Cerebral Ischemia-Hypoxia Induces Intravascular Coagulation and Autophagy
2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.051066
ISSN1525-2191
AutoresFaisal Adhami, Guanghong Liao, Yury M. Morozov, Aryn Schloemer, Vincent J. Schmithorst, John N. Lorenz, R. Scott Dunn, Charles V. Vorhees, Marsha Wills‐Karp, Jay L. Degen, Roger J. Davis, Noboru Mizushima, Pasko Rakić, Bernard J. Dardzinski, Scott K. Holland, Frank R. Sharp, Chia-Yi Kuan,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoHypoxia is a critical factor for cell death or survival in ischemic stroke, but the pathological consequences of combined ischemia-hypoxia are not fully understood. Here we examine this issue using a modified Levine/Vannucci procedure in adult mice that consists of unilateral common carotid artery occlusion and hypoxia with tightly regulated body temperature. At the cellular level, ischemia-hypoxia produced proinflammatory cytokines and simultaneously activated both prosurvival (eg, synthesis of heat shock 70 protein, phosphorylation of ERK and AKT) and proapoptosis signaling pathways (eg, release of cytochrome c and AIF from mitochondria, cleavage of caspase-9 and −8). However, caspase-3 was not activated, and very few cells completed the apoptosis process. Instead, many damaged neurons showed features of autophagic/lysosomal cell death. At the tissue level, ischemia-hypoxia caused persistent cerebral perfusion deficits even after release of the carotid artery occlusion. These changes were associated with both platelet deposition and fibrin accumulation within the cerebral circulation and would be expected to contribute to infarction. Complementary studies in fibrinogen-deficient mice revealed that the absence of fibrin and/or secondary fibrin-mediated inflammatory processes significantly attenuated brain damage. Together, these results suggest that ischemia-hypoxia is a powerful stimulus for spontaneous coagulation leading to reperfusion deficits and autophagic/lysosomal cell death in brain. Hypoxia is a critical factor for cell death or survival in ischemic stroke, but the pathological consequences of combined ischemia-hypoxia are not fully understood. Here we examine this issue using a modified Levine/Vannucci procedure in adult mice that consists of unilateral common carotid artery occlusion and hypoxia with tightly regulated body temperature. At the cellular level, ischemia-hypoxia produced proinflammatory cytokines and simultaneously activated both prosurvival (eg, synthesis of heat shock 70 protein, phosphorylation of ERK and AKT) and proapoptosis signaling pathways (eg, release of cytochrome c and AIF from mitochondria, cleavage of caspase-9 and −8). However, caspase-3 was not activated, and very few cells completed the apoptosis process. Instead, many damaged neurons showed features of autophagic/lysosomal cell death. At the tissue level, ischemia-hypoxia caused persistent cerebral perfusion deficits even after release of the carotid artery occlusion. These changes were associated with both platelet deposition and fibrin accumulation within the cerebral circulation and would be expected to contribute to infarction. Complementary studies in fibrinogen-deficient mice revealed that the absence of fibrin and/or secondary fibrin-mediated inflammatory processes significantly attenuated brain damage. Together, these results suggest that ischemia-hypoxia is a powerful stimulus for spontaneous coagulation leading to reperfusion deficits and autophagic/lysosomal cell death in brain. Hypoxia is a critical factor in cerebral ischemia. Positron emission tomography shows that the regional oxygen extraction fraction is increased in the beginning of cerebral ischemia, compensating for reduction of cerebral blood flow (CBF) to maintain the cerebral metabolic rate of oxygen close to the normal value.1Baron JC Perfusion thresholds in human cerebral ischemia: historical perspective and therapeutic implications.Cerebrovasc Dis. 2001; 11: S2-S8Crossref PubMed Scopus (257) Google Scholar This pattern, originally labeled “misery perfusion,”1Baron JC Perfusion thresholds in human cerebral ischemia: historical perspective and therapeutic implications.Cerebrovasc Dis. 2001; 11: S2-S8Crossref PubMed Scopus (257) Google Scholar is a marker of penumbra that surrounds the ischemic core.2Sobesky J Zaro Weber O Lehnhardt FG Hesselmann V Neveling M Jacobs A Heiss WD Does the mismatch match the penumbra? Magnetic resonance imaging and positron emission tomography in early ischemic stroke.Stroke. 