Reversible Demyelination, Blood-Brain Barrier Breakdown, and Pronounced Neutrophil Recruitment Induced by Chronic IL-1 Expression in the Brain
2004; Elsevier BV; Volume: 165; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63438-4
ISSN1525-2191
AutoresCarina Cintia Ferrari, Amaicha Mara Depino, Federico Prada, Nara I. Muraro, Sandra J. Campbell, Osvaldo L. Podhajcer, V. Hugh Perry, Daniel C. Anthony, Fernando J. Pitossi,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoInterleukin-1β (IL-1) expression is associated with a spectrum of neuroinflammatory processes related to chronic neurodegenerative diseases. The single-bolus microinjection of IL-1 into the central nervous system (CNS) parenchyma gives rise to delayed and localized neutrophil recruitment, transient blood-brain barrier (BBB) breakdown, but no overt damage to CNS integrity. However, acute microinjections of IL-1 do not mimic the chronic IL-1 expression, which is a feature of many CNS diseases. To investigate the response of the CNS to chronic IL-1 expression, we injected a recombinant adenovirus expressing IL-1 into the striatum. At the peak of IL-1 expression (days 8 and 14 post-injection), there was a marked recruitment of neutrophils, vasodilatation, and breakdown of the BBB. Microglia and astrocyte activation was evident during the first 14 days post-injection. At days 8 and 14, extensive demyelination was observed but the number of neurons was not affected by any treatment. Finally, at 30 days, signs of inflammation were no longer present, there was evidence of tissue reorganization, the BBB was intact, and the process of remyelination was noticeable. In summary, our data show that chronic expression of IL-1, in contrast to its acute delivery, can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases. Interleukin-1β (IL-1) expression is associated with a spectrum of neuroinflammatory processes related to chronic neurodegenerative diseases. The single-bolus microinjection of IL-1 into the central nervous system (CNS) parenchyma gives rise to delayed and localized neutrophil recruitment, transient blood-brain barrier (BBB) breakdown, but no overt damage to CNS integrity. However, acute microinjections of IL-1 do not mimic the chronic IL-1 expression, which is a feature of many CNS diseases. To investigate the response of the CNS to chronic IL-1 expression, we injected a recombinant adenovirus expressing IL-1 into the striatum. At the peak of IL-1 expression (days 8 and 14 post-injection), there was a marked recruitment of neutrophils, vasodilatation, and breakdown of the BBB. Microglia and astrocyte activation was evident during the first 14 days post-injection. At days 8 and 14, extensive demyelination was observed but the number of neurons was not affected by any treatment. Finally, at 30 days, signs of inflammation were no longer present, there was evidence of tissue reorganization, the BBB was intact, and the process of remyelination was noticeable. In summary, our data show that chronic expression of IL-1, in contrast to its acute delivery, can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases. Inflammation is a key component of the defense mechanism against infection in the periphery and in the central nervous system (CNS). Inflammation in the CNS has different features than in the periphery. For example, differential induction of cytokines may be involved in the atypical pattern of leukocyte recruitment induced in the brain.1Blond D Campbell SJ Butchart AG Perry VH Anthony DC Differential induction of interleukin-1beta and tumour necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain.Brain Res. 2002; 958: 89-99Crossref PubMed Scopus (64) Google Scholar Importantly, a dysregulated inflammatory response has been associated with many chronic CNS diseases, the prototype of which is multiple sclerosis (MS). However, in comparison with the periphery, many basic facts about inflammation in the CNS and its consequences are still unresolved. One useful approach to shed light on the distinct characteristics of the CNS inflammatory response is to study the response of brain tissue to individual components of inflammation, such as pro-inflammatory cytokines. 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21: 5528-5534Crossref PubMed Google Scholar In MS, the IL-1 levels in the CSF correlates with disease activity and certain IL-1 genotypes or the balance between IL-1 and IL-1ra are associated with disease severity, susceptibility, and/or progression.4Mann CL Davies MB Stevenson VL Leary SM Boggild MD Ko Ko C Jones PW Fryer AA Strange RC Thompson AJ Hawkins CP Interleukin 1 genotypes in multiple sclerosis and relationship to disease severity.J Neuroimmunol. 