little information is available on the age-dependence of the reaction of these cells to an inflammatory challenge. We have previously investigated in young adult rodents the brain response to the i.c.v. administration of interferon (IFN) -␥ (Peng et al., 1998; Robertson et al., 2000) or of this cytokine combined with either LPS or tumor necrosis factor (TNF) -␣ (Kong et al., 2000, 2002). In these studies, we determined that glial cells are strongly activated by such inflammatory mediators, and that this effect persists over several days. IFN-␥ and TNF-␣, which act in synergy (Jeohn et al., 1998; Blasko et al., 2001), are among the cytokines that increase systemically and in the brain in old age (Saurwein-Teissl et al., 2000; Yu et al., 2002; Blasko et al., 2001, 2004), and could therefore prime brain cells. On this basis, the present study was aimed at analyzing whether the acute response of glial cells to cytokine exposure varies with age. We here utilized the paradigm of combined cytokine injection that elicited the most marked glial response in our previous investigation (Kong et al., 2002). We thus investigated qualitatively and quantitatively astrocytes and microglia challenged by i.c.v. administration of IFN-␥ and TNF-␣, comparing mice of three age groups. We also examined whether this treatment could rapidly trigger neuronal death, and therefore a secondary glial activation.

EXPERIMENTAL PROCEDURES Animals Male mice (C57BL/6J), purchased from Harlan-Nossan (Milan, Italy), were maintained under strict veterinarian control, in sameage groups of three or four animals per cage, under a 12-h light/dark cycle (lights on at 7 a.m.), with food and water ad libitum. The observations reported here are based on a total of 47 young mice of 2–3.5 months of age, 33 mice of 10 –11 months (which will be here defined as middle-aged), and 37 mice of 18 –21 months (which will be here defined as old). These ages correspond to generally accepted definitions of aging in laboratory rodents (see Coleman, 2004). Due to the relatively high mortality of old mice after i.c.v. injections (see further), we did not use for these experiments animals older than the age range indicated above. All efforts were made to avoid animal suffering and to minimize the number of animals used. The experiments received institutional approval and authorization by the Italian Ministry of Health, and were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Surgery, treatment and tissue processing The i.c.v. injections were performed in animals deeply anesthetized with chloride hydrate (340 mg/kg, i.p.) and placed on a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) using a mouse adaptor (Kopf 921). A mouse atlas (Franklin and Paxinos, 1997) was used for reference of stereotaxic coordinates. A mixture of 2 ␮l of recombinant murine IFN-␥ and 2 ␮l of recombinant murine TNF-␣ (500 U/␮l for each cytokine; both purchased from PeproTech EC, London, UK) was injected slowly (5–10 min) through a Hamilton microsyringe in the right lateral cerebral ventricle. The dose of cytokines, provided as carrier-free solutions, is the same as that we used in young mice in previous studies (Kong et al., 2000, 2002). Control groups were injected with equivalent volumes of 0.01 M phosphate-buffered saline, pH 7.4 (PBS). All injections were made between 9 a.m. and 2 p.m.. Some of the animals died after the injections (Table 1), and their brain was not examined. The study of glial cells was based on animals allowed to survive 24 h, 2 days, and 4 days (Table 1). As it will be explained further, a survival of 4 days was difficult to achieve in old animals which, therefore, were not sampled at this time point. The mice that were used for the histological analysis were perfused transcardially, under deep anesthesia as above, with PBS followed by freshly prepared 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The brain was removed, postfixed for 3 h in the fixative solution, and then cryoprotected overnight in 30% sucrose in PBS at 4 °C. Forty ␮m-thick coronal sections were obtained with a freezing microtome through the telencephalon and diencephalon, and adjacent series of one in every six sections were collected. Three series of sections were processed free-floating for immunohistochemistry. Sections from an adjacent series were stained with Cresyl Violet for cytoarchitectonic control and verification of the injection needle track.

Study of cell death phenomena FluoroJade B (FJB) histochemistry. In four additional animals, two young (3.5 month-old) and two aged (20 and 21 monthold, respectively), cell death was investigated with FJB labeling. This anionic fluorescein derivative provides a sensitive marker of neurons undergoing degeneration (Schmued and Hopkins, 2000). These animals were perfused 48 h after cytokine injections performed as above, and their brain was processed histologically as already mentioned. Slide-mounted sections were immersed in a solution containing 1% sodium hydroxide in 80% alcohol (20 ml of 5% NaOH added to 80 ml absolute alcohol) for 5 min, and then for 2 min in 70% alcohol and 2 min in distilled water. The sections were then transferred to a solution of 0.06% potassium perman-

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Table 1. Experimental treatment and mortality of mice Age group (mo)

Young (2–3.5)

Middle-aged (10–11)

Old (18–21)

Survival (d)

1 2 4 1 2 4 1 2

N. of injected animals

Mortality

PBS

Cytokines

PBS

5 5 6 4 5 5 7 4

7 8 10 6 6 7 11 8

Cytokines

0/16

0.0%

1/25

4.0%

0/14

0.0%

1/19

5.3%

2/11

18.2%

5/19

26.3%

Abbreviation: cytokines, IFN-␥⫹TNF-␣ mixture.

