The Low-Density Lipoprotein Receptor-Related Protein 1 Mediates Tissue-Type Plasminogen Activator-Induced Microglial Activation in the Ischemic Brain
2009; Elsevier BV; Volume: 174; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.080661
ISSN1525-2191
AutoresChen Zhang, Jie An, Dudley K. Strickland, Manuel Yepes,
Tópico(s)Nuclear Receptors and Signaling
ResumoMicroglia are the immune cells of the central nervous system (CNS) that become activated in response to pathological situations such as cerebral ischemia. Tissue-type plasminogen activator (tPA) is a serine proteinase that is found in the intravascular space and the CNS. The low-density lipoprotein receptor-related protein 1 (LRP1) is a member of the low-density lipoprotein receptor gene family found in neurons, astrocytes, and microglia. The present study investigated whether the interaction between tPA and microglial LRP1 plays a role in cerebral ischemia-induced microglial activation. We found that middle cerebral artery occlusion (MCAO) induces microglial activation in both wild-type and plasminogen-deficient (Plg−/−) mice. In contrast, MCAO-induced microglial activation is significantly decreased in tPA-deficient (tPA−/−) mice and in mice that lack LRP1 in microglial cells (macLRP−). We observed a significant increase in microglial activation when tPA−/− mice received treatment with murine tPA after MCAO. In contrast, treatment of macLRP− mice with tPA did not have an effect on the extent of microglial activation. Finally, both the volume of the ischemic lesion as well as inducible nitric oxide synthase production were significantly decreased in macLRP− mice and macLRP− microglia. In summary, our results indicate that the interaction between tPA and LRP1 induces microglial activation with the generation of an inflammatory response in the ischemic brain, suggesting a cytokine-like role for tPA in the CNS. Microglia are the immune cells of the central nervous system (CNS) that become activated in response to pathological situations such as cerebral ischemia. Tissue-type plasminogen activator (tPA) is a serine proteinase that is found in the intravascular space and the CNS. The low-density lipoprotein receptor-related protein 1 (LRP1) is a member of the low-density lipoprotein receptor gene family found in neurons, astrocytes, and microglia. The present study investigated whether the interaction between tPA and microglial LRP1 plays a role in cerebral ischemia-induced microglial activation. We found that middle cerebral artery occlusion (MCAO) induces microglial activation in both wild-type and plasminogen-deficient (Plg−/−) mice. In contrast, MCAO-induced microglial activation is significantly decreased in tPA-deficient (tPA−/−) mice and in mice that lack LRP1 in microglial cells (macLRP−). We observed a significant increase in microglial activation when tPA−/− mice received treatment with murine tPA after MCAO. In contrast, treatment of macLRP− mice with tPA did not have an effect on the extent of microglial activation. Finally, both the volume of the ischemic lesion as well as inducible nitric oxide synthase production were significantly decreased in macLRP− mice and macLRP− microglia. In summary, our results indicate that the interaction between tPA and LRP1 induces microglial activation with the generation of an inflammatory response in the ischemic brain, suggesting a cytokine-like role for tPA in the CNS. Microglia are the immune cells of the central nervous system (CNS) initially described by del Rio-Hortega1del Rio-Hortega P Microglia.in: Penfield W Cytology and Cellular Pathology of the Nervous System. Hoeber, New York1932: 482-534Google Scholar as a unique cell type that comprises ∼12% of the brain. Microglia become activated in response to changes in the microenvironment induced by multiple pathological situations such as cerebral ischemia.2Hanisch UK Kettenmann H Microglia: active sensor and versatile effector cells in the normal and pathologic brain.Nat Neurosci. 2007; 10: 1387-1394Crossref PubMed Scopus (2697) Google Scholar This process is characterized by a number of features including morphological changes, the acquisition of a phagocytic phenotype, and the release of free radicals and nitric oxide.3Ladeby R Wirenfeldt M Garcia-Ovejero D Fenger C Dissing-Olesen L Dalmau I Finsen B Microglial cell population dynamics in the injured adult central nervous system.Brain Res Rev. 2005; 48: 196-206Crossref PubMed Scopus (272) Google Scholar The onset of cerebral ischemia induces the activation of microglial cells, which results in the generation of a local inflammatory reaction mediated, including the induction of nuclear factor (NF)-κB-regulated pro-inflammatory molecules such as inducible nitric oxide synthase (iNOS).4Iadecola C Zhang F Casey R Nagayama M Ross ME Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene.J Neurosci. 