An in vitro model for studying vascular injury after laser microdissection

Abstract

We have developed an in vitro model for studying vascular injury. After 7-10 days in a three-dimensional collagen gel culture, capillary-like tubes were formed in the collagen gels. We injured these capillary-like tubes with a laser microdissection system or a scrape method with razors and then examined the collagen gel culture by phase contrast and electron microscopy. After laser injury, profuse necrotic cells were observed around the injured capillary-like tubes and within the tubular lumen compared to the razor injury. We then isolated total RNA from these cultures and prepared cDNA for investigations by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real time RT-PCR revealed the up-regulation of transcription factor early growth response-1 (Egr-1) after both laser and razor injury, accompanied by the up-regulation of fibroblast growth factor-2 (FGF-2), a proangiogenic factor downstream of Egr-1. The effective laser energy is concentrated on the minute focal spot only. These methods provide a useful in vitro model for studying vascular injury.

Detection of the Hematopoietic Stem and Progenitor Cell Marker CD133 during Angiogenesis in Three-Dimensional Collagen Gel Culture

“…Using a three-dimensional (3D) collagen gel culture, we have conducted electron microscopic studies [14, 15] and immunohistochemical studies of fibroblast growth factor- (FGF-) 2 and FGF-9 [16]. Additionally, we have used 3D collagen gel cultures to test angiogenic and anti-angiogenic agents (TNF- α and thalidomide) [17, 18] to study vascular injury after laser microdissection [19] and for profiling of DNA microarray gene expression during angiogenesis [20]. The 3D collagen gel culture system provides a simple and rapid method to analyze angiogenesis.…”

Development of In Vitro Method for Assaying Anti-Angiogenic Effect of Drugs

“…We applied the 3D collagen gel culture as an in vitro model of vascular injury [163]. After 7-10 days of culture, capillary tubes were observed in the collagen gels.…”

Recent progress in histochemistry

“…This study revealed that granulosa cells express stable amounts of inhibin-subunit mRNA independently of antral formation in immature rat ovaries. An interesting application of the LMD technique is to induce cell injury as reported in an in vitro model of vascular injury (Fujita et al 2006). Capillary-like tubes formed in culture were injured by a precisely focused laser pulse, making LMD an ideal tool for inducing and studying aspects of vascular injury.…”