2005; 36: 980-985Crossref PubMed Scopus (220) Google Scholar Subsequently, there often occurs a reduction of oxygen extraction fraction and cerebral metabolic rate of oxygen even with the regional CBF maintained in the penumbra range, which indicates imminent infarction.2Sobesky J Zaro Weber O Lehnhardt FG Hesselmann V Neveling M Jacobs A Heiss WD Does the mismatch match the penumbra? Magnetic resonance imaging and positron emission tomography in early ischemic stroke.Stroke. 2005; 36: 980-985Crossref PubMed Scopus (220) Google Scholar These results indicate decreased oxygen metabolism and suggest that hypoxia modulates the fate of ischemic tissues. The combination of hypoxia to ischemia may trigger pathological events that are not induced by ischemia alone. One potential pathological event is hypoxia-induced fibrin deposition that results from altered anti-coagulant properties of endothelial cells as demonstrated in pulmonary vessels.3Yan SF Mackman N Kisiel W Stern DM Pinsky DJ Hypoxia/hypoxemia-induced activation of the procoagulant pathways and the pathogenesis of ischemia-associated thrombosis.Arterioscler Thromb Vasc Biol. 1999; 19: 2029-2035Crossref PubMed Scopus (146) Google Scholar, 4Ten VS Pinsky DJ Endothelial response to hypoxia: physiologic adaptation and pathologic dysfunction.Curr Opin Crit Care. 2002; 8: 242-250Crossref PubMed Scopus (129) Google Scholar Thus, the reduction of CBF in conjunction with hypoxia may induce spontaneous thrombus formation to reduce blood perfusion further. This ischemia/hypoxia-induced microvascular thrombosis may also prevent cerebral reperfusion after the release of the large artery occlusion, similar to the previously described no-reflow phenomenon after brain and cardiac ischemia.5Majno G Ames AI Chiang J Wright R No reflow after cerebral ischaemia.Lancet. 1967; 2: 569-570Abstract Google Scholar, 6Rezkalla SH Kloner RA No-reflow phenomenon.Circulation. 2002; 105: 656-662Crossref PubMed Scopus (533) Google Scholar Another potential consequence of combined ischemia-hypoxia is autophagy (self-eating), which involves the formation of autophagosomes and autophagolysosomes in degrading cellular constituents for energy production in response to nutrient deprivation.7Levine B Klionsky DJ Development by self-digestion: molecular mechanisms and biological functions of autophagy.Dev Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3276) Google Scholar Although autophagy is generally a cell survival mechanism, massive autophagy is associated with cell death.8Clarke PG Developmental cell death: morphological diversity and multiple mechanisms.Anat Embryol (Berl). 1990; 181: 195-213Crossref PubMed Scopus (1545) Google Scholar The involvement of autophagy in ischemic heart and brain has only been described recently.9Yan L Vatner DE Kim SJ Ge H Masurekar M Massover WH Yang G Matsui Y Sadoshima J Vatner SF Autophagy in chronically ischemic myocardium.Proc Natl Acad Sci USA. 2005; 102: 13807-13812Crossref PubMed Scopus (459) Google Scholar, 10Nitatori T Sato N Waguri S Karasawa Y Araki H Shibanai K Kominami E Uchiyama Y Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis.J Neurosci. 1995; 15: 1001-1011Crossref PubMed Google Scholar, 11Zhu C Wang X Xu F Bahr BA Shibata M Uchiyama Y Hagberg H Blomgren K The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia.Cell Death Differ. 2005; 12: 162-176Crossref PubMed Scopus (374) Google Scholar, 12Degterev A Huang Z Boyce M Li Y Jagtap P Mizushima N Cuny GD Mitchison TJ Moskowitz MA Yuan J Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.Nat Chem Biol. 2005; 1: 112-119Crossref PubMed Scopus (2119) Google Scholar We postulate that the combination of ischemia and hypoxia accelerate an energy crisis and precipitate autophagy. Among the animal models of brain ischemia, the Levine/Vannucci procedure is the only paradigm that specifically emphasizes hypoxia.13Levine S Anoxic-ischemic encephalopathy in rats.Am J Pathol. 