1997; 17: 9157-9164Crossref PubMed Google Scholar, 5Parathath SR Parathath S Tsirka SE Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice.J Cell Sci. 2006; 119: 339-349Crossref PubMed Scopus (102) Google ScholarIn vitro studies have indicated that one pathway leading to microglial activation is initiated by tissue-type plasminogen activator (tPA).6Rogove AD Siao C Keyt B Strickland S Tsirka SE Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system.J Cell Sci. 1999; 112: 4007-4016Crossref PubMed Google Scholar tPA, which is produced by endothelial cells, astrocytes, microglia, and neurons,7Krystosek A Seeds NW Plasminogen activator release at the neuronal growth cone.Science. 1981; 213: 1532-1534Crossref PubMed Scopus (320) Google Scholar, 8Krystosek A Seeds NW Plasminogen activator secretion by granule neurons in cultures of developing cerebellum.Proc Natl Acad Sci USA. 1981; 78: 7810-7814Crossref PubMed Scopus (155) Google Scholar, 9Siao CJ Fernandez SR Tsirka SE Cell type-specific roles for tissue plasminogen activator released by neurons or microglia after excitotoxic injury.J Neurosci. 2003; 23: 3234-3242Crossref PubMed Google Scholar is a highly specific serine proteinase and one of the two main plasminogen activators.10Bugge TH Xiao Q Kombrinck KW Flick MJ Holmback K Danton MJ Colbert MC Witte DP Fujikawa K Davie EW Degen JL Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation.Proc Natl Acad Sci USA. 1996; 93: 6258-6263Crossref PubMed Scopus (285) Google Scholar In the intravascular space tPA functions as a thrombolytic enzyme in which its main substrate is plasminogen. Based on these properties, recombinant tPA is the only Food and Drug Administration-approved medication for the treatment of patients with acute ischemic stroke.11The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group Tissue plasminogen activator for acute ischemic stroke.N Engl J Med. 1995; 333: 1581-1587Crossref PubMed Scopus (10026) Google Scholar In contrast, in the CNS tPA initiates multiple physiological and pathological processes via plasminogen-independent pathways, including learning,12Seeds NW Williams BL Bickford PC Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning.Science. 1995; 270: 1992-1994Crossref PubMed Scopus (259) Google Scholar synaptic plasticity,7Krystosek A Seeds NW Plasminogen activator release at the neuronal growth cone.Science. 1981; 213: 1532-1534Crossref PubMed Scopus (320) Google Scholar, 8Krystosek A Seeds NW Plasminogen activator secretion by granule neurons in cultures of developing cerebellum.Proc Natl Acad Sci USA. 1981; 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94: 9779-9781Crossref PubMed Scopus (105) Google Scholar, 17Wang YF Tsirka SE Strickland S Stieg PE Soriano SG Lipton SA Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice.Nat Med. 1998; 4: 228-231Crossref PubMed Scopus (553) Google Scholar, 18Yepes M Sandkvist M Wong MK Coleman TA Smith E Cohan SL Lawrence DA Neuroserpin reduces cerebral infarct volume and protects neurons from ischemia-induced apoptosis.Blood. 2000; 96: 569-576Crossref PubMed Google Scholar regulation of the permeability of the neurovascular unit,19Polavarapu R Gongora MC Yi H Ranganthan S Lawrence DA Strickland D Yepes M Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit.Blood. 2007; 109: 3270-3278Crossref PubMed Scopus (140) Google Scholar, 20Yepes M Sandkvist M Moore EG Bugge TH Strickland DK Lawrence DA Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein.J Clin Invest. 2003; 112: 1533-1540Crossref PubMed Scopus (454) Google Scholar and microglial activation.6Rogove AD Siao C Keyt B Strickland S Tsirka SE Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system.J Cell Sci. 1999; 112: 4007-4016Crossref PubMed Google Scholar, 21Siao CJ Tsirka SE Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II.J Neurosci. 2002; 22: 3352-3358PubMed Google ScholarThe low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a member of the LDL receptor gene family that interacts with multiple ligands including plasminogen activators.22Herz J Strickland DK LRP: a multifunctional scavenger and signaling receptor.J Clin Invest. 