The intravital microscopy (IM) is a powerful tool used in the biomedical field for the in vivo observation and imaging of biological systems, having important application in the study of the leukocyte-endothelial interactions which occur... more
The intravital microscopy (IM) is a powerful tool used in the biomedical field for the in vivo observation and imaging of biological systems, having important application in the study of the leukocyte-endothelial interactions which occur in the microcirculation of various animal tissues under normal and pathological conditions. As the control of the image acquisition conditions in the in vivo analysis is very limited, the presence of blurring and motion artefacts in the acquired video frames sequences, resulting from the animal’s unavoidable breathing and heartbeats, is one of the main obstacles of the IM. This significantly undermines the detection and tracking of leukocytes over the frames, both in visual and automatic (computational) analyses. Therefore, this paper carried out the study and application of two blind deconvolution techniques (deconvblind algorithm and Shan et al.’s method) for the correction of motion artefacts in IM video images, performing a quantitative analysis of how such
restoration affects the performance of two methods of automatic leukocytes detection (template matching and local phase symmetry). Precision versus recall analyses were conducted in order to evaluate the effectiveness of the automatic detection in images with good quality, images degraded by motion and images restored by each technique.
From this study, it was concluded that Shan et al.’s method has the greatest potential to restore the leukocytes and that blind deconvolution techniques are in fact useful as a preprocessing step of IM videos for in vivo analyses, specially when the method of automatic leukocytes detection is more sensitive to errors.
restoration affects the performance of two methods of automatic leukocytes detection (template matching and local phase symmetry). Precision versus recall analyses were conducted in order to evaluate the effectiveness of the automatic detection in images with good quality, images degraded by motion and images restored by each technique.
From this study, it was concluded that Shan et al.’s method has the greatest potential to restore the leukocytes and that blind deconvolution techniques are in fact useful as a preprocessing step of IM videos for in vivo analyses, specially when the method of automatic leukocytes detection is more sensitive to errors.
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Although previous studies have shown that forced exercise modulates inflammation and is therapeutic acutely for experimental autoimmune encephalomyelitis (EAE), the long-term benefits have not been evaluated. In this study, we... more
Although previous studies have shown that forced exercise modulates inflammation and is therapeutic acutely for experimental autoimmune encephalomyelitis (EAE), the long-term benefits have not been evaluated. In this study, we investigated the effects of preconditioning exercise on the clinical and pathological progression of EAE. Female C57BL/6 mice were randomly assigned to either an exercised (Ex) or unexercised (UEx) group and all of them were induced for EAE. Mice in the Ex group had an attenuated clinical score relative to UEx mice throughout the study. At 42 dpi, flow cytometry analysis showed a significant reduction in B cells, CD4(+) T cells, and CD8(+) T cells infiltrating into the spinal cord in the Ex group compared to UEx. Ex mice also had a significant reduction in myelin damage with a corresponding increase in proteolipid protein expression. Finally, Ex mice had a significant reduction in axonal damage. Collectively, our study demonstrates for the first time that a pr...
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Although previous studies have shown that forced exercise modulates inflammation and is therapeutic acutely for experimental autoimmune encephalomyelitis (EAE), the long-term benefits have not been evaluated. In this study, we... more
Although previous studies have shown that forced exercise modulates inflammation and is therapeutic acutely for experimental autoimmune encephalomyelitis (EAE), the long-term benefits have not been evaluated. In this study, we investigated the effects of preconditioning exercise on the clinical and pathological progression of EAE. Female C57BL/6 mice were randomly assigned to either an exercised (Ex) or unexercised (UEx) group and all of them were induced for EAE. Mice in the Ex group had an attenuated clinical score relative to UEx mice throughout the study. At 42 dpi, flow cytometry analysis showed a significant reduction in B cells, CD4(+) T cells, and CD8(+) T cells infiltrating into the spinal cord in the Ex group compared to UEx. Ex mice also had a significant reduction in myelin damage with a corresponding increase in proteolipid protein expression. Finally, Ex mice had a significant reduction in axonal damage. Collectively, our study demonstrates for the first time that a prolonged and forced preconditioning protocol of exercise improves clinical outcome and attenuates pathological hallmarks of EAE at chronic disease. In this study, we show that a program of 6 weeks of preconditioning exercise promoted a significant reduction of cells infiltrating into the spinal cord, a significant reduction in myelin damage and a significant reduction in axonal damage in experimental autoimmune encephalomyelitis (EAE) mice at 42 dpi. Collectively, our study demonstrates for the first time that a preconditioning protocol of exercise improves clinical outcome and attenuates pathological hallmarks of EAE at chronic disease.