ganate for 10 min, rinsed and stained for 20 min in a solution prepared from a 0.01% stock solution of FJB (Histo-Chem Inc., Jefferson, AR, USA) in 0.1% acetic acid. After rinsing and drying, the sections were coverslipped without dehydration. For positive control, FJB histochemistry was simultaneously run on series of sections derived from Wistar rats that had been injected with the convulsant agent pilocarpine as in a previously published study (Fabene et al., 2003). In this protocol, pilocarpine-induced status epilepticus was interrupted pharmacologically after 4 h; the animals were then deeply anesthetized and perfused 24 h or 48 h after seizure onset. With this procedure a considerable number of FJB-stained neurons, indicative of neurodegenerative phenomena, can be seen in several brain regions (Fabene et al., 2003). In situ labeling of DNA fragmentation. The analysis of cells exhibiting DNA fragmentation, which typically occurs during apoptosis, was pursued with terminal deoxynucleotidyl transferasemediated UTP nick-end labeling (TUNEL technique). Four cytokine-treated animals were used for this part of the study (two young and two old, of 20 and 21 months of age, respectively). These animals were injected and perfused 2 days later as described above, and the brain was cut transversely at a cryostat into 10 ␮m-thick sections, which were collected in series of one in every six sections. One series of sections was processed according to the protocol by Gavrieli et al. (1992) to label the doublestranded DNA breaks. Briefly, after washing these sections were incubated for 1 h in 1.2% terminal deoxynucleotidyltransferase (25 units/ml; Roche Diagnostic, Mannheim, Germany) and 2.4% biotinylated 16-dUTP (50 nM, Roche Diagnostic) at 37 °C, and reacted with the standard avidin– biotin peroxidase kit (ABC, Vector) using 3–3=diaminobenzidine as chromogen. These sections were then lightly stained with Thionin, dehydrated and coverslipped. Neuronal cell death consequent to protracted seizures elicited by pilocarpine was used as positive control also for this material, on the basis of previous reports indicating that TUNEL-positive cell nuclei are observed in neurons in this model of temporal seizures (see, for example, Roux et al., 1999). Such control material derived from a Wistar rat injected with pilocarpine as mentioned above, and in which status epilepticus was interrupted after 4 h as indicated above, and the animal was perfused 3 days later. The brain of this animal was cut at the cryostat and sections were processed for TUNEL staining at the same time as those of the cytokine-injected mice.

Immunocytochemistry for glial antigens One series of sections was processed for the visualization of astrocytes using glial fibrillary acidic protein (GFAP) as marker, and two series for the study of microglia, using the F4/80 and CD11b antibodies, respectively. The F4/80 antibody is specifically directed against mature mouse macrophages (Austyn and Gor-

don, 1981), and is an effective marker of macrophages and microglia in the mouse brain (Perry et al., 1985). The CD11b antibody reacts with the 155 kDa ␣M subunit of the CD11b/CD18 heterodimer. This Mac-1 ␣M␤2 integrin functions as a receptor for complement (C3bi) fibrinogen or clotting factor X (Arnaout et al., 1988; Corbi et al., 1988). The CR3bi complement receptor is expressed constitutively in microglia and is upregulated when resting microglia are exposed to factors that activate them to develop into immunoeffector cells (Raivich et al., 1999). Polyclonal rabbit anti-GFAP antibody was purchased from Dako (Glostrup, Denmark) and used at a 1:200 dilution. Monoclonal rat anti-mouse F4/80 (dilution 1: 100) and CD11b antibodies (dilution 1:1000) were purchased from Serotec (Oxford Biomarketing, Oxford, UK). The sections were soaked in 1% H2O2 in PBS for 30 min in order to inactivate endogenous peroxidase activity, and preincubated in a solution of 5% normal goat serum, 0.2% Triton X-100, 0.1% NaN3 in PBS for 1 h. The sections were then incubated overnight, at room temperature, in primary antibodies, diluted in PBS containing 1% normal goat serum, 0.2% Triton X-100, and 0.1% NaN3. Subsequently, the sections were incubated for 2 h with biotinylated secondary antibodies (goat anti-rabbit IgG for GFAP, goat anti-rat IgG for F4/80 and CD11b, all used at a 1:200 dilution and purchased from Vector, Burlingame, CA, USA). The avidin–peroxidase protocol (ABC; Vectastain kit, Vector) was applied in the last step of the procedure, using 3,3=-diaminobenzidine (Sigma, St. Louis, MO, USA) as chromogen. Finally, the sections were mounted on gelatinized slides, dehydrated, cleared, coverslipped. Controls were carried out with the same protocols but omitting the primary antibodies, which did not result in any staining.

Data analysis Selection of histological material for data analysis. The material processed for immunohistochemistry and the sections processed for the TUNEL technique were studied at the microscope under bright-field illumination. The sections processed for FJB staining were examined with a Zeiss LSM 510 confocal scanning laser microscope using the argon (488 nm) excitation beam. Immunolabeling of astrocytes and microglia was examined without the investigator’s knowledge of the animal’s group assignment, according to a procedure similar to that adopted in our previous study (Kong et al., 2002). Preliminary qualitative observations were followed by quantitative evaluation of two parameters: cell number, based on the count of immunostained cell body profiles, and immunosignal intensity, based on the measurement of optical density (OD). On the basis of qualitative screening, we selected for quantitative analysis the most informative time points and brain regions. In particular, we chose to analyze astrocytes in animals which

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survived 2 and 4 days, since 1 day after cytokine injection astrocytes did not show obvious differences with respect to controls. As for microglia, we analyzed these cells in animals which survived 24 h, since microglia appeared activated at this time point, and with a pattern very similar to that observed at subsequent time points. Structures selected for analysis were the hippocampal formation, striatum and septum, in which qualitative examination revealed changes comparable to other periventricular structures. Cell counts were performed on three animals per experimental group, selected on the basis of two criteria: 1) optimally low tissue background after immunohistochemical processing, and 2) perfect placement of the injection in the middle of the lateral cerebral ventricle, as a precaution to ensure a uniform response in the periventricular parenchyma to cytokine exposure.