2001; 108: 779-784Crossref PubMed Scopus (876) Google Scholar, 23Lillis AP Van Duyn LB Murphy-Ullrich JE Strickland DK LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies.Physiol Rev. 2008; 88: 887-918Crossref PubMed Scopus (504) Google Scholar In the CNS, LRP1 is found in perivascular astrocytes, neurons, and microglia19Polavarapu R Gongora MC Yi H Ranganthan S Lawrence DA Strickland D Yepes M Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit.Blood. 2007; 109: 3270-3278Crossref PubMed Scopus (140) Google Scholar, 24Wolf BB Lopes MB VandenBerg SR Gonias SL Characterization and immunohistochemical localization of alpha 2-macroglobulin receptor (low-density lipoprotein receptor-related protein) in human brain.Am J Pathol. 1992; 141: 37-42PubMed Google Scholar where it has been implicated in cellular signal transduction pathways.25Herz J The LDL receptor gene family: (un)expected signal transducers in the brain.Neuron. 2001; 29: 571-581Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar After middle cerebral artery occlusion (MCAO) there is an increase in endogenous tPA activity within the ischemic tissue,17Wang YF Tsirka SE Strickland S Stieg PE Soriano SG Lipton SA Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice.Nat Med. 1998; 4: 228-231Crossref PubMed Scopus (553) Google Scholar, 18Yepes M Sandkvist M Wong MK Coleman TA Smith E Cohan SL Lawrence DA Neuroserpin reduces cerebral infarct volume and protects neurons from ischemia-induced apoptosis.Blood. 2000; 96: 569-576Crossref PubMed Google Scholar, 20Yepes M Sandkvist M Moore EG Bugge TH Strickland DK Lawrence DA Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein.J Clin Invest. 2003; 112: 1533-1540Crossref PubMed Scopus (454) Google Scholar and the association of this tPA with LRP1 has an effect on cerebrovascular tone,26Nassar T Haj-Yehia A Akkawi S Kuo A Bdeir K Mazar A Cines DB Higazi AA Binding of urokinase to low density lipoprotein-related receptor (LRP) regulates vascular smooth muscle cell contraction.J Biol Chem. 2002; 277: 40499-40504Crossref PubMed Scopus (49) Google Scholar NF-κB activation,27Zhang X Polavarapu R She H Mao Z Yepes M Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein mediate cerebral ischemia-induced nuclear factor-{kappa}B pathway activation.Am J Pathol. 2007; 171: 1281-1290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Akt phosphorylation,28An J Zhang C Polavarapu R Zhang X Zhang X Yepes M Tissue-type plasminogen activator and the low density lipoprotein receptor-related protein induce Akt phosphorylation in the ischemic brain.Blood. 2008; 112: 2787-2794Crossref PubMed Scopus (58) Google Scholar and regulation of the permeability of the neurovascular unit.19Polavarapu R Gongora MC Yi H Ranganthan S Lawrence DA Strickland D Yepes M Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit.Blood. 2007; 109: 3270-3278Crossref PubMed Scopus (140) Google Scholar, 20Yepes M Sandkvist M Moore EG Bugge TH Strickland DK Lawrence DA Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein.J Clin Invest. 2003; 112: 1533-1540Crossref PubMed Scopus (454) Google ScholarTo gain insight into mechanisms leading to microglial activation during cerebral ischemia in vivo, we have generated a mouse model in which LRP1 has selectively been deleted in macrophages and microglia. Our results reveal that after MCAO, an interaction between tPA and microglial LRP1 is required for microglial activation with induction of iNOS and accumulation of nitrotyrosine. This novel pathway for cerebral ischemia-induced microglial activation during cerebral ischemia represents a potential target for the treatment of patients with acute ischemic stroke.Materials and MethodsAnimal Model of Cerebral IschemiaMurine strains were wild-type C57BL/6J, tPA-deficient (tPA−/−), and plasminogen-deficient (Plg−/−).29Bugge TH Kombrinck KW Flick MJ Daugherty CC Danton MJ Degen JL Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency.Cell. 1996; 87: 709-719Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar LRP floxed mice30Rohlmann A Gotthardt M Hammer RE Herz J Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.J Clin Invest. 