Mariusz Mielniczuk

Pulsed acoustic cellular treatment induces expression of proangiogenic factors and chemokines in muscle flaps.

BACKGROUND Pulsed Acoustic Cellular Expression (PACE) treatment is a novel technology with potential to improve tissue perfusion, but the mechanism of this action is unknown. We assessed in vivo the effect of PACE therapy on muscle microcirculatory hemodynamics, neovascularization, and proangiogenic and proinflammatory gene expression. METHODS Cremaster muscles were prepared for standard intravital microscopy in 42 Lewis rats divided into five groups: (1) control (n = 10); acute PACE treatment 15 minutes before surgery with (2) 200 impulses (n = 8) and (3) 500 impulses (n = 8); and PACE treatment 24 hours before surgery with (4) 200 impulses (n = 8) and (5) 500 impulses (n = 8).Microcirculatory hemodynamics of red blood cell velocity and capillary perfusion were recorded for 4 hours. Gene expression levels of proinflammatory (inductible nitric oxide synthase [iNOS]) and proangiogenic factors (endothelial nitric oxide synthase [eNOS], vascular endothelial growth factor [VEGF], chemokine (C-X-C motif) ligand 5 [CXCL5], chemokine (C-C motif) ligand 2 [CCL2], and chemokine (C-C motif) receptor 2 [CCR2] were measured using Taqman real-time Polymerase Chain Reaction (PCR). Immunohistochemistry assessed expression of proangiogenic factors: VEGF, von Willebrand factor (vWF), and vessel density by CD31. RESULTS PACE treatment resulted in an increase of arteriolar diameters in acute groups 2 and 3 (p < 0.05). In group 5, vessel densities assessed by CD31, VEGF, and vWF expression increased significantly 24 hours after PACE treatment compared with control (p < 0.05). PACE application downregulated proinflammatory iNOS gene expression and upregulated proangiogenic genes expression of eNOS, VEGF, CXCL5, and CCL2. CONCLUSIONS Application of PACE treatment, applied as short time acting preconditioning and conditioning treatment, resulted in upregulation of proangiogenic chemokines gene expression in the muscle and showed upregulation of expression of proangiogenic factors such as VEGF and vWF on the vessel endothelium.

Pulsed acoustic cellular expression as a protective therapy against I/R injury in a cremaster muscle flap model

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.

Long-term follow up of the effects of extracorporeal shockwave therapy (ESWT) on microcirculation in a denervated muscle flap

UNLABELLED Extracorporeal Shock Wave Therapy (ESWT) is a golden standard for treatment of kidney and urinary calculi. It is also widely used in a number of orthopedic pathologies and other fields of medicine. Although clinical success the exact mechanism of shock wave technology is not well established. Cremaster muscle model used in our experiment is structurally and functionally similar to other skeletal muscles (striated muscle). The aim of the study was to evaluate influence of ESWT treatment on microcirculation and leukocyte-endothelial interactions after longer time period post ESWT application. MATERIAL AND METHODS In experiment we used 34 Lewis rats weighting 125-160 grams. Animals were divided into 4 groups--Group 1 (n = 10) control, without ESWT application, group 2 (n = 8), in which measurements were performed 3 days after application of 500 impulses of ESWT; group 3 (n = 8) in which measurements were performed 7 days after application of 500 impulses of ESWT; group 4 (n = 8), in which measurements were performed 21 days after application of 500 impulses of ESWT. RESULTS The experiment showed a decrease in functional capillaries activity, we also observed the reduction in leukocyte rolling over the endothelium and an increase in flow velocity in V1 venules. CONCLUSIONS ESWT therapy after 3, 7 and 21 days decreases inflammatory process in the muscle, the other of its effect is weakened. This confirms that the treatment had a positive effect if ESWT is applied repeatedly, because only in this case a wave maintains its beneficial effects.

Ringer's lactate solution enhances the inflammatory response during fluid resuscitation of experimentally induced haemorrhagic shock in rats

Introduction Hemorrhagic shock leads to systemic oxygen deficit (hypoxaemia) that results in systemic inflammatory response syndrome (SIRS), a recognised cause of late mortality in this case. The aim of this study was to analyse the impact of fluid resuscitation, using two Ringer solutions, on the microcirculation changes that take place during experimentally induced haemorrhagic shock. Material and methods A model of the rat cremaster muscle was used to assess microcirculation in vivo. The experimental groups (n = 10 each) included: control (CTRL); shock (HSG); Ringer’s acetate (RAG); and Ringer’s lactate (RLG). Microhaemodynamic parameters were measured during the experiment. Results A statistically significantly higher level of leukocytes, both those attached to the endothelium and those located in the extravascular space (p < 0.05), was reported in the lactate Ringer (LR) group compared with the AR group. There were significant differences in the activity of A3 arterioles compared with A1 and A2 arterioles. Ringer’s lactate solution seemed to the inflammation response during fluid resuscitation from haemorrhagic shock. A3 arterioles are likely to play a role as a pre-capillary sphincter in the skeletal muscle. Conclusions The present study revealed that fluid resuscitation with Ringer’s lactate solution exacerbates inflammation in the skeletal muscle. It is worth noting that Ringer’s acetate solution reduces local inflammation and could therefore be recommended as the “first line” crystalloid of the fluid resuscitation during haemorrhagic shock.

Microcirculation and Pace Therapy

Pulsed Acoustic Cellular Expression (PACE) is a novel technology utilizing Extracorporeal Shock Waves (ESW) in as a source of acoustic energy in a pulsed manner capable of delivering a cellular expression response. It has multiple indications in medicine but exact mechanism of PACE remains unknown. In our experiment in a large group of animals we checked influence of PACE on microcirculation in a well-known model of rats’ cremaster muscle. Results revealed that PACE therapy increases circulation in cremaster muscle and causes neovascularization and angiogenesis. It also has anti-inflammatory effect on the muscle. To achieve desired results PACE therapy has to be used periodically.

Cremaster Muscle and Effect of Different Anesthetics

Multiple studies investigating the effect of anesthetics on peripheral and systemic microcirculation indicate that the choice of anesthetic during anesthesia affects basic mechanisms of microcirculation regulation. This issue concerns surgical procedures lasting many hours and microsurgical procedures of organ and tissue transplantation in particular.