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The Neurotropic Parasite Toxoplasma gondii Induces Sustained Neuroinflammation with Microvascular Dysfunction in Infected Mice
2018; Elsevier BV; Volume: 188; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2018.07.007
ISSN1525-2191
AutoresVanessa Estato, Joice Stipursky, Fabiana Gomes, Tally C. Mergener, Edwards Frazão-Teixeira, Silvana Allodi, Eduardo Tibiriçá, Helene Santos Barbosa, Daniel Adesse,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoToxoplasmosis is one of the leading parasitic diseases worldwide. Some data suggest that chronic acquired toxoplasmosis could be linked to behavioral alterations in humans. The parasite infects neurons, forming immunologically silent cysts. Cerebral microcirculation homeostasis is determinant to brain functions, and pathologic states can alter capillarity or blood perfusion, leading to neurodegeneration and cognitive deficits. Albino mice were infected with Toxoplasma gondii (ME49 strain) and analyzed after 10, 40, and 180 days. Infected mice presented decreased cerebral blood flow at 10 and 40 days post infection (dpi), which were restored at 180 dpi, as shown by laser speckle contrast imaging. Intravital microscopy demonstrated that infection led to significant capillary rarefaction, accompanied by neuroinflammation, with microglial activation and increased numbers of rolling and adherent leukocytes to the wall of cerebral capillaries. Acetylcholine-induced vasodilation was altered at all time points, and blood brain barrier permeability was evident in infected animals at 40 dpi. Infection reduced angiogenesis, with a decreased number of isolectin B4–stained blood vessels and a decrease in length and branching of laminin-stained capillaries. Sulfadiazine reduced parasite load and partially repaired microvascular damages. We conclude that T. gondii latent infection causes a harmful insult in the brain, promoting neuroinflammation and microcirculatory dysfunction in the brain, with decreased angiogenesis and can contribute to a neurodegenerative process. Toxoplasmosis is one of the leading parasitic diseases worldwide. Some data suggest that chronic acquired toxoplasmosis could be linked to behavioral alterations in humans. The parasite infects neurons, forming immunologically silent cysts. Cerebral microcirculation homeostasis is determinant to brain functions, and pathologic states can alter capillarity or blood perfusion, leading to neurodegeneration and cognitive deficits. Albino mice were infected with Toxoplasma gondii (ME49 strain) and analyzed after 10, 40, and 180 days. Infected mice presented decreased cerebral blood flow at 10 and 40 days post infection (dpi), which were restored at 180 dpi, as shown by laser speckle contrast imaging. Intravital microscopy demonstrated that infection led to significant capillary rarefaction, accompanied by neuroinflammation, with microglial activation and increased numbers of rolling and adherent leukocytes to the wall of cerebral capillaries. Acetylcholine-induced vasodilation was altered at all time points, and blood brain barrier permeability was evident in infected animals at 40 dpi. Infection reduced angiogenesis, with a decreased number of isolectin B4–stained blood vessels and a decrease in length and branching of laminin-stained capillaries. Sulfadiazine reduced parasite load and partially repaired microvascular damages. We conclude that T. gondii latent infection causes a harmful insult in the brain, promoting neuroinflammation and microcirculatory dysfunction in the brain, with decreased angiogenesis and can contribute to a neurodegenerative process. Toxoplasmosis is one of the most common zoonotic diseases worldwide.1Montoya J.G. Liesenfeld O. Toxoplasmosis.Lancet. 2004; 363: 1965-1976Abstract Full Text Full Text PDF PubMed Scopus (2537) Google Scholar It is estimated that one-third of the world's human population is latently infected by Toxoplasma gondii. 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Cerebral hypoperfusion: a new pathophysiologic concept in multiple sclerosis?.J Cereb Blood Flow Metab. 