1998; 101: 689-695Crossref PubMed Scopus (396) Google Scholar on a LDL receptor (LDLR)-deficient background were kindly provided by Dr. Joachim Herz (University of Texas Southwestern, Dallas, TX). These mice were crossed with LysMCre mice as described,31Clausen BE Burkhardt C Reith W Renkawitz R Forster I Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1535) Google Scholar, 32Lillis AP Greenlee MC Mikhailenko I Pizzo SV Tenner AJ Strickland DK Bohlson SS Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis.J Immunol. 2008; 181: 364-373PubMed Google Scholar and then backcrossed with wild-type C57BL/6J mice to generate LRPflox+/−Cre+/− mice on a LDLR wild-type background. These mice were crossed with each other to generate LRP flox+/+ Cre+ (designated macLRP−) or LRP floxed+/+ Cre− (designated wild-type), which were used as the littermate controls. Transient occlusion of the middle cerebral artery (tMCAO) was induced with a 6-0 silk suture advanced from the external carotid artery into the internal carotid artery until the origin of the middle cerebral artery as described elsewhere.33Belayev L Busto R Zhao W Fernandez G Ginsberg MD Middle cerebral artery occlusion in the mouse by intraluminal suture coated with poly-L-lysine: neurological and histological validation.Brain Res. 1999; 833: 181-190Crossref PubMed Scopus (118) Google Scholar Briefly, after a midline skin incision, the external carotid artery was isolated and ligated proximally with a 6-0 silk suture. A nylon monofilament (6-0; Ethicon, Issy Les Moulineaux, France), coated with a mixture of silicone resin (Xantopren Mucosa; Heraeus Kulzer, Hanau, Grunerweg, Germany) and a hardener (Universal activator; Heraeus Kulzer) was introduced through the incision in the external carotid artery and advanced gently up to the origin of the middle cerebral artery. The suture was tightly fixed at the final position and withdrawn after 60 minutes of cerebral ischemia. Cerebral perfusion in the distribution of the middle cerebral artery was monitored throughout the surgical procedure and after reperfusion with a laser Doppler (Perimed Inc., North Royalton, OH), and only animals with a >70% decrease in cerebral perfusion after occlusion and complete recovery after suture withdrawal were included in this study. The rectal and masseter muscle temperatures were controlled at 37°C with a homoeothermic blanket. Immediately after tMCAO, animals were intracortically injected at bregma, −1 mm; mediolateral, 3 mm; and dorsoventral, 3 mm with 2 μl of either phosphate-buffered saline (PBS) (in wild-type, tPA−/−, Plg−/−, and macLRP− mice) or murine tPA (1 μmol/L, in tPA−/− and macLRP− mice; Molecular Innovations Inc., Royal Oak, MI). A subgroup of tPA−/− mice was intracortically injected with inactive tPA with an alanine for serine substitution at the active site Ser481 (S481A, 1 μmol/L; Molecular Innovations Inc.). Twenty-four hours later the brains of a second subgroup of wild-type and macLRP− mice were harvested and the volumen of the ischemic lesion was measured in brain sections stained with 2,3,5-triphenyltetrazolium chloride as described elsewhere.34Nagai N De Mol M Lijnen HR Carmeliet P Collen D Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice.Circulation. 1999; 99: 2440-2444Crossref PubMed Scopus (212) Google Scholar Statistical analysis was performed with the Student's t-test.Definition of Areas of Interest (AOI) and ImmunohistochemistryThree AOI were previously defined in the ischemic hemisphere by magnetic resonance imaging parameters as described elsewhere.19Polavarapu R Gongora MC Yi H Ranganthan S Lawrence DA Strickland D Yepes M Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit.Blood. 2007; 109: 3270-3278Crossref PubMed Scopus (140) Google Scholar, 27Zhang X Polavarapu R She H Mao Z Yepes M Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein mediate cerebral ischemia-induced nuclear factor-{kappa}B pathway activation.Am J Pathol. 2007; 171: 1281-1290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar In brief, each coronal section of the brain was divided into 16 square areas (150 mm2 each one) that involved the necrotic core and the area of ischemic penumbra, and comparable areas in the nonischemic hemisphere.19Polavarapu R Gongora MC Yi H Ranganthan S Lawrence DA Strickland D Yepes M Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit.Blood. 2007; 109: 3270-3278Crossref PubMed Scopus (140) Google Scholar, 27Zhang X Polavarapu R She H Mao Z Yepes M Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein mediate cerebral ischemia-induced nuclear factor-{kappa}B pathway activation.Am J Pathol. 