1.25; 600⫻ magnification) and the contour of GFAP-immunopositive cell bodies visible within the frame was traced manually. The area of individual cell bodies was then measured with the aid of ImagePro Plus. Between-group differences in the mean numbers of positive cells and mean OD values were statistically evaluated for each structure of interest, and for each marker. The evaluation was based on two-way analysis of variance (ANOVA), with age and treatment as main factors, followed by Bonferroni post hoc tests for pairwise comparisons. Differences in the mean values of the size of astrocytes were also evaluated with two-way ANOVA. Matlab (The Mathworks, Natick, MA, USA) was used for statistical analyses, and P⬍0.05 was chosen as significance threshold.

Image and statistical analysis. Images were acquired, under constant light conditions to standardize the measurements, using a video camera (JVC KY-F58) connected to the microscope. Analysis of digitized images was aided by the software Image Pro Plus 4.5 for Windows (Media Cybernetics, Silver Springs, MD, USA). In each animal, and for each structure, the analysis was performed on three rectangular frames (corresponding to the camera’s acquisition area), placed at a random location within the structure on three separate, equally spaced sections. Care was taken to choose sections at matching anteroposterior levels in all animals and to place frames in comparable locations using anatomical landmarks as a guide (such as the pyramidal cell layer in the hippocampus, or the ventricular border in the septum). Framed images were acquired by the camera and saved as still bitmap files. Immunostained glial cell body profiles found within the captured image were then counted. Correct identification of cell bodies was aided by parallel observation of the same area at the microscope. To avoid bias of edge effects, the upper and left limits of the frame were inclusion lines, and the lower and right limits were exclusion lines. Analysis of GFAP-immunoreactive elements in the hippocampal CA1 field and striatum, and of F4/80-positive cells in CA1 and dentate gyrus (DG) was conducted on image frames of 0.703 mm2 using a 10⫻ objective (NA 0.30; 100⫻ magnification). F4/80- or CD11b-immunoreactive elements in the septum were counted on frames of 0.176 mm2 using a 20⫻ objective (NA 0.50; 200⫻ magnification). Independent of objective and frame size, results are expressed as number of cells per standard unit (1 mm2). Densitometric analysis was performed in a sample of 120 –200 immunostained cells per experimental group and per analyzed structure. Images were collected using a 40⫻ objective (NA 0.75; 400⫻ magnification, yielding a frame of 0.043 mm2) from at least three different sections per structure and per animal. Image frames were placed at a random location in periventricular structures. Individual cell bodies characterized by immunoreactivity were manually traced, and their mean staining intensity was normalized against the respective section’s background, defined as tissue devoid of specific immunostaining. The procedure resulted in arbitrary OD values on a scale of 0 (background staining) to 255. In order to evaluate the potential impact of systematic biases on quantitative analyses (see Guillery, 2002; Schmitz and Hof, 2005), we performed the following additional measurements. First, to ascertain whether tissue shrinkage after histological processing was different between age groups, the area of the sections deriving from all the analyzed animals was measured by digitally acquiring whole sections using a low-power objective and tracing the section outline with the aid of the aforementioned software. Second, we addressed the bias introduced by variations of cell body size on cell count estimates. Qualitative analysis of the material indicated that the effect of treatment on the size of cell bodies was especially marked for GFAP-immunostained astrocytes (see further). We therefore measured the area of 240 randomly sampled astrocytes from each experimental group. To accomplish this, randomly placed images were acquired with a 60⫻ objective (NA:

RESULTS Mortality of mice after i.c.v. injections In the course of the experiments destined to the analysis of glial cells (Table 1), we observed that all animals survived at least 9 h after the i.c.v. injections, but some of them were subsequently found dead in the cage. In particular, all injected animals recovered from anesthesia and were returned to their own cages, although recovery was generally slower for the old mice than for those in the two younger age groups. No systematic behavioral scoring was performed, but direct observation of the animals did not reveal any particular behavioral alterations in the hours following the i.c.v. injections. Eventually, however, a relatively high proportion of the old mice (26.3%, evenly distributed over the entire 18 –21-month age range) did not survive the i.c.v. cytokine injections. This proportion was somewhat higher than that of age-matched controls (18.2%, 19 month-old animals) and of cytokine-injected younger animals (4 –5.3%; Table 1). The majority of deaths (six/nine, represented by two control and four cytokine-injected animals) occurred within 24 h. The three remaining cases, one for each age group, all cytokine-treated, died between 36 h and 48 h post-injection. Study of neuronal cell death after i.c.v. cytokine treatment FJB histochemistry. None of the young or old cytokine-injected mice examined at 2 days postinjection presented any FJB-stained neurons. As expected on the basis of previous findings (Fabene et al., 2003), numerous FJBlabeled neurons were instead evident in several brain regions, including the cerebral cortex, in the simultaneously processed sections which derived from the rat killed 24 h after the onset of pilocarpine-induced seizures. This positive control ensured that the FJB protocol had been applied correctly, and that the absence of FJB-labeled cells in the cytokine-injected animals could not be ascribed to false negative results. TUNEL technique. No TUNEL-positive cell nuclei were observed in the brain of the young and old mice investigated with this method after i.c.v. cytokine injection (Fig. 1G), including areas in which glial cells, and in particular microglia, were found to be activated (see further). TUNEL positivity was instead observed in the sections derived from the rat killed after protracted seizures (Fig. 1H). Also in this case,