2002; 129: 197-204Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 10Hauser SL Doolittle TH Lincoln R Brown RH Dinarello CA Cytokine accumulations in CSF of multiple sclerosis patients: frequent detection of interleukin-1 and tumor necrosis factor but not interleukin-6.Neurology. 1990; 40: 1735-1739Crossref PubMed Google Scholar, 11de Jong BA Huizinga TW Bollen EL Uitdehaag BM Bosma GP van Buchem MA Remarque EJ Burgmans AC Kalkers NF Polman CH Westendorp RG Production of IL-1beta and IL-1Ra as risk factors for susceptibility and progression of relapse-onset multiple sclerosis.J Neuroimmunol. 2002; 126: 172-179Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Moreover, blocking IL-1 action reduces neuronal loss and inflammation induced by experimental brain insults9Boutin H LeFeuvre RA Horai R Asano M Iwakura Y Rothwell NJ Role of IL-1alpha and IL-1beta in ischemic brain damage.J Neurosci. 2001; 21: 5528-5534Crossref PubMed Google Scholar, 12Loddick SA Wong ML Bongiorno PB Gold PW Licinio J Rothwell NJ Endogenous interleukin-1 receptor antagonist is neuroprotective.Biochem Biophys Res Commun. 1997; 234: 211-215Crossref PubMed Scopus (113) Google Scholar, 13Grundy RI Rothwell NJ Allan SM Site-specific actions of interleukin-1 on excitotoxic cell death in the rat striatum.Brain Res. 2002; 926: 142-148Crossref PubMed Scopus (16) Google Scholar and IL-1 has been shown to be cytotoxic for oligodendrocytes both in vitro and in vivo.14Merrill JE Scolding NJ Mechanisms of damage to myelin and oligodendrocytes and their relevance to disease.Neuropathol Appl Neurobiol. 1999; 25: 435-458Crossref PubMed Scopus (95) Google Scholar, 15Brogi A Strazza M Melli M Costantino-Ceccarini E Induction of intracellular ceramide by interleukin-1 beta in oligodendrocytes.J Cell Biochem. 1997; 66: 532-541Crossref PubMed Scopus (47) Google Scholar On the other hand, although most of the evidence points to a detrimental role of IL-1 in MS, it has been shown to promote repair of cuprizone-derived demyelination via IGF-1.16Mason JL Suzuki K Chaplin DD Matsushima GK Interleukin-1beta promotes repair of the CNS.J Neurosci. 2001; 21: 7046-7052PubMed Google Scholar It has been shown that a single-bolus injection of IL-1 into the CNS parenchyma gives rise to delayed and localized neutrophil (PMN) recruitment, but no overt damage to CNS integrity.17Andersson PB Perry VH Gordon S Intracerebral injection of proinflammatory cytokines or leukocyte chemotaxins induces minimal myelomonocytic cell recruitment to the parenchyma of the central nervous system.J Exp Med. 1992; 176: 255-259Crossref PubMed Scopus (145) Google Scholar, 18Perry VH Bell MD Brown HC Matyszak MK Inflammation in the nervous system.Curr Opin Neurobiol. 1995; 5: 636-641Crossref PubMed Scopus (172) Google Scholar However, no study has examined the consequences of chronic expression of IL-1 in the brain parenchyma, which would be more relevant to the pattern of expression observed in chronic inflammatory diseases of the CNS. In this study, we were particularly keen to determine whether the atypical recruitment profile, which is observed after a single-bolus injection of IL-1, is conserved during extended IL-1 expression and whether extended expression would result in CNS damage. To achieve this, we used a recombinant replication-deficient adenovirus vector to overexpress human IL-1 for a transient, but prolonged period in the rat brain. We then analyzed the inflammation, gliosis, and any evidence of tissue damage that might be caused by the chronic expression of IL-1. Adenoviral vectors were generated as already described.19Kolb M Margetts PJ Anthony DC Pitossi F Gauldie J Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.J Clin Invest. 2001; 107: 1529-1536Crossref PubMed Scopus (591) Google Scholar Briefly, for construction of AdhIL-1β, human IL-1β cDNA (in pGEM3Z vector, a gift from British Biotech Pharmaceuticals Ltd., Oxford, United Kingdom) was cloned into a shuttle vector with a human cytomegalovirus promoter and cotransfected on 293 cells with a plasmid containing E1- to E3-deleted type 5 adenoviral genome. The correct recombination was verified with restriction digestions of the purified viral DNA obtained by HIRT, and further confirmed by Southern blot. Transgene expression was observed by Western blot with an anti-human IL-1-specific antibody.20Gallagher S Winston SE Fuller SA Hurrell J Immunoblotting and immunodetection.in: Ausubel FK Brent R Kingston R Moore D Seidman G Smith J Struhl K Current Protocols in Molecular