2015; 35: 1406-1410Crossref PubMed Scopus (74) Google Scholar In vivo approaches provide an understanding on how cerebral microcirculation responds to the parasite, in both the acute and chronic phases of infection. Changes in the different microvascular network components, such as arterioles, capillaries, and venules, may provide insights into the physiopathology of toxoplasmosis and its association with neurologic and psychiatric diseases. Because neuroinflammatory processes are involved in the genesis of neurocognitive alterations and the stability of the blood brain barrier (BBB) is crucial for maintenance of the cerebral blood flow (CBF) and neuronal physiologic mechanisms, we investigated how T. gondii infection in mice affects cerebral microcirculation. Tissue cysts from Toxoplasma gondii, ME49 strain, were obtained from brains of previously infected female C57/bl6 mice. Brains from infected mice were homogenized in a sterile saline solution (0.9% NaCl), and an aliquot was used to count cysts. The experimental model of acquired toxoplasmosis was based on what was described by Hermes et al.14Hermes G. Ajioka J.W. Kelly K.A. Mui E. Roberts F. Kasza K. Mayr T. Kirisits M.J. Wollmann R. Ferguson D.J. Roberts C.W. Hwang J.H. Trendler T. Kennan R.P. Suzuki Y. Reardon C. Hickey W.F. Chen L. McLeod R. Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection.J Neuroinflammation. 2008; 5: 48Crossref PubMed Scopus (153) Google Scholar Forty-day–old female Swiss Webster mice were infected intraperitoneally with 50 T. gondii tissue cysts in a final volume of 100 μL in phosphate-buffered saline (PBS). Controls received the same volume of PBS or brain lysates from uninfected animals. For the sulfadiazine treatment, animals received a 1.25–mg mL−1 solution of sulfadiazine sodium salt (Sigma-Aldrich, St. Louis, MO) diluted in drinking water given ad libitum starting at the eighth day post infection (dpi), when the first clinical symptoms were noticed. All procedures and animal handling were approved by the Committee for the Use of Laboratory Animals of the Oswaldo Cruz Institute (license number L-048/2015). Time points were chosen to represent different times of the disease: 10 dpi, when the mortality rate was approximately 30%, and when mice showed the most severe signs of disease according to the SHIRPA (SmithKline Beecham, Harwell, Imperial College, Royal London Hospital, phenotype assessment) clinical score. A total of 40 dpi corresponded to a recommended 6-week period to ensure the presence of tissue cysts in the brain.46Dubey J.P. Pas A. Rajendran C. Kwok O.C. Ferreira L.R. Martins J. Hebel C. Hammer S. Su C. 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Illness-related behaviors and general appearance were assessed using a modification of the SHIRPA protocol47Reis P.A. Alexandre P.C. D'Avila J.C. Siqueira L.D. Antunes B. Estato V. Tibirica E.V. Verdonk F. Sharshar T. Chretien F. Castro-Faria-Neto H.C. Bozza F.A. Statins prevent cognitive impairment after sepsis by reverting neuroinflammation, and microcirculatory/endothelial dysfunction.Brain Behav Immun. 2017; 60: 293-303Crossref PubMed Scopus (51) Google Scholar, 48Rogers D.C. Fisher E.M. Brown S.D. Peters J. Hunter A.J. Martin J.E. Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment.Mamm Genome. 1997; 8: 711-713Crossref PubMed Scopus (639) Google Scholar to determine the severity of the infection. One point was given for each of the following alterations: piloerection, abdominal writhing, reduced body tone, fecal changes (eg, diarrhea), lacrimation, palpebral closure, low locomotor activity, reflexive escape from touch, spontaneous activity changes, reduced grip strength, hunched posture, and changes in respiration rate (hyperventilation). Control groups that presented any change in clinical score (different from 0) were excluded from the analysis. Animals were weighed as an additional measure of general health, at different time points, whenever the clinical score was assessed. To confirm infection, blood from the mice was collected and the sera separated, removed, and stored at −80°C until tested for T. gondii antibodies by a modified agglutination test (antigen for the modified agglutination test assay kindly provided by Dr. Jitender Prakash Dubey, Animal Parasitic Diseases Laboratory, United States Department of Agriculture), according to Dubey and Desmonts.49Dubey J.P. 