Pattern recognition receptor gene expression in ischemia-induced flap revascularization

The innate immune system is the major contributor to acute inflammation induced by microbial infection or tissue damage. Germline-encoded pattern recognition receptors (PRRs) are responsible for sensing the presence of micro-organisms and endogenous molecules released from damaged cells. We performed microarray analyses on ischemic wound tissue to investigate the temporal relationship between PRR gene expression, wound perfusion, and flap revascularization.

A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of C57BL6 mice (n = 25 total; n = 20 with flap). Laser speckle contrast imaging was utilized to study the pattern of flap ischemia and return of functional revascularization. Flap microvascular density was determined by image analysis of CD-31-immunostained sections. Total RNA was isolated from homogenized flap tissue and was converted to cDNA (RT), which was hybridized to a microarray of pathway-focused genes. Microarray results were validated with quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR).

Laser speckle contrast imaging predicted the spatial and temporal pattern of ischemia and functional revascularization. Histologic analysis demonstrated early leukocyte infiltration and later engraftment, resulting in flap revascularization by new blood vessel growth from the recipient bed and dilatation of preexisting proximal flap vasculature. qRT-PCR demonstrated significant early gene expression of select PRRs, cytokines, chemokines, and growth factors, peaking by 48 hours, and returning toward baseline but remaining elevated at 10 days.

Surgical and ischemic tissue injury resulted in the early gene expression of select PRRs, which may bind with endogenous molecules released from ischemic or necrotic cells, leading to transcription of genes involved in wound inflammation and angiogenesis.