Biology. John Wiley and Sons, Inc., Boston, MA1995: 10.8.7-10.8.24Google Scholar The adenoviral vectors were purified by plaque-formation under agar. Stocks were obtained from large-scale preparations in HEK293 cells by double cesium chloride gradients, and were quantified by plaque assay (final titers: Adβgal = 1.5 × 1010 pfu/μl, AdIL-1 = 1 × 1010 pfu/μl). Stocks had less than 1 ng/ml of endotoxin, assayed with E-TOXATE reagents (Sigma, St. Louis, MO). Viral stocks were free of autoreplicative particles as assessed by PCR and transduction of non-transcomplementary cells (HeLa, ATCC).21Lochmüller H Jani A Huard J Prescott S Simoneau M Massie B Karpati G Acsadi G Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (DE1 + DE3) during multiple passages in 293 cells.Hum Gene Ther. 1994; 5: 1485-1491Crossref PubMed Scopus (171) Google Scholar The adenoviral vector expressing β-galactosidase (Adβgal) was gently provided by Dr. J. Mallet (Hospital Pitie Salpetriere, Paris, France).22Le Gal La Salle G Roberts JJ Berrard S Ridoux V Stratford-Perricaudet LD Perricaudet M Mallet J An adenovirus vector for gene transfer into neurons and glia in the brain.Science. 1993; 259: 988-990Crossref PubMed Scopus (655) Google Scholar Adult male Wistar rats (200 g to 280 g) (Anmat, Buenos Aires), housed in groups of two animals, under controlled temperature (22°C ± 2°C), artificially lit under a 12-hour cycle period and with water and food ad libitum. For central injections, the animals were anesthetized with ketamine chlorhydrate (80 mg/kg) and xylazine (8 mg/kg). The adenovirus were administered with a 50-μm tipped finely drawn glass capillary, stereotactically directed to the center of the right striatum (bregma, +1 mm; lateral, −3 mm; ventral, −4.5 mm).23Paxinos G Watson C The Rat Brain in Stereotaxic Coordinates. Academic Press, Orlando1986Google Scholar Striatal injections of 1 μl of adenoviral vectors or vehicle were infused over a 5-minute period and the pipette was kept in place for additional 2 minutes before removal. All surgical procedures took place in the morning to avoid effects of circadian variations in cytokine expression. Human IL-1β and β-galactosidase expressing adenoviral vectors were diluted in sterile PBS (pH 7.4) and administered at a dose of 107 pfu/rat. The animals were killed at 2, 8, 14, 21, or 30 days post-surgery. All animal procedures were performed according to the rules and standards of the German animal protection law and the regulations for the use of laboratory animals of the National Institutes of Health, USA. The animals were deeply anesthetized, then perfused transcardially with heparinized saline followed by cold 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB) (pH 7.2). After dissecting the brains, they were placed in the same fixative overnight at 4°C. Tissues were then cryoprotected by immersion in 30% sucrose, frozen in isopentane, and serially sectioned in a cryostat (14 μm and 40 μm, alternating). The 14-μm sections were mounted on gelatin-coated slides and the 40-μm sections were used for free-floating immunohistochemistry. Serial sections (14 μm) were stained with Luxol fast blue/cresyl violet stain to determine demyelination in the nervous tissue. Serial free-floating sections were used to detect β-galactosidase activity, using X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside) as substrate.22Le Gal La Salle G Roberts JJ Berrard S Ridoux V Stratford-Perricaudet LD Perricaudet M Mallet J An adenovirus vector for gene transfer into neurons and glia in the brain.Science. 1993; 259: 988-990Crossref PubMed Scopus (655) Google Scholar To identify neurons we used immunohistochemistry to detect the neuronal marker NeuN in unfixed tissue rats were deeply anesthetized and perfused transcardially with heparinized saline. The brain was then dissected and quickly frozen in isopentane. Serial sections (10 μm) were cut in a cryostat, mounted on gelatin-subbing slides and kept at −20°C. Polymorphonuclear neutrophil (PMN) cells were identified by their nuclear morphology appearance in 40-μm thick cresyl violet-stained sections and confirmed by immunohistochemistry with MBS II antibody.24Anthony DC Bolton SJ Fearn S Perry VH Age-related effects of interleukin-1 beta on polymorphonuclear neutrophil-dependent increases in blood-brain barrier permeability in rats.Brain. 