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Beyond the classical actions of the renin-angiotensin system on the regulation of cardiovascular homeostasis, several studies have shown its involvement in acute and chronic inflammation. The G protein-coupled receptor Mas is a functional... more
Beyond the classical actions of the renin-angiotensin system on the regulation of cardiovascular homeostasis, several studies have shown its involvement in acute and chronic inflammation. The G protein-coupled receptor Mas is a functional binding site for the angiotensin-(1-7); however, its role in the immune system has not been fully elucidated. In this study, we evaluated the effect of genetic deletion of Mas receptor in lipopolysaccharide (LPS)-induced systemic and cerebral inflammation in mice. Inflammatory response was triggered in Mas deficient (Mas(-/-)) and C57BL/6 wild-type (WT) mice (8-12 weeks-old) by intraperitoneal injection of LPS (5mg/kg). Mas(-/-) mice presented more intense hypothermia compared to WT mice 24h after LPS injection. Systemically, the bone marrow of Mas(-/-) mice contained a lower number of neutrophils and monocytes 3h and 24h after LPS injection, respectively. The plasma levels of inflammatory mediators KC, MCP-1 and IL-10 were higher in Mas(-/-) mice 24h after LPS injection in comparison to WT. In the brain, Mas(-/-) animals had a significant increase in the number of adherent leukocytes to the brain microvasculature compared to WT mice, as well as, increased number of monocytes and neutrophils recruited to the pia-mater. The elevated number of adherent leukocytes on brain microvasculature in Mas(-/-) mice was associated with increased expression of CD11b - the alpha-subunit of the Mac-1 integrin - in bone marrow neutrophils 3h after LPS injection, and with increased brain levels of chemoattractants KC, MIP-2 and MCP-1, 24h later. In conclusion, we demonstrated that Mas receptor deficiency results in exacerbated inflammation in LPS-challenged mice, which suggest a potential role for the Mas receptor as a regulator of systemic and brain inflammatory response induced by LPS.
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Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically... more
Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation...
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S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or... more
S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or pathologic BBB disruption (BBBD). Elevated S100B matches the clinical presence of indices of BBBD (gadolinium enhancement or albumin coefficient). After repeated sub-concussive episodes, serum S100B triggers an antigen-driven production of anti-S100B autoantibodies. We tested the hypothesis that the presence of S100B in extracranial tissue is due to peripheral cellular uptake of serum S100B by antigen presenting cells, which may induce the production of auto antibodies against S100B. To test this hypothesis, we used animal models of seizures, enrolled patients undergoing repeated BBBD, and collected serum samples from epileptic patients. We employed a broad array of techniques, including immunohistochemistry, RNA analysis, tracer injection and serum analysis. mRNA for S100B was segregated to barrier organs (testis, kidney and brain) but S100B protein was detected in immunocompetent cells in spleen, thymus and lymph nodes, in resident immune cells (Langerhans, satellite cells in heart muscle, etc.) and BBB endothelium. Uptake of labeled S100B by rat spleen CD4+ or CD8+ and CD86+ dendritic cells was exacerbated by pilocarpine-induced status epilepticus which is accompanied by BBBD. Clinical seizures were preceded by a surge of serum S100B. In patients undergoing repeated therapeutic BBBD, an autoimmune response against S100B was measured. In addition to its role in the central nervous system and its diagnostic value as a BBBD reporter, S100B may integrate blood-brain barrier disruption to the control of systemic immunity by a mechanism involving the activation of immune cells. We propose a scenario where extravasated S100B may trigger a pathologic autoimmune reaction linking systemic and CNS immune responses.
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Research Interests: Skeletal muscle biology, Fluorescence Microscopy, Microcirculation, Oxidative Stress, Microvascular Physiology, and 17 moreBrain, Video microscopy, Enalapril, Blood Glucose, Hypertension, Insulin, Capillaries, Renin Angiotensin Aldosterone System, Animals, Male, Clinical Sciences, Rats, Coronary Circulation, Imidazoles, Heart Ventricles, Cerebrovascular Disorders, and Angiotensin Converting Enzyme Inhibitors
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Research Interests: Immunology, Multiple sclerosis, Cytokines, Brain, Mice, and 14 moreFemale, Animals, Spinal Cord, Neuroinflammation, Central Nervous System, Glycoproteins, Clinical Sciences, Chemokines, Experimental Autoimmune Encephalomyelitis, Neurosciences, Leukocytes, Intravital Microscopy, Microvessels, and Experimental Model
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Research Interests: Immunology, Multiple sclerosis, Microcirculation, Cell Adhesion, Neuroimmunology, and 13 moreAntibodies, Mice, Female, Animals, Glycoproteins, Analysis of Variance, Time Factors, Body Weight, Experimental Autoimmune Encephalomyelitis, Neurosciences, Leukocytes, Enzyme Linked Immunosorbent Assay, and Intravital Microscopy
Although it is well known that regular exercise may promote neuroprotection, the mechanisms underlying this effect are still not fully understood. We investigated if swim training promotes neuroprotection by potentiating antioxidant... more
Although it is well known that regular exercise may promote neuroprotection, the mechanisms underlying this effect are still not fully understood. We investigated if swim training promotes neuroprotection by potentiating antioxidant pathways, thereby decreasing the effects of oxidative stress on glutamate and nitric oxide release. Male Wistar rats (n=36) were evenly randomized into a trained group (TRA) (5 days/week, 8 weeks, 30 min) and a sedentary group (SED). Forty-eight hours after the last session of exercise, animals were killed and brain was collected for in vitro ischemia. Cortical slices were divided into two groups: a group in which oxidative stress was induced by oxygen and glucose deprivation (OGD), and a group of non-deprived controls (nOGD). Interestingly, exercise by itself increased superoxide dismutase activity (nOGD, SED vs. TRA animals) with no effect on pro-oxidative markers. In fact, TRA-OGD slices showed lowered levels of lactate dehydrogenase when compared with SED-OGD controls, reinforcing the idea that exercise affords a neuroprotective effect. We also demonstrated that exercise decreased glutamate and nitrite release as well as lipid membrane damage in the OGD cortical slices. Our data suggest that under conditions of metabolic stress, swim training prevents oxidative damage caused by glutamate and nitric oxide release.