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therefore, the positive control ensured that no technical bias could have affected the lack of TUNEL positivity in the cytokine-treated mice. Effect of i.c.v. treatment on astrocytes After PBS injection, GFAP-positive astrocytes exhibited a distribution consistent with our previous findings (Kong et al., 2002) in the brain of all age groups. Briefly, immunopositive elements were numerous both in fiber tracts and in the gray matter, including the cerebral cortex (particularly in its superficial layers), the septum, the hippocampal formation, and the hypothalamic parenchyma surrounding the third ventricle. Positive cells were also found scattered throughout the striatum. In vehicle-treated subjects of the younger age groups, immunostained astrocytes mainly exhibited the features of resting cells with long, slender processes, although a few hypertrophic elements with relatively thick processes were also seen (Fig. 1A–D). In the old mice, at all time points after vehicle injections, the density of GFAP-immunopositive elements seemed to be higher than in the younger animals at a qualitative screening of the sections, especially in the hippocampus (Fig. 1E, F), septum, and striatum. In addition, numerous immunolabeled astrocytes in the old mice appeared hypertrophic, with a relatively large cell body and very thick, highly ramified processes which resulted in an immunopositive meshwork in the neuropil (Fig. 1E, F). At 24 h after the injections of IFN-␥⫹TNF-␣, no obvious differences in GFAP immunoreactivity were evident as compared with control animals of all ages. At 2 days, a marked hypertrophy of GFAP-positive astrocytes was instead seen in the material derived from the cytokine-treated animals of all age groups (Fig. 2). This was accompanied by an increment of GFAP-labeled elements, compared with the PBS-injected controls, especially evident in the hippocampus, septum, striatum, and cortex, including the frontal and cingulate cortical fields. As shown in the CA1 field of the hippocampus in Fig. 2E and F, the phenotypic features of astrocytic activation were especially marked in the old animals. In all age groups, immunolabeled astrocytes exhibited a relatively large cell body. In the old animals, they gave off numerous, very thick, highly ramified, and intensely immunostained branches which filled the neuropil. Such findings were consistent across animals. The features of activation of GFAP-positive elements described above persisted in the brain of the young and middle-aged animals 4 days after cytokine injections (at which point, as mentioned above, old animals were not examined). Treatment- and age-related differences in the number and immunostaining intensity of astrocytes were confirmed by the quantitative evaluation (Fig. 3). The ANOVAs (see Tables 2 and 3 for a listing of F and P values) revealed highly significant main effects of cytokine treatment in all studied structures. Post hoc tests confirmed that cytokine injection caused robust increases both in the number of labeled cells and in OD values in all age groups, and at both the analyzed time points (P⬍0.0001 for both cell number and immunostaining intensity in all groups; not

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shown in Fig. 3). For the comparisons that included old mice, 2 days after the injections, highly significant main effects of age on both cell count and OD values were also found. Interestingly, as shown in Fig. 3, cytokine-induced increases of GFAP reactivity were not equal in the different age groups. Rather, astrocytes were in general more numerous and more intensely stained in the old mice compared with the younger age groups, after cytokine treatment. Indeed, significant interactions between age and treatment were found for the number and immunostaining intensity of GFAP-reactive cells both in the hippocampus and in the striatum. Post hoc tests (Fig. 3) confirmed that the source of the interaction was a significantly larger cytokine-dependent increase of GFAP immunoreactivity in the old mice relative to the increases observed in the other age groups. In addition, it should be noted that GFAP immunoreactivity in the CA1 field of the hippocampus was also enhanced in vehicle-treated old mice compared with younger cohorts. In this region, pairwise post hoc tests showed that immunostaining intensity was significantly higher in old mice than in either young or middle-aged mice, and the number of immunopositive cells was significantly higher in the old versus young mice, with the comparison between old and middle-aged mice showing a similar trend. None of the other analyses showed agedependent effects of vehicle injections. The correlates of astrocytic activation in both young and middle-aged mice were still present 4 days after i.c.v. cytokine injection in terms of quantitative measures of GFAP reactivity (Fig. 3). The results were similar in the young and middle-aged animals, without, therefore, a significant effect of age in these groups (Tables 2 and 3). Control of systematic biases on cell counts As mentioned previously, of the known sources of bias in counting cells (Guillery, 2002; Schmitz and Hof, 2005), we specifically investigated those potentially affecting to a different degree subjects from different age groups, thus contributing to the age-related effects reported above. First, the mean area of whole brain sections was essentially identical in all experimental groups, which allowed to conclude that treatment- and age-related differences in glial cell counts were not due to different degrees of tissue shrinkage during fixation and histology. Second, the mean area of GFAP-immunostained cell bodies in cytokine-treated, middle-aged and old subjects (28.0⫾10.1 ␮m2) was significantly larger than that of vehicle-injected animals (16.1⫾8.7 ␮m2) (two-way ANOVA, main effect of treatment, P⬍0.01), but the analysis revealed no main effect of age or interaction between factors (Fig. 3). Since larger cell bodies may result in overestimated cell counts (Schmitz and Hof, 2005), it is possible that the treatment-related effects we report are slightly overestimated. The lack of age-related effects on cell size, however, shows that the bias must have equally affected all cytokine-treated age groups and could not, therefore, account for the age-related differences in cell counts.

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Fig. 1. The plate illustrates the immunoreactivity of astrocytes in the CA1 field of the hippocampus in representative images derived from the three different age groups at 2 days after control injections of vehicle (A–F), and the data obtained in the study of in situ nick-end labeling of DNA fragmentation (G, H). The images B, D, F in the right column represent at higher power details of the fields shown in A, C, E, respectively, and the asterisks indicate the same points as reference. Note that some astrocytes exhibit hypertrophic and intensely stained processes in the old animal. G and

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Fig. 2. The plate illustrates the findings revealed by GFAP immunoreactivity in the CA1 field of the hippocampus at 2 days after i.c.v. injections of a mixture of IFN-␥ and TNF-␣ in experiments which match the control experiments shown in Fig. 1. Note that in all age groups the astrocytes appear markedly activated, with increase of the cell body size, by the cytokine treatment compared with the respective controls shown in Fig. 1. In addition, the comparison between the features observed at different ages reveals that the hypertrophy of astrocytes, with numerous intensely stained processes, is more marked in the old animal (E, F), compared with the younger ones (A–D). Scale bars⫽45 ␮m in A (applies also to C, E); B, 20 ␮m (applies also to D, F).

