title: Pulsed acoustic cellular expression as a protective therapy against I/R injury in a cremaster muscle flap model.
abstract: BACKGROUND
  Tissue ischemia and reperfusion (I/R) affects blood flow restoration and oxygen delivery to the damaged tissues contributing to tissue morbidity and microcirculatory compromise. Pulsed acoustic cellular expression (PACE) technology is known to support tissue neovascularization. The aim of this study was to test PACE conditioning mechanism of action on microcirculatory hemodynamics in ischemia-reperfusion injury model.
  
  
  METHODS
  34 rat cremaster muscle flaps were monitored under intravital microscopy system in 4 experimental groups: 1) non-ischemic controls (n=10), 2) 5h ischemia without conditioning (n=8), 3) pre-ischemic (5h) PACE conditioning (n=8), 4) post-ischemic (5h) PACE conditioning (n=8). Standard microcirculatory hemodynamics of RBC velocity, vessel diameters and functional capillary perfusion were recorded for 2h after I/R. Immunohistochemistry assessed expression of proangiogenic factors: VEGF and vWF, whereas real-time PCR assessed proangiogenic (VEGF, eNOS) and proinflammatory factors (iNOS; chemokines: CCL2, CXCL5 and chemokine receptor CCR2).
  
  
  RESULTS
  Pre-ischemic PACE conditioning (group 3) resulted in increased RBC velocity of second (A-2) and third order arterioles (A-3) and venule (V-1) by 40%, 15% and 24% respectively comparing to ischemic group without conditioning (p<0.05). Post-ischemic PACE conditioning (group 4) revealed: 1) increase in RBC velocity in second (A-2) and third order arterioles (A-3) by 65% and 31% respectively comparing to ischemia without conditioning (group 2), 2) 33% increase in first order arterioles diameter (A-1) (p<0.05) compared to ischemic controls, 3) 21% increase in number of functional capillaries compared to ischemia without conditioning (group 2) (P<0.05). Immunostaining assays showed that PACE postconditioning up-regulated proangiogenic factors vWF and VEGF protein expression. This correlated with increased gene expression of VEGF (up to 180%). In contrast, gene expression of proinflammatory factors (iNOS, CCL2, CXCL5) decreased compared to ischemic controls. Pre-ischemic PACE conditioning decreased gene expression of proinflammatory chemokines (CCL2 and CXCL5), compared to ischemic controls without conditioning.
  
  
  CONCLUSIONS
  As expected 5h ischemia resulted in deterioration of microcirculatory hemodynamics confirmed by decreased vessels diameters and RBC velocities. This was alleviated by pre- and post-ischemic PACE conditioning which improved functional capillary density and stimulated angiogenesis as confirmed by up-regulated VEGF expression. Furthermore, post-ischemic PACE conditioning correlated with decreased expression of early proinflammatory factors (iNOS, CCL2, CXCL5). Both types of PACE conditioning ameliorated deleterious effect of ischemia-reperfusion injury on microcirculatory hemodynamics of muscle flaps.
authors.affiliation: Department of Plastic Surgery, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Krokowicz
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Klimczak
authors.forename: Joanna
authors.initials: J
authors.lastname: Cwykiel
authors.forename: Mariusz
authors.initials: M
authors.lastname: Mielniczuk
authors.forename: Christopher
authors.initials: C
authors.lastname: Grykien
authors.forename: Maria
authors.initials: M
authors.lastname: Siemionow
mesh_terms: D042501:Angiogenic Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001783:Blood Flow Velocity; D002196:Capillaries / Q000378:metabolism / Q000503:physiopathology*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D006439:Hemodynamics; D019220:High-Energy Shock Waves; D018836:Inflammation Mediators / Q000378:metabolism; D036904:Leukocyte Rolling; D008297:Male; D008833:Microcirculation; D018715:Microscopy, Video; D009132:Muscles / Q000098:blood supply* / Q000601:surgery*; D018919:Neovascularization, Physiologic / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D012039:Regional Blood Flow; D015427:Reperfusion Injury / Q000209:etiology / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy*; D013524:Surgical Flaps / Q000009:adverse effects*; D013997:Time Factors; D057705:Transendothelial and Transepithelial Migration
chemical_list: D042501:Angiogenic Proteins; D015415:Biomarkers; D018836:Inflammation Mediators