2007; 171: 1281-1290Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar AOI-1 and AOI-2 were localized in the area of ischemic penumbra in the fronto- and temporo-parietal lobes, whereas AOI-3 corresponded to the necrotic zone in the parietal lobe. For the immunohistochemistry studies, 20 frozen brain sections 10 μm each, were obtained 24 hours after reperfusion in wild-type, tPA−/−, Plg−/−, and macLRP− mice and co-stained with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA), the glial cell marker β-isolectin (Sigma-Aldrich, St. Louis, MO), and antibodies against F4/80 (Serotec, Raleigh, NC). A subset of brains was stained with DAPI and antibodies against Mac-1 and nitrotyrosine (1:200; Cayman Chemical, Ann Arbor, MI). Activated microglia was identified by the simultaneous presence of ameboid morphology and immunoreactivity for β-isolectin and F4/80. Images were digitized in a Zeiss Axioplan 2 microscope (20-fold objective) with a Zeiss AxioCam and imported into AxioVision (Carl Zeiss Microimaging, Thornwood, NY). Images were then viewed at 150% of the original ×20 images with an Image MetaMorph Software (Molecular Devices, Sunnyvale, CA). The number of cells with ameboid morphology and positive for both, β-isolectin and F4/80, was expressed as a percentage of the total number of cells in each field in each AOI. Each observation was repeated 10 times. Statistical analysis was performed with a one-way analysis of variance test.Cell Cultures and Laser Confocal Microscopy StudiesMicroglial cells were cultured from 1-day-old wild-type and macLRP− C57BL/6J mice as described elsewhere.35Fedoroff S Richardson A Cultures of astroglia and microglia from primary cultures of mouse neopallium.in: Fedoroff S Richardson A Protocols for Neuronal Cell Culture. Humana Press, New Jersey2001: 139-148Crossref Google Scholar, 36Yepes M Moore E Brown SA Hanscom HN Smith EP Lawrence DA Winkles JA Progressive ankylosis (Ank) protein is expressed by neurons and Ank immunohistochemical reactivity is increased by limbic seizures.Lab Invest. 2003; 83: 1025-1032Crossref PubMed Scopus (23) Google Scholar Briefly, cells were dissociated into a single-cell suspension by tritration through a Pasteur pipette and plated onto either 12-mm glass coverslips or six-well plates coated with 0.05 mg/ml poly-d-lysine and grown in Dulbecco's modified Eagle's medium media (Life Technologies, Inc., Grand Island, NY) supplemented with 25 mmol/L glucose, 10% heat-inactivated horse serum, 10% heat-inactivated fetal bovine serum, 2 μmol/L glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin. At the end of day 12, microglia were microscopically identified floating in the medium of the stationary cultures and centrifuged at 80 × g for 5 minutes to obtain a pellet of nearly pure microglia, which was then plated directly into poly-d-lysine-coated coverslips and stained with DAPI and antibodies against Mac-1 (Serotec, Oxford, UK) and LRP1. As controls, a separate set of coverslips was incubated with an IgG isotype control or with the secondary antibody only. The determination of the co-expression of LRP1 and Mac-1 was performed with a laser confocal microscope (Carl Zeiss Microimaging).Western Blot AnalysisPolyclonal antibodies to nitrotyrosine were purchased from Cayman Chemical. Polyclonal antibodies to β-actin were obtained from Sigma-Aldrich. Wild-type and macLRP− mice underwent tMCAO and brains were extracted 24 hours later. Tissue was processed and gels were loaded as described.37Polavarapu R Gongora MC Winkles JA Yepes M Tumor necrosis factor-like weak inducer of apoptosis increases the permeability of the neurovascular unit through nuclear factor-kappaB pathway activation.J Neurosci. 2005; 25: 10094-10100Crossref PubMed Scopus (107) Google Scholar A total of three observations were made for each time point.Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) AnalysisWild-type and macLRP− mice underwent tMCAO and brains were extracted 24 hours later. A subset of macLRP− mice was injected directly into the ischemic area immediately after MCAO with murine tPA as described above. Wild-type and macLRP− microglial cultures were maintained under oxygen-glucose deprivation conditions for 3 hours as described elsewhere.19Polavarapu R Gongora MC Yi H Ranganthan S Lawrence DA Strickland D Yepes M Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit.Blood. 