Effect of i.c.v. treatment on microglia F4/80 immunoreactivity. In the brain of PBS-injected control animals of all ages, at 24 h F4/80-positive cells prevailed in the cortex around the needle track and in the parenchyma surrounding the lateral and third ventricles,

i.e. in the septum, the hippocampal formation (Fig. 4A–D), anterior hypothalamus, and fiber tracts (corpus callosum, anterior commissure, fimbria). In control brains of the three age groups, most of the F4/80-positive elements exhibited the features of resting

H are derived from sections processed for TUNEL labeling and counterstained with Thionin. The image in G shows a region of the periventricular thalamus (the laterodorsal nucleus) of a cytokine-injected mouse of 21 months of age; as explained in the text, glial activation was prominent in this area after cytokine injections. The image in H shows the same region (the laterodorsal thalamic nucleus) in a rat subjected to pilocarpine-induced protracted seizures. Note in G the absence of TUNEL-stained elements, which is instead evident in H in the positive control showing TUNEL-labeled cell nuclei (which appear as dark round elements). Scale bars⫽45 ␮m in A (applies also to C, E); B, 20 ␮m (applies also to D, F); G, H, 50 ␮m.

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Fig. 3. The bar graphs illustrate the results of quantitative analysis of GFAP-positive cells in the different age groups (Y⫽young, M⫽middle-aged, O⫽old) and in the two treatment paradigms (vehicle and cytokine injections, respectively). Left-hand charts represent the average number of astrocytes in the CA1 field of the hippocampus and striatum at two different post-injection survival times, and right-hand charts their mean immunostaining intensity (OD) evaluated in CA1. The graph at the bottom right illustrates the results obtained in the evaluation of the size of GFAP-immunoreactive cell bodies, whose increase after cytokine treatment did not show significant age-related differences (see text). Error bars represent the standard deviation of the mean. Asterisks highlight statistically significant post hoc pairwise between-age⫹comparisons following the two-way ANOVAs (* P⬍0.05; ** P⬍0.005; *** P⬍0.0005). Since cytokine treatment was invariably accompanied by highly significant increases of immunoreactivity at all ages (see text), the corresponding asterisks are omitted for the sake of clarity.

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Table 2. Results of two-way ANOVA statistic performed on cell counts Marker

GFAP

F4/80

CD11b

Structure (survival)

CA1 (2 d) CA1 (4 d) Striatum (2 d) Striatum (4 d) CA1 (1 d) DG (1 d) Septum (1 d) Septum (1 d)

Main effect of treatment

Main effect of age

Treatment⫻age interaction

F (1,17)

P

F (2,16)

P

F (2,16)

P

266.21 28.12 235.13 40.95 492.41 708.33 4314.34 15233.21

⬍0.0001 ⬍0.001 ⬍0.0001 ⬍0.0005 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001

61.26 0.00 14.35 0.29 8.31 26.83 20.95 14.69

⬍0.0001 n.s. ⬍0.001 n.s. ⬍0.01 ⬍0.0001 ⬍0.0005 ⬍0.001

10.87 0.01 4.29 0.00 5.31 19.66 18.66 16.59

⬍0.005 n.s. ⬍0.05 n.s. ⬍0.05 ⬍0.0005 ⬍0.0005 ⬍0.0005

Abbreviation: n.s., not significant.

microglial cells, with long, slender processes and several thin side-branches (Fig. 4A–D). In certain regions, including the hippocampus, F4/80-stained cell processes appeared denser in the old mice than in the younger cohorts (Fig. 4C, D). After cytokine injections, F4/80-positive cells exhibited phenotypic changes indicative of microglial cell activation, which were well evident at 24 h (Fig. 4E–H). Thus, within the brain parenchyma, F4/80-immunostained cells showed a ramified appearance in all the cytokine-treated age groups. However, at variance with the data observed in the matched control animals, after cytokine injections these immunostained cells were characterized by hypertrophic cell bodies with thicker and stouter processes and intense immunostaining (Fig. 4F, H). The most prominent changes in F4/80-positive cell reactivity were detected in periventricular regions, including the hippocampal formation (Fig. 4E–H), septum, striatum, cerebral cortex, periventricular thalamic regions, and anterior hypothalamus, but were also found more deeply in the parenchyma, for example in the bed nucleus of the stria terminalis. 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 (Fig. 4E–H). At 2 days, such features of activation were still evident in the septum, hippocampus, hypothalamic structures close to the third ventricle and corpus callosum, as well as throughout the cerebral cortex. In addition, numerous F4-80-immunostained ameboid cells were detected in aged animals at 24 h and 2 days after cytokine injection, in periventricular diencephalic structures, as illustrated in the medial habenular nucleus in