Research Interests: Neurochemistry, Oxidative Stress, Swimming, Glucose, Cerebral Cortex, and 6 moreAnimals, Male, Rats, Oxygen, Wistar Rats, and Neurosciences
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Research Interests: Immune response, Fluorescence Microscopy, Malaria, Microcirculation, Inflammation, and 16 moreCell Adhesion, Immunohistochemistry, Biological Sciences, Brain, Tuberculosis, Mice, Blood brain barrier, Female, Animals, Vascular endothelium, Experimental Infection, Species Specificity, Chemokines, Leukocytes, Intravital Microscopy, and Plasmodium berghei
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Detection of rolling and adhered leukocytes in intravital microscopy image sequences is an important task in studies of leukocyte-endothelial interactions in the microcirculation of living small animals under different inflammatory... more
Detection of rolling and adhered leukocytes in intravital microscopy image sequences is an important task in studies of leukocyte-endothelial interactions in the microcirculation
of living small animals under different inflammatory conditions. This procedure is usually performed by visual assessment of the image sequences. However, despite being tedious and time consuming, this procedure is prone to the inter- and intra-observer variability. In this work, we developed an automated computer system for the detection of leukocytes in intravital video microscopy. First, the video frames were processed by the bilateral filter to reduce noise while preserving sharp edges. Then, a demons-based image registration technique was applied to minimize animal motion. Finally, the detection of leukocytes was performed by local analysis of Hessian matrix eigenvalues. Quantitative and qualitative evaluation of the proposed method were conducted by using 220 video frames obtained from an experimental study performed on the brain microvasculature of mice. The proposed method was compared with the template matching technique using precision, recall and F1-Score measures. For the Hessian-based method, the results of precision, recall and F1-Score were, respectively, equal to 0.81, 0.86, and 0.83. For direct comparison, the results obtained for the template matching technique were 0.86, 0.73 and 0.79.
of living small animals under different inflammatory conditions. This procedure is usually performed by visual assessment of the image sequences. However, despite being tedious and time consuming, this procedure is prone to the inter- and intra-observer variability. In this work, we developed an automated computer system for the detection of leukocytes in intravital video microscopy. First, the video frames were processed by the bilateral filter to reduce noise while preserving sharp edges. Then, a demons-based image registration technique was applied to minimize animal motion. Finally, the detection of leukocytes was performed by local analysis of Hessian matrix eigenvalues. Quantitative and qualitative evaluation of the proposed method were conducted by using 220 video frames obtained from an experimental study performed on the brain microvasculature of mice. The proposed method was compared with the template matching technique using precision, recall and F1-Score measures. For the Hessian-based method, the results of precision, recall and F1-Score were, respectively, equal to 0.81, 0.86, and 0.83. For direct comparison, the results obtained for the template matching technique were 0.86, 0.73 and 0.79.
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Resumo: A detecção e o rastreamento automáticos de leucócitos em imagens de vídeo de MI podem garantir análises mais precisas e, consequentemente, auxiliar os pesquisadores no desenvolvimento de estratégias terapêuticas mais eficazes.... more
Resumo: A detecção e o rastreamento automáticos de leucócitos em imagens de vídeo de MI podem garantir análises mais precisas e, consequentemente, auxiliar os pesquisadores no desenvolvimento de estratégias terapêuticas mais eficazes. Entretanto, na análise in vivo, a respiração e a atividade cardíaca do animal ocasionam a perda momentânea do foco dos vasos, gerando imagens borradas e distorcidas. Esse fato dificulta consideravelmente a detecção e o rastreamento dos leucócitos. Portanto, este trabalho propõe um método multirresolução para a estabilização de sequências de imagens de MI que utiliza a técnica demons simétrica, com transformações afins e deformáveis. Os resultados obtidos da aplicação do método proposto indicam uma melhoria visual significativa na estabilização das imagens, quantificada pelo aumento no valor médio da métrica PSNR de 35 para 40dB, aplicada sobre os resíduos resultantes da subtração de imagens consecutivas do vídeo. Palavras-chave: estabilização de imagem...