1960; 36: 1-17PubMed Google Scholar, 14Rice III, JE Vannucci RC Brierley JB The influence of immaturity on hypoxic-ischemic brain damage in the rat.Ann Neurol. 1981; 9: 131-141Crossref PubMed Scopus (1952) Google Scholar Although focal ischemia undoubtedly can induce local hypoxia, the use of systemic hypoxia in the Levine/Vannucci model produces more uniform tissue hypoxia that helps control this variable. The Levine/Vannucci model consists of unilateral common carotid artery occlusion and then subjecting animals to a hypoxic stress for a predetermined time. This model is unique in that neither the unilateral carotid occlusion nor the hypoxia alone produces brain injury, but the combination of both causes infarction.13Levine S Anoxic-ischemic encephalopathy in rats.Am J Pathol. 1960; 36: 1-17PubMed Google Scholar, 14Rice III, JE Vannucci RC Brierley JB The influence of immaturity on hypoxic-ischemic brain damage in the rat.Ann Neurol. 1981; 9: 131-141Crossref PubMed Scopus (1952) Google Scholar The Levine/Vannucci procedure had been extensively used to mimic perinatal hypoxic-ischemic brain injury14Rice III, JE Vannucci RC Brierley JB The influence of immaturity on hypoxic-ischemic brain damage in the rat.Ann Neurol. 1981; 9: 131-141Crossref PubMed Scopus (1952) Google Scholar but recently has also been applied to adult animals.15Vannucci SJ Willing LB Goto S Alkayed NJ Brucklacher RM Wood TL Towfighi J Hurn PD Simpson IA Experimental stroke in the female diabetic, db/db, mouse.J Cereb Blood Flow Metab. 2001; 21: 52-60Crossref PubMed Scopus (129) Google Scholar, 16Basu A Lazovic J Krady JK Mauger DT Rothstein RP Smith MB Levison SW Interleukin-1 and the interleukin-1 type 1 receptor are essential for the progressive neurodegeneration that ensues subsequent to a mild hypoxic/ischemic injury.J Cereb Blood Flow Metab. 2005; 25: 17-29Crossref PubMed Scopus (96) Google Scholar It was shown that controlling the body core temperature of adult animals at 37.5 to 37.7°C during hypoxia, typically performed by infusing hypoxic gas into a jar containing animals and submerged in a water-bath maintained at 35.5°C, produces consistent brain injury.15Vannucci SJ Willing LB Goto S Alkayed NJ Brucklacher RM Wood TL Towfighi J Hurn PD Simpson IA Experimental stroke in the female diabetic, db/db, mouse.J Cereb Blood Flow Metab. 2001; 21: 52-60Crossref PubMed Scopus (129) Google Scholar Although controlling the environmental temperature influences body temperatures of the animals inside the jar, this method may not prevent fluctuations of body temperature during the hypoxia interval. This is undesirable because it is known that small differences in brain temperature during ischemia can influence the extent of brain injury.17Busto R Dietrich WD Globus MY Valdes I Scheinberg P Ginsberg MD Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury.J Cereb Blood Flow Metab. 1987; 7: 729-738Crossref PubMed Scopus (1486) Google Scholar Furthermore, placing the animals inside an isolated jar precludes the ability to monitor CBF during hypoxia. In the present study, we set out to further our understanding of the consequences of combined ischemia-hypoxia in adult brains. We have used a modified Levine/Vannucci model by controlling the core temperature of the mouse at 36.5 to 37.5°C using a heating pad on the surgical table and directly delivering hypoxic gas with anesthesia to individual animals immediately after carotid occlusion via a face mask. This modification prevents fluctuations of body temperature and allows us to monitor the changes in brain perfusion during hypoxia. We found that the combination of ischemia and hypoxia induces acute thrombosis leading to reperfusion deficits and autophagic/lysosomal processes. Taken together, these results shed new insight into the mechanism of ischemic brain injury in stroke. The following mouse strains were used in the present study: C57BL/6 wild-type mice, GFP-LC3 transgenic mice,18Mizushima N Yamamoto A Matsui M Yoshimori T Ohsumi Y In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.Mol Biol Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1954) Google Scholar and fibrinogen-null mice19Suh TT Holmback K Jensen NJ Daugherty CC Small K Simon DI Potter S Degen JL Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice.Genes