Mickaël Tobalem

Nonactivated versus thrombin-activated platelets on wound healing and fibroblast-to-myofibroblast differentiation in vivo and in vitro.

Background: Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing. Methods: The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control. Results: Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced two-fold higher myofibroblast differentiation and contraction compared with activated platelets. Conclusions: Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered “on demand” rather than in an initial bolus.

Erythropoietin-induced upregulation of endothelial nitric oxide synthase but not vascular endothelial growth factor prevents musculocutaneous tissue from ischemic damage

Recent findings have attested the protective effects of erythropoietin (EPO) in ischemically challenged organs. We therefore aimed at elaborating the underlying mechanism of EPO-mediated protection in musculocutaneous tissue undergoing persistent ischemia after acute injury. Mice were assigned to five experimental groups equipped with a randomly perfused flap fixed in a dorsal skinfold chamber, whereas the sixth group did not undergo flap preparation: EPO, L-Name, EPO and L-Name, EPO and bevacizumab, untreated flap, and nonischemic chamber (control). Intravital fluorescence microscopic analysis of microhemodynamics, apoptotic cell death, macromolecular leakage and angiogenesis was carried out over a 10-day period. Further, immunohistochemical analysis was used to study the protein expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF). Increased expression of eNOS in EPO-administered mice correlated with significant arteriolar dilation and thus increased blood flow resulting in a maintained functional capillary density (FCD) at day 10. In addition, EPO induced a VEGF upregulation, which was associated with newly formed capillaries. In addition, EPO was able to reduce ischemia-induced apoptotic cell death and finally to significantly reduce flap necrosis. In contrast, coadministration of L-Name abolished EPO-mediated tissue protection by abrogating the dilatory effect resulting in reduced FCD and tissue survival, without counteracting angiogenesis and apoptotic cell death, whereas additional administration of bevacizumab did not influence the beneficial effect of EPO on flap survival despite abrogating angiogenesis. Macromolecular leakage was found to be increased in all treatment groups. This study shows that EPO administration prevents musculocutaneous tissue from ischemic necrosis as a consequence of an eNOS-dependent arteriolar hyperperfusion maintaining capillary perfusion, thus representing a promising approach to pharmacologically protect ischemically challenged tissue.

Images in clinical medicine. Evolution of a diabetic foot infection.

An obese 50-year-old man with no known medical history presented with a necrotizing infection of his right foot that had begun 10 days previously with lesions that he attributed to wearing new shoes. He was found to have diabetes with peripheral neuropathy.

Hyperglycemia Increases Susceptibility to Ischemic Necrosis

Diabetic patients are at risk for spontaneous foot ulcers, chronic wounds, infections, and tissue necrosis. Current theories suggest that the development and progression of diabetic foot ulcers are mainly caused by arteriosclerosis and peripheral neuropathy. Tissue necrosis plays a primordial role in the progression of diabetic foot ulcers but the underlying mechanisms are poorly understood. The aim of the present study was to investigate the effects of hyperglycemia per se on the susceptibility of ischemic tissue to necrosis, using a critical ischemic hind limb animal model. We inflicted the same degree of ischemia in both euglycemic and streptozotocin-induced hyperglycemic rats by resecting the external iliac, the femoral, and the saphenous arteries. Postoperative laser Doppler flowmetry of the ischemic feet showed the same degree of reduction in skin perfusion in both hyperglycemic and euglycemic animals. Nevertheless, we found a significantly higher rate of limb necrosis in hyperglycemic rats compared to euglycemic rats (71% versus 29%, resp.). In this study, we revealed that hyperglycemia per se increases the susceptibility to limb necrosis in ischemic conditions. Our results may help to better understand the physiopathology of progressive diabetic wounds and underline the importance of strict glycemic control in patients with critical limb ischemia.

Secondary burn progression decreased by erythropoietin.

