RESUMO
Intestinal injury owing to inflammation, severe trauma, and burn is a leading cause of morbidity and mortality. Currently, animal models employed to study the intestinal response to injury and inflammation depend on outdated methods of analysis. Given that these classic intestinal assays are lethal to the experimental animal, there is no ability to study the gut response to injury in the same animal over time. We postulated that by developing an in vivo assay to image intestinal injury using fluorescent dye, it could complement other expensive, time-consuming, and semiquantitative classic means of detecting intestinal injury. We describe a novel in vivo, noninvasive method to image intestinal injury using a charge-coupled device (CCD) camera that allows for serial visual and quantitative analysis of intestinal injury. Our results correlate with traditional, time-consuming, semiquantitative assays of intestinal injury, now allowing the noninvasive, nonlethal assessment of injury over time.
Assuntos
Fluorometria/métodos , Intestinos/lesões , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Queimaduras/metabolismo , Dextranos/química , Dextranos/metabolismo , Modelos Animais de Doenças , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Histocitoquímica , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estatísticas não Paramétricas , Imagem Corporal Total/métodosRESUMO
Because there are few reports using gene delivery in clinically-approved synthetic matrices, we examined the feasibility of using a noninvasive imaging system to study the kinetics of luciferase gene expression when delivered in an adenoviral vector. Using a mouse model of full thickness injury, we quantified the kinetics of gene expression, determined the optimal dose of particle delivery, and established the temporal importance of drug delivery in obtaining optimal gene expression. Specifically, we found that the ideal time to deliver adenovirus to a graft is during the early phase of graft wound closure (days 0-3 post-operatively) for a peak of gene expression to occur 7 days after delivery. Under these conditions, there is a saturating dose of 6 x 10(8) adenoviral particles per graft. In light of these findings, we examined whether the efficacy of delivery could be increased by modulating the composition of the grafts. When a collagen gene-activated matrix (GAM) containing basic fibroblast growth factor (FGF2) was compared to matrix alone, a significant increase in gene expression is observed when identical amounts of vector are delivered (p<0.05). Taken together, these results show how a noninvasive and quantitative assessment of gene expression can be used to optimize gene delivery and that the composition of matrices can dramatically influence gene expression in the wound bed.