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Influence of innate immune signaling and dexamethasone (DEX) administration on the degeneration and neororegeneration of the olfactory epithelium (OE)

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Umberto Crisafulli
Total Authors: 1
Document type: Doctoral Thesis
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
Defense date:
Examining board members:
Isaias Glezer; Niels Olsen Saraiva Câmara; Fábio Luís Forti; Fábio Papes; Alexander Henning Ulrich
Advisor: Isaias Glezer

The innate immune response signaling plays a key role in the elimination of pathogens and in the recruitment of new cells to recover the injured nervous tissues. Our preliminary studies on the roles of the inflammatory activity in OE degeneration and neuroregeneration process showed that the topical administration of lipopolysaccharide (LPS) generates the loss of olfactory sensorial neurons (OSNs), Myd88 gene-deficient mice exhibit a higher neuronal replacement rate after injury than wild-type mice, and that the consecutive use of DEX provokes retarding olfactory neuroregeneration over the short term. We seek now to clarify these results in three ways: The first describes the OE response in wild-type animals Tlr4-/-, Myd88-/-, Ticam1-/-, Il1r1-/-, Casp1-/-/Casp4-/- and Casp1-/-/Casp4-/-/Casp4tg animals to inflammation through the intranasal infusion (IN) of ligands and cytokines associated with Toll-Like Receptors (TLR) signaling. This was acomplished through histological analysis of the thickness and anatomical changes in DAPI stained OE, Il1b and Nfκbia expression analysis by in situ hybridization (ISH), and TUNEL-positive cells counting after treatment. In this study we propose an inflammatory lesion for neuroregeneration studies using immunofluorescence (IF) analysis of the OE in function of time after the intranasal infusion of LPS. The second part analyzes the Myd88 gene loss in the OE gene expression of after a lesion using microarray. This is followed by the histological analysis of regeneration using IF in transgenic mice with the deletion of specific genes (microarray versus literature review). Finally, the third study evaluates the OE degeneration and neuroregeneration under the influence of consecutive topical use of the DEX. The corticosteroid is co-administered with the inflammatory stimulus in order to evaluate its protective effect and consecutively for three days in two models of lesion to assess their influence on neuroregeneration 14 days after treatment. The IF analysis was performed in order to quantify the neuronal volumes, the thickness of the OE, cell proliferation, protein synthesis (by incorporation the identification of puromycin) and cell death by TUNEL. The topical use of LPS promotes the degeneration of OE by TLR4 from sites that feature an overexpression of Il1b and Nfκbia and a high number of TUNEL-positive cells. This effect is MyD88-dependent. The Myd88 gene is as crucial in this degeneration of the epithelium as in those generated by rmIL-1β and rmTNFα. Their absence does not promote cytoprotection against the cytotoxic gas nitric oxide (NO). It is possiby that CASP1 and IL-1R are also involved. The immunologic lesion model for neuroregeneration studies is fast and effective. The absence of Myd88 gene reduces the expression of insulin-degrading enzyme (IDE) in the post-lesion OE. Mice without this enzyme show a rapid cellular restoration in the epithelium. The Co-IN of DEX with LPS prevents the degeneration EO. The doses adopted by us are nontoxic; however its short-term consecutive use promotes anatomical aberrations in the immunological lesion model, and interferes with the dynamics of neural replacement by impairing both the rate of protein synthesis and proliferation of the epithelium without halting their differentiation. (AU)

FAPESP's process: 11/13134-0 - Study of the influence of signaling Toll-Like Receptors (TLRs) and the gene MyD88 on olfactry epithelium neuronal regeneration
Grantee:Umberto Crisafulli
Support Opportunities: Scholarships in Brazil - Doctorate (Direct)