Subject(s)
Ear Diseases/diagnosis , Osteogenesis Imperfecta/complications , Tympanic Membrane Perforation/diagnosis , Child , Ear Canal/pathology , Ear Diseases/genetics , Humans , Male , Osteogenesis Imperfecta/genetics , Osteogenesis Imperfecta/pathology , Otoscopy , Tympanic Membrane/pathology , Tympanic Membrane Perforation/geneticsABSTRACT
OBJECTIVES: The goal of this work was to identify genes, known to be involved in the skin wound healing, that express differentially in the healthy and injured tympanic membrane (TM), and designate the molecules potentially beneficial for treatment of TM perforation. The molecular mechanisms controlling the course of TM regeneration are far from being elucidated. METHODS: Twenty rats had their tympanic membranes perforated, while four served as a control. Animals were sacrificed on either days 1, 2, 3, 5 and 10 post injury, and TMs were immediately dissected and frozen in liquid nitrogen. Total TM RNA was isolated and reversely transcribed. qPCR was performed using Rat Wound Healing RT(2) Profiler PCR Array (QIAGEN) containing primers for 84 genes. RESULTS: Statistically significant changes in the expression of 42 genes were found in various stages of TM healing. The increased expression of genes taking part in the inflammatory reaction (interleukin 6, granulocyte and macrophage chemotactic proteins) was observed from day 2. The expression of several genes of extracellular matrix components and their remodeling enzymes was also changed. Among growth factor genes: Vegfa, Igf1 and Hbegf showed increased expression at the beginning of the healing process, while Hgf expression was highest on day 3. CONCLUSIONS: Several changes in the expression of genes involved in remodeling of extracellular matrix point to important role of connective tissue in TM healing. The molecules accelerating this process, like HbEGF and HGF, seem to be good candidates for further evaluation of their possible use in clinical treatment.
Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation , Polymerase Chain Reaction/methods , Tympanic Membrane Perforation/genetics , Wound Healing/genetics , Animals , Disease Models, Animal , Male , Random Allocation , Rats , Rats, Wistar , Reference Values , Sensitivity and Specificity , Tympanic Membrane Perforation/physiopathology , Wound Healing/physiologyABSTRACT
OBJECTIVES/HYPOTHESIS: The aim of this study is to elucidate transcriptional changes that occur in response to tympanic membrane (TM) perforation in rats and to infer key genes and molecular events in the healing process. STUDY DESIGN: A prospective cohort study of 393 male Sprague-Dawley (Rattus norvegicus) rats. METHODS: Sprague-Dawley rats were randomly allocated into either control or perforation groups spanning a 7-day time period. Perforation groups consisted of 12-hour, 24-hour, 36-hour, 2-day, 3-day, 4-day, 5-day, six-day, and 7-day time points. The left TMs of all perforation groups were perforated and the RNA extracted at the specified time point postperforation. Subsequent analysis was performed using Agilent's 4 × 44 k whole rat genome arrays (40 in total) to assess wound-healing gene expression over a 7-day time period. RESULTS: Over a 7-day time course and at nine time points that encompassed the wounding and progression of healing, a total of 3,262 genes were differentially expressed. In this study the transcripts most upregulated occurred at 12 hours. These were Stefin A2 (344-fold), Stefin 2 (143-fold), and Natriuretic peptide precursor type B (222-fold). Those most downregulated also occurred at 12 hours. These were alcohol dehydrogenase 7 (13.1-fold) and gamma-butyrobetaine hydroxylase (10.4-fold). Results were validated by quantitative real-time polymerase chain reaction. CONCLUSIONS: The findings of this study provide a baseline against which to identify disease-related molecular signatures, biomarkers, and to develop new treatments for TM conditions based on molecular evidence.
Subject(s)
Gene Expression Profiling , Tympanic Membrane Perforation/genetics , Wound Healing/genetics , Acute Disease , Animals , Chronic Disease , Confidence Intervals , Cystatin A/genetics , Cystatin A/metabolism , Desmocollins/genetics , Desmocollins/metabolism , Disease Models, Animal , Down-Regulation , Gene Expression Regulation , Lipocalin-2 , Lipocalins/genetics , Lipocalins/metabolism , Male , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Microarray Analysis , Random Allocation , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tympanic Membrane Perforation/metabolism , Up-Regulation , Wound Healing/physiologyABSTRACT
The aim of this study was to provide a transcriptome profile of Keratinocyte Growth Factor (KGF)-1, Fibroblast Growth Factor (FGF) 2 and FGF10 (KGF2) in the healing rat tympanic membrane (TM) over 7 days and an immunohistochemical account over 14 days following perforation. KGF1, FGF2, and FGF10 play important roles in TM wound healing. The tympanic membranes of rats were perforated and sacrificed at time points over a 14-day period following perforation. The normalized signal intensities and immunohistochemical protein expression patterns at each time point for KGF1, FGF2, and FGF10 are presented. The primary role of both KGF1 and FGF2 appeared to be in the proliferation and migration of keratinocytes. Whereas the role of KGF1 appeared to be exclusively concerned with increased proliferation and migration at the perforation site, the continued expression of FGF2, beyond perforation closure, suggested it has an additional role to play. FGF10 (KGF2), whilst possessing the highest sequence homologous to KGF1, has a different role in TM wound healing. The effect of FGF10 on keratinocytes in wound healing appeared to emanate from the connective tissue layer.