Objective:To investigate whether systemic erythropoietin administration can prevent secondary burn progression in an experimental model and to elucidate the underlying mechanisms. Design:Prospective study. Setting:University-based laboratory research. Subjects:Twenty-one male Wistar rats. Interventions:The burn comb model creates four rectangular burned surfaces that are intercalated by three unburned zones (interspaces) prone to secondary necrosis. Twenty-one animals were randomized to three experimental groups: 1) Local cooling with water for 20 minutes (control, 17°C); 2) and 3) local cooling with water and intraperitoneal erythropoietin once a day for five days starting 45 minutes after burn injury (500 IU/kg body weight: EPO 500 or 2500 IU/kg body weight: EPO 2500). Measurements and Main Results:Secondary burn progression—both in depth (histology) and in surface (planimetry)—as well as interspace perfusion (laser Doppler flowmetry) and hematocrit were analyzed. Further, dilatory response (inducible nitric oxide synthase expression), inflammation (leukocyte count), and angiogenesis (CD31 expression) were assessed. Finally, wound healing time and contracture rate were reported. Burn progression resulted in complete dermal destruction as well as in important interspace necrosis in control animals, whereas burn progression was significantly reduced in a dose-dependent manner in animals treated with erythropoietin. Tissue protection was associated with an increased interspace perfusion with EPO 500, but not with EPO 2500, and was paralleled by a significant increase in inducible nitric oxide synthase expression and decreased inflammation, independent of the erythropoietin dosage. EPO 2500 led to a significant increase of hematocrit at day 4. Finally, faster wound healing and less contracture were observed in animals treated with EPO 500 only. Conclusions:Erythropoietin represents an easy-to-use therapeutic approach to prevent secondary burn progression, i.e., to control damage after burn injury. It preserves microcirculatory perfusion within the endangered areas in a dose-dependent manner.

First-aid with warm water delays burn progression and increases skin survival

INTRODUCTION First aid treatment for thermal injuries with cold water removes heat and decreases inflammation. However, perfusion in the ischemic zone surrounding the coagulated core can be compromised by cold-induced vasoconstriction and favor burn progression. The aim of this study is to evaluate the effect of local warming on burn progression in the rat comb burn model. METHODS 24 male Wistar rats were randomly assigned to either no treatment (control) or application of cold (17 °C) or warm (37 °C) water applied for 20 min. Evolution of burn depth, interspace necrosis, and microcirculatory perfusion were assessed with histology, planimetry, respectively with Laser Doppler flowmetry after 1 h, as well as 1, 4, and 7 days. RESULTS Consistent conversion from a superficial to a deep dermal burn within 24 h was obtained in control animals. Warm and cold water significantly delayed burn depth progression, however after 4 days the burn depth was similar in all groups. Interspace necrosis was significantly reduced by warm water treatment (62±4% vs. 69±5% (cold water) and 82±3% (control); p<0.05). This was attributed to the significantly improved perfusion after warming, which was present 1 h after burn induction and was maintained thereafter (103±4% of baseline vs. 91±3% for cold water and 80±2% for control, p<0.05). CONCLUSION In order to limit damage after burn injury, burn progression has to be prevented. Besides delaying burn progression, the application of warm water provided an additional benefit by improving the microcirculatory perfusion, which translated into increased tissue survival.

Risks and benefits of using an absorbable mesh in one-stage immediate breast reconstruction: a comparative study

Background: Complete implant coverage by pectoralis major muscle in immediate breast reconstruction prevents implant exposure but restricts implant volume, increases the risk of a high-riding implant, and prevents natural ptosis. The authors hypothesized that extension of the subpectoral pocket with Vicryl mesh may produce more satisfactory results. The safety and efficiency of this technique were assessed by comparing complication rates and clinical results of the two reconstruction techniques: with and without mesh. Methods: The authors reviewed 161 immediate breast reconstructions in 139 patients from 2002 to 2010. A complete submuscular pocket was performed in 46 breasts and a partial submuscular pocket with Vicryl mesh extension was performed in 115 breasts. Complications, surgical revisions rates, implant size, and contralateral breast procedures were compared between the two groups. Results: Early and late surgical revisions were similar between the mesh and nonmesh groups (11.6 percent versus 4.3 percent at 90 days, p = 0.09; and 34.3 percent versus 41.4 percent at 5 years, p = 0.41). There were fewer revisions for implant malposition in the mesh group (8.9 percent versus 21.7 percent, p = 0.05). Patients with mesh could have a larger implant in comparison with those without mesh (329 g versus 284 g, p = 0.01) and had fewer contralateral mastopexies (12 percent versus 30 percent, p = 0.01). Conclusions: Larger implants and perhaps better control of implant position were possible using the Vicryl mesh extension without increasing complications. Because the mesh technique also recreates a slightly ptotic breast, fewer contralateral mastopexies were needed. The Vicryl mesh extension is a low-cost alternative to biological matrices or tissue expanders. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Erythropoietin in the prevention of experimental burn progression