2007; 109: 3270-3278Crossref PubMed Scopus (140) Google Scholar Briefly, cultures were incubated with serum-free media and washed with PBS three times. The culture medium was then replaced by glucose-free Earle's balanced salt solution previously saturated with 95% N2/5% CO2 at 37°C. Cultures were placed in an anaerobic chamber (Billups-Rothenberg, Inc., Del Mar, CA) equipped with inlet and outlet valves, and equilibrated for 15 minutes with a continuous flux of gas (5% CO2/95% N2). With this setting the concentration of oxygen in the media drops to <1%. The chamber was then sealed and placed in an incubator at 37°C for 3 hours. As a control, a similar group of cells was kept under normoxic conditions. For quantitative measurement of mRNA, 2 μg of DNase I-treated total RNA was used for cDNA synthesis. Reverse transcription was performed with a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) with random oligonucleotide primers. TaqMan Gene Express assays of TaqMan probes and primers for iNOS (Mm00440485-m1) and LRP1 (Mm00464608-m1) were purchased from Applied Biosystems. Polymerase chain reactions were performed in an ABI Prism 7000 system (Applied Biosystems) under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Each observation was repeated six times. PCR results were analyzed as described elsewhere38Pfaffl MW A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (24916) Google Scholar and statistical analysis was performed with the Student's t-test.ResultsTissue-Type Plasminogen Activator Mediates Cerebral Ischemia-Induced Microglial Activation via a Plasminogen-Independent MechanismTo study the role of tPA on cerebral ischemia-induced microglial activation in vivo, we investigated the presence of cells with ameboid morphology positive for both F4/80 and β-isolectin (activated microglial cells) in each AOI in wild-type and tPA−/− mice 24 hours after MCAO. The contralateral, nonischemic hemisphere was used as a negative control (Figure 1A, d, h, j, and p). We observed that compared with wild-type mice (Figure 1A, a–c), genetic deficiency of tPA resulted in a significant decrease in the number of activated microglial cells in the ischemic tissue (Figure 1A, e–g). In contrast, the extent of microglial activation in tPA−/− mice treated with tPA (Figure 1A, i–k) and Plg−/− animals (Figure 1A, m–o) was indistinguishable from that observed in wild-type mice. To quantify these results we counted the number of β-isolectin-positive cells with ameboid morphology and immunoreactive for F4/80 in each AOI in wild-type, tPA−/− and Plg−/− mice. These data reveal that the average percentage of activated microglial cells in each AOI was 45.99 ± 2.43% in wild-type mice, 11.2 ± 3.53% in tPA−/− mice, and 33.32 ± 4.92% in Plg−/− animals. Additionally, when tPA−/− mice were injected with either active or inactive tPA directly into the ischemic area immediately after the onset of the ischemic insult, the average percentage of activated microglial cells in each AOI increased to 36.67 ± 3.80% and 29 ± 2.50%, respectively (Figure 1B; n = 10, P < 0.001 when wild-type mice were compared with sham and untreated tPA−/− animals, and not significant when wild-type mice are compared with either tPA−/− mice treated with tPA or with Plg−/− animals).LRP1 Is Required for tPA-Mediated Microglial Activation during Cerebral IschemiaPrevious studies confirmed an effective deletion of LRP1 in macrophages by crossing into the LysMCre mice.39Lillis AP Greenlee MC Mikhailenko I Pizzo SV Tenner AJ Strickland DK Bohlson SS Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis.J Immunol. 2008; 181: 364-373PubMed Google Scholar Because microglia and macrophages share the same mesenchymal origin,3Ladeby R Wirenfeldt M Garcia-Ovejero D Fenger C Dissing-Olesen L Dalmau I Finsen B Microglial cell population dynamics in the injured adult central nervous system.Brain Res Rev. 2005; 48: 196-206Crossref PubMed Scopus (272) Google Scholar, 40Cuadros MA Navascues J The origin and differentiation of microglial cells during development.Prog Neurobiol. 1998; 56: 173-189Crossref PubMed Scopus (253) Google Scholar, 41Dalmau I Finsen B Tonder N Zimmer J Gonzalez B Castellano B Development of microglia in the prenatal rat hippocampus.J Comp Neurol. 1997; 377: 70-84Crossref PubMed Scopus (88) Google Scholar, 42Dalmau I Finsen B Zimmer J Gonzalez B Castellano B Development of microglia in the postnatal rat hippocampus.Hippocampus. 1998; 8: 458-474Crossref PubMed Scopus (107) Google Scholar, 43Dalmau I Vela JM Gonzalez B Finsen B Castellano B Dynamics of microglia in the developing rat brain.J C
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