Novel biomarkers of arterial and venous ischemia in microvascular flaps.

The field of reconstructive microsurgery is experiencing tremendous growth, as evidenced by recent advances in face and hand transplantation, lower limb salvage after trauma, and breast reconstruction. Common to all of these procedures is the creation of a nutrient vascular supply by microsurgical anastomosis between a single artery and vein. Complications related to occluded arterial inflow and obstructed venous outflow are not uncommon, and can result in irreversible tissue injury, necrosis, and flap loss. At times, these complications are challenging to clinically determine. Since early intervention with return to the operating room to re-establish arterial inflow or venous outflow is key to flap salvage, the accurate diagnosis of early stage complications is essential. To date, there are no biochemical markers or serum assays that can predict these complications. In this study, we utilized a rat model of flap ischemia in order to identify the transcriptional signatures of venous congestion and arterial ischemia. We found that the critical ischemia time for the superficial inferior epigastric fasciocutaneus flap was four hours and therefore performed detailed analyses at this time point. Histolgical analysis confirmed significant differences between arterial and venous ischemia. The transcriptome of ischemic, congested, and control flap tissues was deciphered by performing Affymetrix microarray analysis and verified by qRT-PCR. Principal component analysis revealed that arterial ischemia and venous congestion were characterized by distinct transcriptomes. Arterial ischemia and venous congestion was characterized by 408 and 1536>2-fold differentially expressed genes, respectively. qRT-PCR was used to identify five candidate genes Prol1, Muc1, Fcnb, Il1b, and Vcsa1 to serve as biomarkers for flap failure in both arterial ischemia and venous congestion. Our data suggests that Prol1 and Vcsa1 may be specific indicators of venous congestion and allow clinicians to both diagnose and successfully treat microvascular complications before irreversible tissue damage and flap loss occurs

Validation of microarray data using qRT-PCR analysis.

<p>qRT-PCR performed on 5 differentially expressed genes (<i>Prol1, Muc1, Vcsa1, Fcnb, Il1b</i>) for flaps undergoing both arterial ischemia and venous congestion for 4 hours compared to control. <i>n = </i>5 and performed in duplicate. Data are expressed as means ± SE and analyzed by unpaired <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, cf. control flaps; ^§<i>P<</i>0.05, cf. arterial ischemia flaps.</p

Global transcriptome profiles in ischemic flaps.

<p>A) PCA diagram B) Venn diagram of differentially expressed genes upon 4 hours of arterial ischemia and venous congestion C) Hierarchical cluster analysis of the differentially expressed genes in rats following 4 hours of arterial ischemia and venous congestion.</p

Top GO classes of differentially expressed genes.

<p>Top GO classes of differentially expressed genes.</p

Temporal expression profile of selected genes under venous congestion.

<p>qRT-PCR performed on <i>Prol1, Muc1, Vcsa1, Fcnb, Il1b</i> for flaps undergoing venous congestion compared to control at <i>t = </i>1, 3, 4 hrs. Data are expressed as means ± SE and analyzed by unpaired <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Microarray data of genes demonstrating highest and lowest fold change.

<p>Microarray data of genes demonstrating highest and lowest fold change.</p

Ingenuity Pathway Analysis results for differentially expressed genes in rat composite microvascular flaps undergoing 4 hours of arterial ischemia and venous congestion compared to control flaps.

<p>Ingenuity Pathway Analysis results for differentially expressed genes in rat composite microvascular flaps undergoing 4 hours of arterial ischemia and venous congestion compared to control flaps.</p

Phenotype analysis, temporal profile of flap survival following vessel occlusion.

<p>A) Bar graph of percent flap survival of ischemia-reperfusion vs. time on postoperative day 6 of arterial ischemia and venous congestion. Data are expressed as means ± SE and analyzed by unpaired <i>t</i>-test. *<i>P</i><0.05. B) Gross photos of flaps at 3.5 vs. 4 hours of venous congestion C) From left to right, gross photos of flaps subjected to 4 hours of arterial ischemia and venous congestion compared to control D) Histology showing flaps subjected to 4 hours of venous congestion showing extravasation of blood, while control specimen and arterial ischemia lack such findings.</p

Diagram of operative procedure, phenotype analysis.

<p>A) Diagram of lower left inguinal anatomic landmarks of flap with labeling of superficial inferior epigastric artery, vein, and inguinal ligament.</p