Dev. 1995; 9: 2020-2033Crossref PubMed Scopus (360) Google Scholar that have been back-crossed to the C57BL/6 strain for six generations. Male mice (8 to 12 weeks of age) were used for stroke surgery. Animals were anesthetized using 0.5 to 2% isoflurane while maintaining the respiration rate at 80 to 120/minute. The right common carotid artery was occluded (RCCAO) reversibly with a releasable suture or permanently by transecting the common carotid artery between two sutures. After this, hypoxia was maintained for 30, 35, 40, or 60 minutes under anesthesia by administering 7.5% O2 balanced with 92.5% N2 through a gas mask. Temperature was maintained at 37 ± 0.5°C with a temperature controller (model 89000-00; Cole Parmer, Vernon Hills, IL) with a rectal probe and heating pad. After recovery from anesthesia, mice were returned to the animal care facility and inspected daily. These animal procedures were approved by the Institutional Animal Care and Use Committee and conform to the National Institutes of Health Guide for Care and Use of Laboratory Animals. Postischemia/hypoxia, animals were scored on a 6-point scale for neurological deficits based on a previous publication.20Longa EZ Weinstein PR Carlson S Cummins R Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke. 1989; 20: 84-91Crossref PubMed Scopus (6880) Google Scholar These are: 0, no detectable neurological deficit; 1, ptosis of eyelid ipsilateral to the occluded CCA side and/or failure to extend ipsilateral forepaw; 2, animal persistently walks in large circles toward the ipsilateral side; 3, animal persistently walks in small circles and/or rolls over repeatedly toward ipsilateral side; 4, animal lies nearly motionless on the contralateral side; 5, animal dies after recovery from the anesthesia. For fixation, mice were killed under deep anesthesia by transcardiac perfusion with phosphate-buffered saline containing 4% paraformaldehyde. For cryosectioning, brains were taken through graded sucrose solutions and then frozen in tissue-freezing medium. Serial coronal floating sections (100 μm for Golgi stain, 50 μm for Nissl and silver stains, 30 μm for immunohistochemistry) were cut using a vibratome or cryostat, and the eight sections corresponding to +1.98, +1.18, 0.50, 0.22, −1.06, −1.82, −2.46, and −3.28 mm to the Bregma point21Franklin KBJ Paxinos G The Mouse Brain in Stereotaxic Coordinates. Academic Press, San Diego1997Google Scholar were mounted on slides. Nissl stain was done with cresyl violet, and infarct size was measured as percent area of the ipsilateral hemisphere using a digital image analysis system (MCID, Imaging Research Inc.; or ImageJ, National Institutes of Health). 2,3,5-Triphenylterazolim chloride (TTC) staining was done on 1-mm fresh brain sections in 2% TTC in distilled water for 30 minutes at 37°C. The silver and Golgi stains were performed with kits following the manufacturer's instruction (FD Neuro Technologies, Ellicott City, MD). Cerebral capillary permeability was tested by intraperitoneal injection of 2% Evans blue (Sigma, St. Louis, MO) at designated times after hypoxia, followed 2 hours later by fixation as above. The TUNEL labeling was performed as previously described.22Kuan CY Schloemer AJ Lu A Burns KA Weng WL Williams MT Strauss KI Vorhees CV Flavell RA Davis RJ Sharp FR Rakic P Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain.J Neurosci. 2004; 24: 10763-10772Crossref PubMed Scopus (254) Google Scholar The DNA PANT labeling was performed on paraffin-embedded sections (10 μm) using a previously described method.23Chen J Jin K Chen M Pei W Kawaguchi K Greenberg DA Simon RP Early detection of DNA strand breaks in the brain after transient focal ischemia: implications for the role of DNA damage in apoptosis and neuronal cell death.J Neurochem. 