Fig. 5E, F. Ameboid microglial cells were also observed in the brain of young and middle-aged animals after cytokine treatment, but they were much more numerous in the cytokine-treated aged mice with respect to the younger age groups. Such findings were consistent across different cases. The results of the quantitative analyses performed, as mentioned above, at 1 day post-injection, are shown in Fig. 6 and Tables 2 and 3. In all the analyzed structures (CA1, DG, and septum), two-way ANOVA analyses demonstrated highly significant effects of both treatment and age. As shown in Fig. 6, cytokine treatment was indeed associated with a dramatic increase in the number of F4/80stained cells (roughly three-fold in all studied structures) at all ages. Accordingly, pairwise post hoc comparison documented in each age group highly significant increases of this cell phenotype after cytokine treatment with respect to the matched control experiments (P⬍0.0001 in all groups; not shown in Fig. 6). On the other hand, the main effects of age were due to selectively higher increases of immunoreactive cells in the old mice, compared with the younger age groups, as demonstrated by post hoc tests (Fig. 6). Quantification of the intensity of F4/80 immunoreactivity of microglia showed a similar pattern of results (Fig. 6). In particular, cytokine injection resulted in highly significant, substantial increases in OD values at all ages and in all studied regions, compared with controls. Furthermore, old mice showed overall more intense F4/80 microglial immunostaining compared with younger mice. Interestingly, the analysis of microglia activation also revealed significant age-by-treatment interactions in terms of both cell counts and OD evaluation. In particular, cyto-

Table 3. Results of two-way ANOVA statistic performed on OD measurements Marker

GFAP F4/80

CD11b

Structure (survival)

CA1 (2 d) CA1 (4 d) CA1 (1 d) DG (1 d) Septum (1 d) Septum (1 d)

Abbreviation: n.s., not significant.

Main effect of treatment

Main effect of age

Treatment⫻age interaction

F (1,17)

P

F (2,16)

P

F (2,16)

P

185.78 123.80 2122.94 695.99 773.29 532.44

⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001

42.13 0.29 57.05 28.85 11.30 4.43

⬍0.0001 n.s. ⬍0.0001 ⬍0.0001 ⬍0.005 ⬍0.05

0.04 0.39 45.13 26.67 5.27 1.94

n.s. n.s. ⬍0.0001 ⬍0.0001 ⬍0.05 n.s.

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Fig. 4. The plate illustrates F4/80-positive cells in the CA1 field of hippocampus in middle-aged and old mice at 1 day post-injection. A–D illustrate the control experiments, and E–H the cytokine treatment. Note the cytokine-induced activation of microglia, with a bushy appearance of processes which is very marked in the old animal (G, H). The images in the right column (B, D, F, H) represent at higher power details of the fields shown in the left column (A, C, E, G, respectively), and the asterisks provides spatial reference. Scale bars⫽45 ␮m in A (applies also to C, E, G); B, 20 ␮m (applies also to D, F, H).

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Fig. 5. The plate illustrates the features of CD11b immunoreactivity observed in a representative periventricular structure (the septum) in middle-aged and old animals compared with the respective controls, at 1 day after vehicle (A, C) or cytokine (B, D) injections. The insets in A–D illustrate at higher magnification the cells marked by the asterisk in the respective low power views. Note that the cytokine-induced activation of microglial cells is more marked in the old than in the middle-aged animal. In addition, the plate illustrates the occurrence of numerous rod-shaped and ameboid microglial cell observed in the brain of a 21 month-old mice at 1 day after cytokine injections. (E, F) F4/80-positive cells in the medial habenular nucleus; F illustrates a detail of the area marked by the asterisk in E. (G, H) H shows CD11b-positive cells in the laterodorsal nucleus of the thalamus, and G illustrates a detail of the area marked in H by the asterisk. Scale bars⫽45 ␮m in B (applies also to A, C, D); inset in B: 12 ␮m (the other insets are at the same magnification). E, 48 ␮m, H, 25 ␮m; F and G, 8 ␮m. D3V, dorsal third ventricle.

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Fig. 6. The bar graphs illustrate the results of quantitative analysis of immunoreactivity of microglial cells labeled by the F4/80 immunoreactivity in the hippocampal formation (CA1 field and DG), and by F4/80 and CD11b antibodies in the septum in the different age groups (Y⫽young, M⫽middle-aged, O⫽old) and in the two treatment paradigms, at 1 day post-injection. Left-hand and right-hand charts represent the average number of stained cells per group, and their mean immunostaining intensity (OD) per group, respectively. Error bars represent the standard deviation of the mean. Asterisks highlight the statistically significant post hoc pairwise between-age comparisons following the two-way ANOVAs (* P⬍0.05; ** P⬍0.005; *** P⬍0.0005). The asterisks indicating the highly significant increases of immunoreactivity elicited by cytokine treatment in each age group (see text) are omitted for the sake of clarity.

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kine-dependent increases in the number of activated microglial cells were selectively larger in old mice than in young or middle-aged mice. The effect was significant in the hippocampus and in the septum (Fig. 6), while a similar trend observed in the striatum did not reach statistical significance. Likewise, the immunostaining intensity of microglial cells (F4/80 OD values) increased significantly more in the old than in younger mice in all analyzed structures. CD11b immunoreactivity. The results observed with CD11b immunohistochemistry were similar to those obtained with F4/80 antibodies. Features indicative of activation of CD11b-immunostained elements were seen in the brain parenchyma, and especially in periventricular structures, after cytokine i.c.v. injections, and these were very marked in the old animals (Fig. 5A–D). These features included an increased cell density at the qualitative screening of the sections (compare B and D in Fig. 5), with hypertrophy of the immunostained cells (Fig. 5D), although with some inter-individual variability. Consistently with the observations performed in the study of F4/80 immunoreactivity, numerous CD11b-immunostained cells showing ameboid features were also observed, and these were much more numerous in the old mice than in the younger ones. Ameboid elements were detected especially in periventricular diencephalic structures, including the medial habenular nucleus, the laterodorsal (Fig. 5G, H) and paraventricular thalamic nuclei, the medial preoptic area and suprachiasmatic nucleus of the hypothalamus. Two-way ANOVA analysis of the number and immunostaining intensity of CD11b-positive microglial cells in the septum at 24 h postinjection (Tables 2 and 3; Fig. 6) confirmed significant effects of treatment and age. Similarly to what observed with the other microglial marker, a significant interaction showed that cytokine treatment increased the number of CD11b-immunoreactive cells more in the old mice than in younger ones. At variance with F4/80, however, the CD11b immunostaining intensity in the studied age groups was not differentially affected by cytokine treatment.