1997; 120: 435-444Crossref PubMed Scopus (221) Google Scholar Leukocytes were classified as: “marginated”, those cells that appeared to be adherent to the luminal side of the endothelium; “cuffed”, those that appeared on the abluminal side of the vessels; and “recruited”, those cells that had crossed the vascular endothelium and the basement membrane and were located in the parenchyma.24Anthony DC Bolton SJ Fearn S Perry VH Age-related effects of interleukin-1 beta on polymorphonuclear neutrophil-dependent increases in blood-brain barrier permeability in rats.Brain. 1997; 120: 435-444Crossref PubMed Scopus (221) Google Scholar Cells of the mononuclear phagocyte lineage, microglia and macrophages, were identified using the lectin Griffonia simplicifolia (GSA-1B4)25Kaur C Ling EA Study of the transformation of amoeboid microglial cells into microglia labelled with the isolectin Griffonia simplicifolia in postnatal rats.Acta Anat (Basel). 1991; 142: 118-125Crossref PubMed Scopus (66) Google Scholar and the antibody ED1 which stains recently recruited monocytes/macrophages and activated but not quiescent microglia. Free-floating sections were incubated in blocking buffer (1% donkey serum, 0.1% Triton in 0.1 mol/L PB) for 45 minutes, rinsed in 0.1% Triton in 0.1 mol/L PB and incubated overnight with primary antibodies diluted in blocking solution. The antibodies used were: anti-human IL-1β (that recognizes both human and rat IL-1β) (1:100; Peprotech, Mexico), ED1 (1:200; Serotec, Raleigh, NC), MBS II (1:100; specific for neutrophils,24Anthony DC Bolton SJ Fearn S Perry VH Age-related effects of interleukin-1 beta on polymorphonuclear neutrophil-dependent increases in blood-brain barrier permeability in rats.Brain. 1997; 120: 435-444Crossref PubMed Scopus (221) Google Scholar ED2 (1:200; Serotec), anti-GFAP (1:700; Dako, Carpinteria, CA), and anti-NeuN (1:100; Chemicon, Temecula, CA). We used the biotinylated lectin Griffonia simplicifolia (GSA-1B4, 1:50; Vector Laboratories, Burlingame, CA), that specifically binds to the terminal α-D-galactose residues in the plasma membrane of transforming microglial cells.25Kaur C Ling EA Study of the transformation of amoeboid microglial cells into microglia labelled with the isolectin Griffonia simplicifolia in postnatal rats.Acta Anat (Basel). 1991; 142: 118-125Crossref PubMed Scopus (66) Google Scholar After three 5-minute washes with 0.1 mol/L PB, the sections were incubated with either indocarbocyanine Cy3 (Cy3) conjugated donkey anti-mouse antibody (1:250; Jackson ImmunoResearch Laboratories Inc., West Grove, PA), cyanine Cy2 (Cy2) conjugated donkey anti-rabbit antibody (1:250; Jackson), or Cy2 conjugated streptavidin (1:250; Jackson) for 2 hours, rinsed in 0.1 mol/L PB, and mounted in Mowiol (Calbiochem, San Diego, CA). Digital images were collected in a Zeiss LSM 510 laser scanning confocal microscope equipped with a krypton-argon laser. For the quantification of neutrophils, cell types were identified by their morphology on cresyl violet staining under ×40 magnification. Every sixth 40-μm thick serial section was counted and the area of the striatum was measured using the Image Pro image analysis system (Media Cybernetics, Silver Spring, MD). Graphs show the number of cells per mm3. For the quantification of activated macrophages, we measured the ED1-positive area in every sixth 40-μm serial section using Image Pro image analysis system. For the quantification of the total number of neurons in the striatum, NeuN-positive cells were counted in every tenth 10-μm thick serial section under ×20 magnification. In addition, the areas in which we counted the cells were measured with the Image Pro image analysis software and the striatal volume was calculated based on these measurements and the distance among areas. Nuclear staining for NeuN was counted as positive only for those nuclei showing an intact morphology. False-positive cells (mostly composed of PMN cells) were discarded based on cellular morphology. All measurements were performed throughout the striatum. Graphs show the total number of neurons in the whole striatum. Anesthetized animals were intracardially perfused with a modified saline solution (0.8% NaCl, 0.8% sucrose, 0.4% glucose) followed by a fixative made of 2% glutaraldehyde, 2% paraformaldehyde in 0.1 mol/L cacodylate buffer (pH 7.2). The 1-mm3 samples of striatum were then immersed in the same fixative at 4°C overnight. The tissue was stored in 0.1 mol/L cacodylate buffer (CB) with 0.2 mol/L sucrose. After 5-minute washes in CB, they were post-fixed in OsO4 in CB for 1 hour, washed in CB, dehydrated in ethanol series, cleared in acetone, and embedded in Araldite. Semi-thin and ultra-thin sections were cut on a Sorvall-Porter-Blum ultramicrotome and stained with toluidine blue and double-stained with uranyl acetate and lead citrate, respectively. Ultra-thin sections were examined in either a JEOL 1200EX2 or Zeiss EM10-C electron microscopes. The electronic photomicrograhs were obtained by using standard photographic and darkroom procedures. Thirty minutes before perfusion the animals were injected intravenously with 104 U/kg of type II horseradish peroxidase (HRP, Sigma, St. Louis, MO). Then, the animals were perfused as previously described with Karnovsky′s fixative and their brains processed as previously described. HRP was detected in free-floating sections by a modified Hanker-Yates method.26Perry VH Linden R Evidence for dendritic competion in the developing retina.Nature. 