1997; 69: 232-245Crossref PubMed Scopus (294) Google Scholar For mitochondrial/cytosol fractionation, brain samples (cortex or hippocampus from lesion or contralateral sides) were gently homogenized in a glass grinder in cold buffer [containing 20 mmol/L HEPES, pH 7.4, 250 mmol/L sucrose, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.7% protease inhibitor cocktail (Sigma), 1 mmol/L Na3VO4]. The mixture was centrifuged at 750 × g for 10 minutes to separate the pellet (P1) fraction containing the nuclei and the supernatant (S1) with mitochondria/cytosol. The nuclear fraction was resuspended in TLB (containing 20 mmol/L Tris, pH 7.4, 137 mmol/L NaCl, 25 mmol/L β-glycerophosphate, 25 mmol/L Na-pyrophosphate, 2 mmol/L EDTA, 1 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 1 mmol/L phenylmethyl sulfonyl fluoride, 0.7% protease inhibitor cocktail), cooled on ice for 20 minutes, sonicated three times for 10 seconds, and then centrifuged for 20 minutes at maximum speed. The supernatant (S1) was removed and centrifuged at 8000 × g for 20 minutes. This pellet (P2) containing the mitochondrial fraction was resuspended in TLB, and cooled in ice and centrifuged at 140 × g for 10 minutes. The remaining supernatant (S2) was removed and spun in an airfuge (Beckman, Fullerton, CA) at 20 psi (100 × g) for 10 minutes. This supernatant (S3) contained the cytosolic fraction. For whole-cell protein purification, brain samples were homogenized as above in TLB buffer, cooled on ice for 20 minutes, sonicated, and centrifuged at 14,000 × g for 10 minutes. The proteins were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedures, electrotransferred onto a polyvinylidene fluoride microporous membrane (Bio-Rad, Hercules, CA), and immunoblotted with designated antibodies followed by enhanced chemiluminescence detection (Amersham Biosciences, Arlington Heights, IL). The antibodies used in immunoblots include Hsp70 (SPA810; Stressgen, Victoria, ON, Canada), p-ERK (no. 9101; Cell Signaling, Beverly, MA), ERK1/2 (no. sc93 and sc154; Santa Cruz Biotechnology, Santa Cruz, CA), p-Akt (Ser 473, no. 9271; Cell Signaling), Akt (no. 9272; Cell Signaling), β-actin (no. A5441; Sigma), cytochrome c (no. 4272; Cell Signaling), AIF (no. sc9416, Santa Cruz), cytochrome oxidase subunit IV (no. A21348; Molecular Probes, Invitrogen, Carlsbad, CA), caspase-9 (no. 9504; Cell Signaling), caspase-3 (no. 9662; Cell Signaling), LC3 (generated by N. Mizushima), and LAMP-1 (no. sc5570; Santa Cruz Biotechnology). Whole-cell lysates from brain tissues were prepared as above and the concentrations were determined by Bradford assay. Equal amounts of samples were used for measuring the cleavage of Z-DEVD-AMC (for caspase-3, EnzChek caspase-3 assay kit; Molecular Probes, Invitrogen) with an excitation wavelength of 368 nm and an emission wavelength of 467 nm. The cleavage of IETD-AFC (for caspase 8) and IEHD-AFC (for caspase 9; Alexis Corp., San Diego, CA) was measured with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Every sample was analyzed three times and the average value was used, the relative fluorescence units were shown with the value in untreated samples set as 100%. Quantification of the cytokine transcript levels was performed by RNase protection assay following the manufacturer's instructions. Briefly, [33P]-labeled anti-sense riboprobes were synthesized using T7 MAXIscript (Ambion, Austin, TX) based on multiprobe templates (mCK2b and mCK3b from BD PharMingen, Franklin Lakes, NJ). RNA of cerebral cortex was extracted using Trizol reagent (Life Technologies, Inc., Grand Island, NY) and purified by phenol-chloroform extraction, then hybridized overnight at 56°C with the probes. The RNase digestion and precipitation of protected fragments were completed using the RPA III kit (Ambion). After separation via electrophoresis with a 5% acrylamide, 8 mol/L urea, and 1× TBE gel, the radioactivity was measured by exposure to a phosphorimager screen (Molecular Devices, Sunnyvale, CA) and quantified. Measurements of protein levels for interleukin (IL)-1β and IL-6 in the mouse brain were performed using ELISA and following the manufacturer's instructions (R&D Systems, Minneapolis, MN). Briefly, 96-well plates were coated with the capture antibody overnight and then blocked (1% bovine serum albumin, 5% sucrose, 0.05% NaN3). Standards of IL-1β and IL-6 (5000 to 0 pg/ml in triplicates) or samples [homogenates from each mouse hemisphere in 500 μl of phosphate-buffered saline (PBS), using 50 μl per well in duplicates] were then added to the plates for 2 hours. Protein concentrations were determined using a streptavidin-peroxidase substrate system and biotinylated detection