DISCUSSION Vulnerability of old mice to the i.c.v. treatment Despite the low number of animals on which the present observations on the postinjection mortality of the animals were made, these observations suggest not only an increased mortality of the old mice after the i.c.v. injections, but also high vulnerability to cytokine injections with respect to younger mice. Such observations supplement previous data (Kalehua et al., 2000) on a marked ageassociated increase in mortality following i.c.v. injection of the endotoxin LPS in mice (about 22% of 19 –20 month-old mice, and about 61% of 24 –30 month-old mice), with the majority of deaths occurring, as in our study, within 1 day after the i.c.v. inflammatory challenge. In the study of Kalehua et al. (2000) centrally administered LPS resulted

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in a significant age-associated increase in brain and serum TNF-␣, along with plasma levels of interleukins 1␤ and 12, and the enhanced vulnerability to LPS was ascribed to the age-related increase in TNF-␣ expression. It has been shown in mice that levels of inflammatory molecules, including TNF-␣, increase during aging also after a different paradigm of brain injury (Sandhir et al., 2004). A recent study based on i.p. injection of LPS has reported an amplification of the neuroinflammatory reaction in aged mice after such stimulation of the peripheral innate immune system (Godbout et al., 2005), confirming an increased sensitivity of the aged brain to inflammatory challenges. However, compared with previous investigations which reported the mortality of old mice following LPS i.p. administration (Chorinchath et al., 1996; Tateda et al., 1996), Kalehua et al. (2000) concluded that the aged CNS is more susceptible to a direct intracerebral inflammatory insult than to a systemic insult. The mechanisms and sites of action underlying such effect remain to be clarified. Although the i.c.v. injections of inflammatory mediators could influence the stress response directly, without glial activation as a mediating step, it is interesting to note that the cytokine-induced mortality in the old mice appeared to parallel in our study an age-related enhancement of the glial response to the administered cytokines, which, as discussed below, was not detected in the study by Kalehua et al. (2000). In particular, the early timing of the animals’ death could correlate with early microglial activation, although the strong reactivity of microglial cells elicited in the old brain by cytokines could have been influenced by survival selection, not being, therefore, toxic per se. Acute inflammatory challenge and neuronal death In our paradigm of inflammatory insult, the strategies we adopted to investigate neurodegeneration did not reveal ongoing neuronal death at 2 days, when overt activation of both astrocytes and microglia was evident. Although this does not exclude that the administration of cytokine mixture could exert neurotoxic effects after a longer survival, this part of the study indicated that the glial cell activation we observed was not secondary to the onset of neurodegenerative phenomena in either the young or the old brain. When administered individually, cytokines do not evoke neural cell death directly, but coadministration of inflammatory mediators can exert synergistic effects resulting in neurotoxicity (Allan and Rothwell, 2001). Although in vivo studies indicate that certain cytokines, such as TNF-␣, induce cell death in certain conditions, the primary neurotoxic or neuroprotective influences seem to be exerted through their effects on glial function (Allan and Rothwell, 2001). This is at variance with chronic inflammation, whose role in neurodegeneration has been repeatedly inferred (see, for example, Hauss-Wegrzyniak et al., 1998, 2002; Blasko et al., 2004; Perry, 2004; Mrak and Griffin, 2005). On the other hand, acute inflammation has been found to affect chronic neurodegenerative conditions. For example, increased neuronal death in the CNS after both central

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and peripheral LPS administration was found in chronic neurodegeneration represented by a murine model of prion disease (Cunningham et al., 2005). It is also noteworthy that the neuroinflammatory response to i.v. administration of LPS was exacerbated in a murine model of Alzheimer’s disease amyloidosis, further suggesting that the exogenous inflammatory insult could amplify the neurodegenerative process (Sly et al., 2001). In the present study, however, we investigated the acute response of the “healthy” old brain to an inflammatory challenge. We used FJB staining because of its sensitivity in labeling irreversibly damaged neurons (Poirier et al., 2000), and in labeling both dying and “suffering” neurons regardless of whether the insult is of an apoptotic or necrotic nature (Schmued and Hopkins, 2000). Cell death was also investigated with in situ labeling of DNA fragmentation. Our data, therefore, suggest that central cytokine administration does not trigger rapidly neuronal death in the normal brain. Microglial and astrocytic response of the aging brain to acute i.c.v. administration of proinflammatory cytokines The main finding we have obtained in the study of the astrocytic and microglial response to cytokine exposure is that the glial activation elicited by this treatment is more marked in the old brain than at younger ages. Overall, the data we have obtained in both young and middle-aged mice are consistent with the findings reported previously in the same experimental paradigm in young mice (Kong et al., 2002). Interestingly, the comparison of three age groups reported in the present study suggests that the intensity of the response to the inflammatory challenge is not a linear function of age, but appears to characterize the advanced age in particular. The cytokineinduced activation of glial cells included phenotypic changes of both astrocytes and microglia, an increase in the number of these cells, and increases in protein expression as reflected by increased immunoreactivity documented quantitatively with image analysis. The enhancement of such response in the old brain involved all of these parameters. The data observed in the control brains after saline i.c.v. injections indicate some regional differences in the astrocytic reaction to the acute perturbation of the cerebrospinal fluid, to which some CNS areas could be especially sensitive. An increase of the astrocytic response was found in the present investigation in the CA1 field of the hippocampus of control saline-injected old mice, in which previous stereological analysis of the number of astrocytes and microglia in male mice of the same strain did not reveal statistically significant age-related differences (Long et al., 1998; Mouton et al., 2002). On the other hand, RNAs and protein markers of astrocytic and microglial activation show regional variation in the aged rat brain (Morgan et al., 1999). Phenotypic changes of glial cells in the old brain, e.g. in rat astrocytes (Landfield et al.,1977; Genisman et al., 1978; Lindsey et al.,1979; Pilegaard and Ladefoged, 1996) and human microglia (Streit et al., 2004), have also