1982; 297: 683-685Crossref PubMed Scopus (289) Google Scholar Fourteen-μm sections mounted on gelatin slides were stained with Fluoro-Jade B (Histo-Chem, Jefferson, AR) according to the technique previously described.27Schmued LC Hopkins KJ Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration.Brain Res. 2000; 874: 123-130Crossref PubMed Scopus (1074) Google Scholar As a positive control, the substantia nigra of a rat brain, which had been injected intrastrially with 6OH-dopamine, was used. The animals were perfused as previously described with heparinized saline to clear the blood from the brain samples. The brains were quickly removed, sliced into 3-mm sections and both the injected and uninjected striatum were punched out, snap-frozen in liquid nitrogen, and stored at −80°C. The tissue was homogenized on ice in 400 μl of Tris-HCl buffer (pH 7.3) containing protease inhibitors (10 μg/ml aproteinin, 5 μg/ml peptastin, 5 μg/ml leupeptin, 1 mmol/L PMSF, Sigma). Homogenates were centrifuged at 10000 × g at 4°C for 10 minutes and the supernatants were then ultracentrifuged at 40000 rpm for 2 hours. The supernatants were stored at −80°C until used. Bradford protein assays were performed to determine total protein concentration in each sample. A commercially available human IL-1β (hIL-1β) ELISA kit (R&D, Minneapolis, MN) was used according to the manufacturer's instructions to quantify hIL-1β with high sensitivity (2 pg/ml). To estimate protein recovery, hIL-1β standard was added exogenously to brain homogenates and the ELISA was performed as described before. A recovery of 85 to 90% of the added hIL-1β standard was found. Statistical differences among treatments were determined using one-way analysis of variance test, followed by Newman-Keuls Multiple Comparison test. Alternatively, Student's t-tests were used. We achieved chronic expression of hIL-1β in the rat striatum with the administration of a low dose (107 pfu) of a replication-deficient, recombinant adenoviral vector (AdIL-1). We chose this dose because it was the maximal dose of control vector (Adβgal) that failed to cause an inflammatory response assessed by the presence or abscence of inflammatory infiltrate in Nissl-stained histological sections. In AdIL-1β injected animals, hIL-1β expression started 2 days after administration, peaked between 8 and 14 days and was still detected by ELISA 30 days post-inoculation (Figure 1A). hIL-1β expression was not detected in control animals, reflecting the specificity of the ELISA kit. The calculated mean amounts of total hIL-1β in the whole striatum (2.5 ng, 41.1 ng, 40.6 ng, and 2.1 ng, at 2, 8, 14, and 30 days, respectively) are within the range that produces an inflammatory response when injected in the periphery in rodents and humans.28Dinarello CA Interleukin-1.Cytokine Growth Factor Rev. 1997; 8: 253-265Abstract Full Text PDF PubMed Scopus (596) Google Scholar At 2 and 8 days post-AdIL-1 injection, ramified IL-1β-positive cells were restricted to the injection site and surrounding vessels (data not shown). At 14 days, most of the IL-1β-positive cells were located close to the injection region within the coronal sections through the injection site (Figure 1B). However, scattered IL-1β-positive cells were widespread throughout the antero-posterior axis of the striatum. No IL-1β staining was observed outside the striatum. Thirty days post-injection, expression of IL-1β was not detected by immunofluorescence, probably due to a lack of sensitivity. After the inoculation of control vector (Adβgal), β-galactosidase activity was detected in the injected striatum only by X-gal staining at all the analyzed time points (data not shown). Little or no recruitment of any type of leukocyte or vasodilatation was observed in the uninjected hemispheres or in animals injected with similar doses of Adβgal or saline solution at any of the time points studied (Figure 2A and data not shown). Two days after AdIL-1 injection, marginated leukocytes filled