antibodies, and the plates were read at 450 nm with 595 nm background subtraction. Animals were exposed to ischemia-hypoxia, and after 6 hours of recovery, anesthetized, and perfused transcardially with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 mol/L phosphate buffer. The brains were removed and coronal 100-μm-thick sections were cut by a vibratome. The sections were postfixed with 1% OsO4, dehydrated, and embedded in Durcupan (ACM; Fluka, Buchs, Switzerland) on a microscope slide and coverslipped. Fragments of cerebral cortex from damaged and contralateral hemispheres were re-embedded into Durcupan blocks and cut by a Reichert ultramicrotome into 70-nm-thick sections. The ultrathin sections were then stained with uranil acetate and lead citrate and evaluated in a JEOL 1010 electron microscope. Mice expressing the GFP-LC3 fusion protein have been characterized.18Mizushima N Yamamoto A Matsui M Yoshimori T Ohsumi Y In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.Mol Biol Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1954) Google Scholar These mice have been used to monitor autophagy in cells because the GFP-LC3 protein normally has a diffuse, cytoplasmic distribution but is shifted to bright, punctate dots on autophagy. These mice were subjected to ischemia-hypoxia as described above for 40 minutes and analyzed for immunohistochemistry as described above after either 6 or 18 hours of recovery. Sections were stained with a rabbit monoclonal antibody against GFP (no. A11122; Molecular Probes, Invitrogen) and ethidium homodimer (2 μmol/L, no. E1169; Molecular Probes) and sections were analyzed using a Zeiss (Zeiss, Jena, Germany) confocal microscope (LSM-510). Each time point was analyzed in four different animals. The CBF was measured using a Periflux 5010 laser Doppler perfusion system (Perimed, Jarfalla, Sweden) with a model 407 probe mounted directly on the skull over the right forebrain. Data were recorded during a 2-minute baseline period, for 2 minutes after the suture was tightened, throughout 40 minutes of hypoxia, and for 20 minutes after restoration to normoxia. Mice that died during hypoxia were excluded from average due to an exaggerated drop in perfusion. Data are presented as a percentage of baseline and were averaged as follows: three mice each for hypoxia, reversible ischemia, or permanent ischemia alone; seven mice for permanent ischemia with hypoxia; five for reversible ischemia with hypoxia. For studies in fibrinogen-deficient and heterozygous mice, three mice for each genotype were analyzed and averaged, focusing on three data points: blood flow averaged throughout 1 minute of right common carotid occlusion alone, throughout 10 minutes during hypoxia (25 to 35 minutes), and throughout 10 minutes during recovery (10 to 20 minutes recovery). Mice were anesthetized as described with isoflurane. The right common carotid was isolated and occluded using a single suture. Then, on the side of the suture proximal to the heart, the right common carotid artery was cannulated with polyethylene tubing. The catheter was connected to a COBE CDXIII fixed-dome pressure transducer (COBE Cardiovascular, Arvada, CO) for measurement of arterial blood pressure. To determine carotid blood flow to the brain after RCCAO, the left common carotid artery was isolated and a Transonics Systems (Ithaca, NY) 0.5SB series perivascular flow probe placed over the artery. Flow signals from a Transonic System model TS420 transit time flowmeter and pressure signals from the mentioned pressure transducer were recorded at a sampling rate of 1000 samples per second per channel and analyzed using a MacLab 4/s (ADInstruments, Colorado Springs, CO) data acquisition system. Heart rate and cerebrovascular resistance was calculated using the software from recordings. Data obtained were averaged from three mice. Cerebral hemispheric ischemia/hypoxia was performed as described above. Blood was collected by the tail clip method. Mice were heated to 39°C just before the collection times to induce flushing of the tail with predominantly arterial blood. Approximately 150 μl of blood was collected in heparinized capillary tubes before hypoxia, 2 minutes before the end of hypoxia, and 30 minutes into