Damage control is essential in first aid of burn lesions. The aim of the present study was to investigate whether systemic erythropoietin (EPO) administration could prevent secondary burn progression in an experimental model.

Hyperglycemia Interacts with Ischemia in a Synergistic Way on Wound Repair and Myofibroblast Differentiation.

Background: Hyperglycemia is known to adversely affect the outcome of ischemic insults, but its interaction with ischemia has not been investigated in wound repair yet. In this study, we develop a new animal model allowing to investigate the interaction between hyperglycemia and ischemia during the wound repair process. We focus on myofibroblast differentiation, a key element of wound repair. Methods: Ischemia was inflicted in Wistar rats by resection of the femoral to popliteal arteries on the left side, whereas arteries were dissected without resection on the right side. Full-thickness skin wounds (1 cm2) were created on both feet. Hyperglycemia was induced by injection of streptozotocin. Normoglycemic animals served as control (n = 23/group). Blood flow, wound closure, and myofibroblast expression were measured. Results: Wound closure was significantly delayed in ischemic compared with nonischemic wounds in all rats. This delay was almost 5-fold exacerbated in hyperglycemic rats compared with normoglycemic rats, while hyperglycemia alone showed only a slight effect on wound repair. Delayed wound repair was associated with impaired wound contraction and myofibroblast differentiation. Conclusions: Our model allows to specifically quantify the effect of hyperglycemia and ischemia alone or in combination on wound repair. We show that hyperglycemia amplifies the inhibitory effect of ischemia on wound repair and myofibroblast expression. Our data reveal for the first time the synergic aspect of this interaction and therefore stress the importance of a strict glycemic control in the management of ischemic wounds.

Local shockwave-induced capillary recruitment improves survival of musculocutaneous flaps

BACKGROUND Shockwave (SW) application has been shown to limit flap necrosis. However, the underlying microhemodynamic mechanisms remain unclear. Therefore, the objective of this study was to analyze the effect of SW application on a microcirculatory level. METHODS We treated 12 C57BL/6 mice with local SW application (500 shockwave impulses at 0.15 mJ/mm(2)) either 24 h before (preconditioning [PRE]) or 30 min after (postconditioning [POST]) flap elevation. Animals with an untreated flap (CON) or without a flap served as controls. We applied dorsal skinfold chambers to the animals and performed epifluorescence microscopy over a 10-d period to assess microcirculatory parameters (arteriolar diameter, red blood cell velocity, blood flow, functional capillary density, and intercapillary distance) as well as inflammation, apoptotic cell death, and necrosis. RESULTS SW application significantly decreased tissue necrosis independently of the application time point (PRE: 29% ± 7%; POST: 25% ± 7% versus CON: 47% ± 2%; day 10, P < 0.05). Arteriolar diameter, red blood cell velocity, and blood flow were not statistically significantly different among the 3 flap groups. However, SW (PRE and POST) resulted in an early and persistent increase in functional capillary density and consequently decreased intercapillary distance compared with CON and the group without a flap (P < 0.05). Also, SW resulted in a significantly decreased inflammatory response (P < 0.05) and induced an angiogenic response, as indicated by new functional microvessel formation observed 5 d after therapy. CONCLUSIONS Local SW application improved tissue survival by recruitment of sleeping capillaries within the non ischemic tissue and maintenance of capillary perfusion within the critically perfused tissue after induction of ischemia, which was independent of the application time point. Neoangiogenesis occurred beyond the ischemic tolerance of the tissue, and therefore does not seem to contribute to improved tissue survival.