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Effects of cholinergic function deficiency on pulmonary mechanics and histopathology in an experimental model of acute inflammation induced by LPS instillation in genetically modified mice

Grant number: 13/02881-4
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): December 01, 2013
Effective date (End): March 31, 2016
Field of knowledge:Biological Sciences - Physiology - Physiology of Organs and Systems
Principal researcher:Carla Máximo Prado
Grantee:Nathalia Montouro Pinheiro Menegasso
Home Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil


Acute Lung Injury (ALI) is characterized by extensive lung inflammation, recruitment of polymorphonuclear cells and release of proinflammatory mediators. It is a severe condition that progresses with death in approximately 40% of cases. Despite several studies that elucidate the pathophysiology of ALI, the treatment is still unsatisfactory. Recently the anti-inflammatory cholinergic system has been described in the lung and it is related to a reflex via the vagus nerve which inhibits the release of inflammatory cytokines by stimulation of nicotinic receptors. In the first stage of this study we found that animals with systemic cholinergic deficiency present the same degree of acute lung inflammation induced by LPS compared to wild-type. In order to understand the mechanisms involved in this response, we intend to use pharmacological stimuli to isolate the action of muscarinic and nicotinic receptors in these animals. Furthermore, in order to elucidate whether the cholinergic anti-inflammatory pathway is involved in ALI in naive animals, we will use the nicotinic receptor agonist. Objective: 1. To evalaute the VAChT in animals and determine whether the induction of ALI interfere with it; 2. To evaluate mechanisms involved in the response to LPS in animals with cholinergic deficiency; 3. To evaluate the potential therapeutic effect of PNU agonist of nicotinic alpha-7 receptor, in a model of LPS in C57Bl6. Methodology: We will use genetically modified male mice that have a reduction in VAChT protein expression, which is associated with decreased acetylcholine release. These animals will be divided according to their genotype for homozygous (HOM) and wild type (WT) and receive intratraqueal instillation of LPS or saline 24 hours before the experimental protocol. These animals will be treated with PNU, nicotinic receptor agonist, pilocarpine or agonist of muscarinic receptors. Also in naive C57BL6 animals that receive instillation of saline or LPS, we use the PNU, performing a dose response curve at different times after the installation of LPS. We will evaluate: respiratory mechanics, lung inflammation through bronchoalveolar lavage as well as flow citometry and macrophage response in the lung tissue, the extracellular matrix remodeling, evaluating the content of collagen and elastic fibers, the expression of TNF-alpha and oxidative stress through quantifying the content of the isoprostane PGF2. All histopathological parameters are quantified by morphometric technique. Depending on the answers, other cytokines are also quantified. Furthermore, we evaluate by ELISA or western blot, cholinergic components in the lung in order to elucidate how these components are in VAChT animals and also to understand if ALI interferes with this system (muscarinic and nicotinic receptors, VAChT and AChE). Statistical analysis will be performed using the SigmaStat. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
PINHEIRO, NATHALIA M.; SANTANA, FERNANDA P. R.; ALMEIDA, RAFAEL RIBEIRO; GUERREIRO, MARINA; MARTINS, MILTON A.; CAPERUTO, LUCIANA C.; CAMARA, NIELS O. S.; WENSING, LISLAINE A.; PRADO, VANIA F.; TIBERIO, IOLANDA F. L. C.; PRADO, MARCO ANTONIO M.; PRADO, CARLA M. Acute lung injury is reduced by the alpha 7nAChR agonist PNU-282987 through changes in the macrophage profile. FASEB JOURNAL, v. 31, n. 1, p. 320-332, JAN 2017. Web of Science Citations: 11.
GAMES, ELLEN; GUERREIRO, MARINA; SANTANA, FERNANDA R.; PINHEIRO, NATHALIA M.; DE OLIVEIRA, EMERSON A.; LOPES, FERNANDA D. T. Q. S.; OLIVO, CLARICE R.; TIBERIO, IOLANDA F. L. C.; MARTINS, MILTON A.; LAGO, JOAO HENRIQUE G.; PRADO, CARLA M. Structurally Related Monoterpenes p-Cymene, Carvacrol and Thymol Isolated from Essential Oil from Leaves of Lippia sidoides Cham. (Verbenaceae) Protect Mice against Elastase-Induced Emphysema. Molecules, v. 21, n. 10 OCT 2016. Web of Science Citations: 8.

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