Reconstruction of Perineal Defects: A Comparison of the Myocutaneous Gracilis and the Gluteal Fold Flap in Interdisciplinary Anorectal Tumor Resection.

Introduction: Resection of anorectal malignancies may result in extensive perineal/pelvic defects that require an interdisciplinary surgical approach involving reconstructive surgery. The myocutaneous gracilis flap (MGF) and the gluteal fold flap (GFF) are common options for defect coverage in this area. Here we report our experience with the MGF/GFF and compare the outcome regarding clinical key parameters. Methods: In a retrospective chart review, we collected data from the Department of Plastic Surgery of the University of Freiburg from December 2008-18 focusing on epidemiological, oncological, and therapy-related data including comorbidities (ASA Classification) and peri-/postoperative complications (Clavien-Dindo-System). Results: Twenty-nine patients were included with a mean follow-up of 17 months. Of the cases, 19 (65.5%) presented with recurrent disease, 21 (72.4%) received radiochemotherapy preoperatively, 2 (6.9%) received chemotherapy alone. Microscopic tumor free margins were achieved in 25 cases (86.2%). 17 patients (7 men, 10 women, rectal adenocarcinoma n = 11; anal squamous cell carcinoma n = 6; mean age 58.5 ± 10.68, mean BMI 23.1, mean ASA score 2.8) received a MGF (unilateral n = 10; bilateral n = 7). Twelve patients (7 men, 5 women, rectal adenocarcinoma n = 7; anal squamous cell carcinoma n = 4, proctodeal gland carcinoma n = 1, mean age 66.2 ± 9.2, mean BMI 23.6, mean ASA score 2.6) received coverage with a GFF (unilateral n = 4; bilateral n = 8). Mean operation time of coverage was 105 ± 9 min for unilateral and 163 ± 11 for bilateral MGFs, 70 ± 13 min for unilateral and 107 ± 14 for bilateral GFFs. Complications affected 62%. There was no significant difference in the complication rate between the MGF- and GFF-group. Complications were mainly wound healing disorders that did not extend the hospital stay. No flap loss and no complication that lead to long-lasting disability was documented (both groups). Pain-free sitting took more time in the GFF-group due to the location of the donor site. Conclusion: MG-flaps and GF-flaps prove to be reliable and robust techniques for perineal/pelvic reconstruction. Though flap elevation is significantly faster for GF-flaps, preoperative planning and intraoperative Doppler confirmation are advisable. With comparable complication rates, we suggest a decision-making based on distribution of adipose tissue for dead space obliteration, intraoperative patient positioning, and perforator vessel quality/distribution.

A Conformational Change in C-Reactive Protein Enhances Leukocyte Recruitment and Reactive Oxygen Species Generation in Ischemia/Reperfusion Injury.

Introduction: C-reactive protein circulates as a pentameric protein (pCRP). pCRP is a well-established diagnostic marker as plasma levels rise in response to tissue injury and inflammation. We recently described pro-inflammatory properties of CRP, which are mediated by conformational changes from pCRP to bioactive isoforms expressing pro-inflammatory neo-epitopes [pCRP* and monomeric C-reactive protein (mCRP)]. Here, we investigate the role of CRP isoforms in renal ischemia/reperfusion injury (IRI). Methods: Rat kidneys in animals with and without intraperitoneally injected pCRP were subjected to IRI by the time of pCRP exposure and were subsequently analyzed for monocyte infiltration, caspase-3 expression, and tubular damage. Blood urea nitrogen (BUN) was analyzed pre-ischemia and post-reperfusion. CRP effects on leukocyte recruitment were investigated via intravital imaging of rat-striated muscle IRI. Localized conformational CRP changes were analyzed by immunohistochemistry using conformation specific antibodies. 1,6-bis(phosphocholine)-hexane (1,6-bisPC), which stabilizes CRP in its native pentameric form was used to validate CRP effects. Leukocyte activation was assessed by quantification of reactive oxygen species (ROS) induction by CRP isoforms ex vivo and in vitro through electron spin resonance spectroscopy. Signaling pathways were analyzed by disrupting lipid rafts with nystatin and subsequent ROS detection. In order to confirm the translational relevance of our findings, biopsies of microsurgical human free tissue transfers before and after IRI were examined by immunofluorescence for CRP deposition and co-localization of CD68+ leukocytes. Results: The application of pCRP aggravates tissue damage in renal IRI. 1,6-bisPC reverses these effects via inhibition of the conformational change that leads to exposure of pro-inflammatory epitopes in CRP (pCRP* and mCRP). Structurally altered CRP induces leukocyte-endothelial interaction and induces ROS formation in leukocytes, the latter can be abrogated by blocking lipid raft-dependent signaling pathways with Nystatin. Stabilizing pCRP in its native pentameric state abrogates these pro-inflammatory effects. Importantly, these findings are confirmed in human IRI challenged muscle tissue. Conclusion: These results suggest that CRP is a potent modulator of IRI. Stabilizing the native pCRP conformation represents a promising anti-inflammatory therapeutic strategy by attenuation of leukocyte recruitment and ROS formation, the primary pathomechanisms of IRI.