the vessels located near the injection site. This leukocyte population was composed predominantly of monocytes/macrophages and only scarce PMN were observed (Figure 2, B and F). No cuffed leukocytes were observed at this time point (Figure 2B). However, at 8 days, cuffed and recruited neutrophils to the brain parenchyma were found to be widespread throughout the whole striatum (Figure 2F and data not shown). At 14 days, a very large number of neutrophils were observed throughout the striatum (Figure 2, C, D, and F) and the blood vessels were vasodilated and filled with marginated PMN and few monocytes/macrophages (Figure 2, C, D, and G). However, there was no inflammatory response in the meninges of either hemisphere at this time point (see supplementary data 1). The inflammatory response in the parenchyma was totally resolved by 30 days after surgery, when no signs of vasodilatation or inflammatory infiltrate were visualized (Figure 2E). Control animals injected either with Adβgal or PBS exhibited minor microglial/macrophage activation 2 and 8 days after injection, as seen by GSA and ED1 immunostaining (Figure 3, A, D, E, and data not shown). Microglial activation was evident close to the injection site, reflecting the non-specific response of the tissue to the surgery (Figure 3, A, E, and data not shown). The contribution of peripheral monocytes/macrophages to the total number of activated microglial cells was not possible to determine based on morphology or immunostaining. No GSA-positive (GSA+) or ED-1-positive (ED-1+) microglia was observed in control animals at 14, 21, or 30 days post-injection (Figure 3, D, H, and data not shown). No GFAP-positive (GFAP+) astrocytes were detected in control animals at any time point studied (Figure 3, I, L, and data not shown). Two days after the injection of AdIL-1, activated microglia defined as GSA+ or ED-1+ cells, with either thick and stout processes or round-shaped morphology were detected (Figure 3, B, D, and F). The ED-1+ cells were more restricted to the needle tract (Figure 3B). An extensive and widespread astrogliosis, defined as GFAP+ ramified cells, was observed in the striatum (Figure 3J). At 8 and 14 days, immunoreactive cells for ED1 and with morphological signs of phagocytic activity (round shape and vacuolated cytoplasm) were observed in the whole striatum (Figure 3C). The area covered by ED1+ cells reached a maximum at these stages (Figure 3, C and D, and data not shown). The GSA+ cells located nearest to the injection site exhibited a typical appearance of phagocytic cells, ie, round shape with vacuolated cytoplasm, while the GSA+ cells distant to the injection site exhibited the typical ramified morphology of microglial cells (Figure 3G). Finally, the whole striatum was filled with GFAP+ cells at this stage, reflecting an intense astrogliosis after the inflammatory insult (Figure 3K). At 30 days, the inflammatory response had resolved and there were no signs of GSA+, ED-1+, or GFAP+ cells in the striatum of the animals injected with AdIL-1 (Figure 3D). To evaluate whether neutrophil recruitment was accompanied by BBB damage, we examined the permeability of the BBB by the intravenous injection of HRP in treated animals, followed by the HRP detection as described in the methods. No breakdown of the BBB was observed in the animals injected with Adβgal or in the contralateral striatum of AdIL-1β injected rats at any time point studied (Figure 4A). At 2 days, no marked breakdown of the BBB was observed in animals injected with AdIL-1 (Figure 4, B and G). However damage to the BBB was evident at 8 days, and was conspicuous at 14 days, as demonstrated by extravasation of HRP from vessels in the parenchyma of the striatum (Figure 4, C, D, and G). A large number of HRP-positive neutrophils and phagocytic cells were also evident in the brain parenchyma with this technique (data not shown). The integrity of the BBB was still not restored by 21 days after AdIL-1 injection, but it was totally restored by 30 days post-injection (Figure 4, E, F, and G). The effects of the chronic expression of IL-1 on the integrity of myelin were also studied. No evidence of myelin damage or demyelination was observed in Adβgal-injected animals or in the uninjected striatum of AdIL-1 injected rats by Luxol staining, in semi-thin sections and at the ultrastructural level at any time point studied (Figure 5, A, D, and L). At 2 days, no myelin damage was seen in the striatum of AdIL-1-injected animals (data not shown). At 8 days post-injection, an evident loss of the regular d
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