been repeatedly documented, and the present data on glial cell features in the old mouse brain further support agerelated variation of the astroglial and microglial cell morphology. This study is the first to report an increased activation of glial cells in response to an inflammatory insult in the old age. In previous investigations based on acute LPS injection in the lateral cerebral ventricle of mice of different ages, i.e. with a paradigm similar to that we used, and in the same mouse strain (Kalehua et al., 2000), or based on chronic LPS infusion in the fourth ventricle of rats of different ages (Hauss-Wegrzyniak et al., 1999), neuroinflammation induced by LPS treatment was not found to elicit enhanced activation of glial cells in the brain parenchyma of the old animals. However, in the study by Kalehua et al. (2000) microglia was labeled by B4 isolectin histochemistry, and Hauss-Wegrzyniak et al. (1999) labeled microglia with the OX6 antibody which recognizes major histocompatibility class II antigens. The discrepancies between the present and previous data could, thus, be due to the strategies adopted for microglial labeling, and/or to the different paradigm of i.c.v. injection, which was based in our study on the administration of mixed cytokines. The present investigation pointed out that the enhanced activation of both astrocytes and microglia in the old brain compared with younger ones involves changes in both cell phenotype and cell number. Expansion of the microglial cell population is a highly characteristic feature of the activation of this cell type and reactive microgliosis (Ladeby et al., 2005). Increased number of microglia can be due to several mechanisms, which can act separately or in combination. Such mechanisms include proliferation of resident microglia, regulated also by neurotrophins expressed by activated microglia (Elkabes et al., 1996), migration of microglia from adjacent areas, and recruitment of microglial progenitors from the blood (Ladeby et al., 2005). In our investigation, the increase of microglia also implicated ameboid microglial cells, which could derive from hypertrophic microglia with stout processes or could also reflect migration of macrophages from blood vessels (see, for example, Dihné et al., 2001), or proliferation of hypertrophic microglial cells. The mechanisms underlying the increased number of activated microglial cells in the old brain remain to be investigated. The timing of such glial activation, which in general has been investigated in relation to neuronal degeneration, corresponds to that shown by previous studies (reviewed by Raivich et al., 1999; Ladeby et al., 2005) and overall the present data indicate that such activation occurs very rapidly also in the old brain after cytokine exposure. Increased numbers of astrocytes could be due to cell proliferation and/or differentiation of astrocyte precursors (Norton et al., 1992; Dihné et al., 2001). It is interesting to note in this respect that inflammatory mediators, including TNF-␣, are mitogens for astrocytes (Norton et al., 1992). Glial proliferation mechanisms have not been hitherto investigated in detail in the aging brain, but their molecular regulation could undergo changes during senescence, as

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shown by age-related alterations of growth factors in hippocampal astrocytes (Shetty et al., 2005). Data obtained with i.p. administration of LPS in mice of different ages indicated enhancement of immune-related gene expression in the hippocampus of old mice, suggesting T cell activation (Terao et al., 2002). Such findings suggest that lymphocytes, besides glial cells, could also take part in the enhanced response to an inflammatory stimulus in the aged brain, and this additional parameter of neuroimmune interaction will be addressed in future studies. It has been hypothesized that constant basal expression of cytokines may create in glial cells of the aged brain a level of tolerance such that an increase in cytokine expression of a related phenotypic change may require greater levels of stimulation than younger glia (Yu et al., 2002). A dose/response study was beyond the scope of the present investigation, and we therefore do not know if the age-related variation of neuroinflammation in the context of our paradigm has a threshold. However, the cytokine-induced enhancement of glial cell activation we have observed in the aged brain was consistent and significant, indicating that the response elicited by an inflammatory insult in glial cells primed by aging is amplified. Primed microglia can be morphologically similar to activated microglia, but can produce an exaggerated response to cytokines when further solicited (Godbout et al., 2005). The present study therefore suggests that the age-related enhanced response brings about marked transcriptional activation. Chronic overexpression of cytokines derived from activated glial cells in pathological conditions and in normal aging has been proposed to drive neurodegeneration leading to increased risk of Alzheimer’s disease during aging (Mrak and Griffin, 2005). It has also been suggested that glial cells, and in particular microglia, can be primed by aging for further activation through different mechanisms, including systemic inflammatory insults that can affect the brain through several routes (Perry et al., 2003; Perry, 2004). The present study shows that exposure to cytokines amplifies the activation of astrocytes and microglia during senescence, and could thus increase the potency of a vicious cycle between this process and neuronal injury. Acknowledgments—The authors are grateful to Guo-Ying Kong and Ze-Chun Peng for their advice in the initial steps of this study, to Anna Andrioli for having supplied material from pilocarpineinjected rats as positive control for the study of neuronal cell death, and to Raffaella Mariotti for her help with the TUNEL technique. This work was supported by grants of the Italian Ministry of Health and by the European Commission (grant QLK6-CT2002-02258).

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(Accepted 13 April 2006) (Available online 30 May 2006)