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Serological responses of equids fed Toxoplasma gondii oocysts.Equine Vet J. 1987; 19: 337-339Crossref PubMed Scopus (667) Google Scholar Laser speckle contrast imaging (Perimed, Järfälla, Sweden) flowmetry is a reliable tool that provides a microvascular perfusion index proportional to the concentration and mean velocity of red blood cells. This noninvasive technique for imaging the microvascular blood flow has been used mostly in clinical studies to assess microcirculation reactivity and accurately quantify relative changes in skin perfusion.50Roustit M. Cracowski J.L. Assessment of endothelial and neurovascular function in human skin microcirculation.Trends Pharmacol Sci. 2013; 34: 373-384Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar In the present study, this method allowed the measurement of the cortical CBF with high temporal and spatial resolution, providing selected-field analysis and determination of the relative changes in the cortical CBF. Mice were anesthetized with a mixture of xylazine and ketamine hydrochloride (200 and 10 mg kg−1 intraperitoneally, respectively) and positioned at a fixed working distance of 10 cm, under the laser light at 785 nm for the continuous measurement of tissue blood flow. To determine the microvascular CBF of the cortex, a region of interest was defined in the projected image on the monitor. The region of interest consists of an ellipse with two axes that measure 0.9 cm anteroposteriorly and 0.7 cm laterolaterally, selected on the surface area of both hemispheres, and the mean cerebral microcirculation rate was quantified and expressed as arbitrary perfusion units. Care was taken not to disturb the preparation during image acquisition. Analysis of 16 laser speckle images per second and relative CBF of all animal groups were acquired for 10 minutes using Perisoft software version 1.5 (Perimed) and expressed as arbitrary perfusion units. After the laser speckle imaging, the anesthetized mice had their tail veins cannulated for the injection of fluorescent tracers and additional anesthesia. Core temperature was monitored with a rectal probe and maintained at 37°C with a homoeothermic blanket system (Harvard Apparatus, Cambridge, UK). Intravital microscopy was performed as described previously.51Reis P.A. Estato V. da Silva T.I. d'Avila J.C. Siqueira L.D. Assis E.F. Bozza P.T. Bozza F.A. Tibirica E.V. Zimmerman G.A. Castro-Faria-Neto H.C. Statins decrease neuroinflammation and prevent cognitive impairment after cerebral malaria.PLoS Pathog. 2012; 8: e1003099Crossref PubMed Scopus (78) Google Scholar Briefly, animals were fixed in a stereotaxic frame, the left parietal bone was exposed by a midline skin incision, a cranial window overlying the left parietal bone (1 to 5 mm lateral, between the coronal and the lambdoid sutures) was created with a high-speed drill, and the dura mater and the arachnoid membranes were excised and withdrawn to expose cerebral microcirculation. The cranial window was suffused with artificial cerebrospinal fluid (NaCl, 132 mmol; KCl, 2.95 mmol; CaCl2, 1.71 mmol; MgCl2, 0.64 mmol; NaHCO3, 24.6 mmol; dextrose, 3.71 mmol; and urea, 6.7 mmol at 37°C, pH 7.4). This procedure does not cause changes in permeability under baseline conditions. Animals were then placed under an upright fixed-stage intravital microscope equipped with a mercury lamp (BX51/WI, Olympus, NY) coupled to a CCD digital video camera system (Optronics, Tokyo, Japan). Olympus 10× and 20× objectives were used in the experiments and produced total magnifications of ×100 and ×200, respectively. Visualization of brain microvessels of the cortical layer near the pial surface was facilitated by the intravenous administration of 0.1 mL of 2% fluorescein isothiocyanate–labeled dextran (molecular weight, 150,000 Da) and by epi-illumination at 460 to 490 nm, using a 520-nm emission filter. To analyze the interactions between leukocytes and the endothelium on postcapillary venules, leukocytes were labeled by intravenous injection of rhodamine 6G (0.3 mg/kg) and visualized by epi-illumination at 510 to 550 nm, using a 590-nm emission filter. Five randomly selected venular segments (30 to 100 μm in diameter) in each preparation were observed for 30 seconds to assess leukocyte recruitment. Leukocyte-endothelial interactions were evaluated by determining the number of leukocytes adhering to the venular wall (100 μm long) for a period of 30 seconds and expressed as the number of cells per minute per 100 μm. Rolling leukocytes were defined as movement of white blood cells into the venular segment at a speed slower than that of the circulating red blood cells and expressed as the number of cells per minute. Microvascular endothelial function was evaluated by the pial arteriolar responses to the topical application of the endothelium-dependent vasodilator a
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