recovery and analyzed with an i-Stat portable clinical analyzer with a CG8+ cartridge (Abbott Laboratories, Abbott Park, IL). Data obtained were averaged from five mice. All images were acquired on a Bruker 7T/30 cm imaging spectrometer with a 20 G/cm gradient coil insert. Radiofrequency (RF) excitation was with a 72-mm-volume coil and RF reception was with a surface coil. Details for the three imaging sequences are described below. Spin-echo RARE sequence, TR/TE = 5000/83 ms, rare factor = 6, imaging BW = 75 kHz, FOV = 25.6 × 25.6 mm, matrix = 256 × 192, slice thickness = 0.75 mm, NEX = 2. FLASH sequence, TR/TE = 25/3.5 ms, imaging BW = 50 kHz, FOV = 25.6 × 25.6 mm, matrix = 128 × 128, slice thickness = 2.0 mm, tagging slab thickness = 7 mm. Forty-eight total images were acquired, with 24 tagged images alternated with 24 control images. In the tagged images, the tagging slab was placed inferior to the imaging plane to saturate the incoming blood; in the control images, the tagging was placed superior to the imaging plane (to make any magnetization transfer effects identical for tagged and control images). Percent change maps were calculated from the difference in signal intensities between the tagged and control images. Four-segment diffusion-weighted spin-echo EPI sequence, TR/TE = 2500/26 ms, imaging BW = 250 kHz, FOV = 25.6 × 25.6 mm, matrix = 128 × 128, slice thickness = 1.0 mm. Three images were acquired with no diffusion weighting. Twelve diffusion-weighed images were acquired with δ = 5 ms, D = 13 ms, b value = 1000 seconds/mm2, and diffusion gradient directions of (1.0, 0.5, 1.0), (1.0, −0.5, 1.0), (0.5, 1.0, 1.0], (−0.5, 1.0, 1.0), (1.0, 1.0, 0.5), (1.0, 1.0, −0.5) (two images acquired for each direction). Components of the diffusion tensor were calculated from a least-squares fit of the logarithm of the signal intensities and the directionally averaged apparent diffusion coefficient (ADC) was obtained by averaging the diagonal elements. Perfusion status was measured using a modification of a published procedure.24Ding G Jiang Q Zhang L Zhang ZG Li L Knight RA Ewing JR Wang Y Chopp M Analysis of combined treatment of embolic stroke in rat with r-tPA and a GPIIb/IIIa inhibitor.J Cereb Blood Flow Metab. 2005; 25: 87-97Crossref PubMed Scopus (37) Google Scholar Briefly, fluorescein isothiocyanate (FITC)-dextran (5 mg in 100 μl PBS of 2 × 106 MW; Sigma) was injected with a small-bore needle into the left ventricle of mice 1 or 3 hours after ischemia/hypoxia. After 2 minutes, the animal was rapidly decapitated and the brain was removed and postfixed for 48 hours. Sections were cut as above and analyzed with fluorescence microscopy. For quantification in fibrinogen studies, images obtained were analyzed using ImageJ software. An index of perfusion was measured in an area excluding the midline (matching the maximal possible infarction) as the number of FITC pixels per number of total pixels in maximal infarct area of ipsilateral hemisphere. This was then normalized to the equivalent measurement on the contralateral side to account for potential animal global perfusion variation. Immunohistochemistry was performed using a rat monoclonal antibody against P-selectin (no. 550289; BD PharMingen, San Diego, CA), a rabbit polyclonal antibody against fibrin/fibrinogen,19Suh TT Holmback K Jensen NJ Daugherty CC Small K Simon DI Potter S Degen JL Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice.Genes Dev. 1995; 9: 2020-2033Crossref PubMed Scopus (360) Google Scholar and a rat monoclonal antibody against glycoprotein IIb (CD41, no. 553847; BD PharMingen). Quantitative data were compared between experimental and control groups using Microsoft Excel's two-sample (unpaired) t-test assuming equal variance. Male C57BL/6 mice (8 to 12 weeks old) were subjected to combined cerebral ischemia-hypoxia. Mice were anesthetized with isoflurane delivered with pure oxygen through a face mask, and the right common carotid artery (RCCA) was ligated while the core temperature of the animal was maintained at 36.5 to 37.5°C using a rectal thermometer and a heating pad. Immediately after occlusion of the RCCA and closure of the surgical wound, the gas was switched to 7.5% oxygen and 92.5% nitrogen to initi
Referência(s)