Comparison of symmetry after smile reconstruction for flaccid facial paralysis with combined fascia lata grafts and functional gracilis transfer for static suspension or gracilis transfer alone.

PURPOSE: Facial paralysis has a profound impact on functionality and esthetics of the oral region. In patients with strong skin laxity and soft tissue ptosis, functional smile reconstruction is challenging due to the accentuated asymmetry at rest. Thus, the purpose of the study was to analyze facial symmetry in this patient clientele following a combination of dynamic reanimation with fascial strips for static suspension compared to functional gracilis transfer alone. METHODS: In 2014, we altered the single-stage approach for microsurgical smile reconstruction in patients with significant soft tissue ptosis by adding fascia lata grafts for static support. We evaluated 6 patients (mean age 57.8 ± 5.2, group A) who underwent the combined procedure, and compared their results to 6 patients with flaccid facial paralysis who were treated before 2014 and received a functional gracilis transfer alone (mean age 52.5 ± 7.5, group B). To test the efficacy of the technique, we retrospectively analyzed the correction of the oral asymmetry as well as nasal and philtral deviation by computer-assisted photograph analysis 6 months postoperatively. RESULTS: The comparative analysis revealed a significant postoperative improvement of the oral asymmetry (A: 90.0 ± 5.0% relative correction at rest vs. B: 62.6 ± 17.2%, P < .05), nasal (A: 0.4 ± 0.2 vs. B: 0.7 ± 0.4 mm, P < .05), and philtral deviation (A: 0.5 ± 0.6 vs. B: 2.8 ± 1.8 mm, P < .05) in group A. CONCLUSIONS: The combined procedure for dynamic facial reanimation allows for immediate correction of the oral asymmetry and improves overall outcome in patients with advanced soft tissue ptosis and oral asymmetry at rest.

[Results After Distal Digital Replantation - Is It Worth The Effort?] / Ergebnisse nach Fingerendgliedreplantation ­ Ist der Aufwand gerechtfertigt?

There are only relative indications for distal digital replantation in zones 1 and 2 according to Tamai. In contrast to primary closure for fingertip amputations, replantation is a complex procedure that requires skills in supermicrosurgical techniques, as vessels with diameters between 0.3-0.8 mm are connected. In addition the time spent in hospital and the time off from work are longer. Distal digital replantation is thus only indicated, if the expected functional and aesthetic benefits surmount those of primary closure. We retrospectively analysed all fingertip amputations in zone 1 and 2 according to Tamai between 9/2009 and 7/2014 where we attempted distal digital replantation. The success of replantation, wound healing and functional results were evaluated according to Yamano. We performed 11 distal digital replantations in the study period. There were 6 total amputations, 4 subtotal amputations and 1 avulsion of the digital pulp. Revascularisation with long-term reattachment of the amputated tissues was possible in 8 cases (73%). In 3 cases (27%) secondary amputation closure was necessary. The mean operating time was 3 h 56 min. 6 patients, which had a successful replantation, were available for follow-up examinations after a mean period of 19 months. 5 patients were satisfied with the result and would again prefer replantation over primary amputation closure. 4 patients reported a good function of the replanted digits and did not complain about any limitations in their use. 2 patients complained about restricted function. All patients could return to their previous places of employment and were free of pain. Of the 12 affected digital nerves 11 nerves had a 2-point discrimination (2-PD) of ≤15 mm, 3 of them had a 2-PD between 7 and 10 mm and 4 of them of <6 mm. Soft tissue atrophy was obvious in 3 replanted digits and nail deformities in 2 patients. Distal digital replantation is complex and technically challenging. It leads to high patient satisfaction with only minimal functional limitations, if successful. Due to the good results that can be obtained by these procedures, fingertip replantation should be attempted, if operative risks are minimal and if requested by the patient.

Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites.

C-reactive protein (CRP) concentrations rise in response to tissue injury or infection. Circulating pentameric CRP (pCRP) localizes to damaged tissue where it leads to complement activation and further tissue damage. In-depth knowledge of the pCRP activation mechanism is essential to develop therapeutic strategies to minimize tissue injury. Here we demonstrate that pCRP by binding to cell-derived microvesicles undergoes a structural change without disrupting the pentameric symmetry (pCRP*). pCRP* constitutes the major CRP species in human-inflamed tissue and allows binding of complement factor 1q (C1q) and activation of the classical complement pathway. pCRP*-microvesicle complexes lead to enhanced recruitment of leukocytes to inflamed tissue. A small-molecule inhibitor of pCRP (1,6-bis(phosphocholine)-hexane), which blocks the pCRP-microvesicle interactions, abrogates these proinflammatory effects. Reducing inflammation-mediated tissue injury by therapeutic inhibition might improve the outcome of myocardial infarction, stroke and other inflammatory conditions.

Microsurgical reconstruction of oncological scalp defects in the elderly.

The incidence of scalp tumors requiring radical excision increases with age. Free flap surgery is the standard reconstructive option for large defects; however, there is an ongoing uncertainty about its safety in the elderly. We conducted a review of data and report on 19 patients aged ≥75 years and 13 < 75 years. Data regarding patient demographics, types of tumors, surgery, size of defect, flaps used, and prevalence of complications were analyzed. The patients in the elderly group had more accompanying medical conditions and a higher ASA score. Squamous cell carcinoma was the predominant tumor in the elderly and sarcoma in the younger patients. The defect size was similar in both groups. The latissimus dorsi (LD) and the anterolateral thigh (ALT) flaps were flaps of first choice, with temporal vessels most commonly used for anastomosis. Surgery lasted longer in the younger patients. There was no difference in the duration of hospital stay. No significant correlation was found between age and the flap-related or medical complications. Revision surgery was more often required in the younger patients. We conclude that free flap surgery is safe and reliable in the elderly population. The LD and the ALT are the most commonly used flaps. Advanced age should not be considered a risk factor for free flap surgery in these patients.

MicroRNA-155 aggravates ischemia-reperfusion injury by modulation of inflammatory cell recruitment and the respiratory oxidative burst.

The inflammatory sequelae of ischemia-reperfusion injury (IRI) are a major causal factor of tissue injury in various clinical settings. MicroRNAs (miRs) are short, non-coding RNAs, which regulate protein expression. Here, we investigated the role of miR-155 in IR-related tissue injury. Quantifying microRNA-expression levels in a human muscle tissue after IRI, we found miR-155 expression to be significantly increased and to correlate with the increased expression of TNF-α, IL-1ß, CD105, and Caspase3 as well as with leukocyte infiltration. The direct miR-155 target gene SOCS-1 was downregulated. In a mouse model of myocardial infarction, temporary LAD ligation and reperfusion injury resulted in a smaller area of necrosis in miR-155-/- animals compared to wildtype animals. To investigate the underlying mechanisms, we evaluated the effect of miR-155 on inflammatory cell recruitment by intravital microscopy and on the generation of reactive oxygen species (ROS) of macrophages. Our intravital imaging results demonstrated a decreased recruitment of inflammatory cells in miR-155-/- animals during IRI. The generation of ROS in leukocytic cells of miR-155-/- animals was also reduced. RNA silencing of the direct miR-155 target gene SOCS-1 abrogated this effect. In conclusion, miR-155 aggravates the inflammatory response, leukocyte infiltration and tissue damage in IRI via modulation of SOCS-1-dependent generation of ROS. MiR-155 is thus a potential target for the treatment or prevention of IRI.

Salvage procedures after failed facial reanimation surgery using the masseteric nerve as the motor nerve for free functional gracilis muscle transfer.

IMPORTANCE: Free muscle transfer innervated by a cross-facial nerve graft represents the criterion standard for smile reconstruction in facial paralysis. If primary reconstruction fails, a second muscle transfer is usually needed. Herein, we investigated the possibility of avoiding a second free muscle transfer by in situ coaptation of the gracilis muscle to the masseteric nerve. OBSERVATIONS: We report a series of 3 failed free muscle transfers for facial reanimation among 21 free flap transfers performed for facial reanimation between March 2008 and August 2013. To salvage the muscle, we performed coaptation of the neural pedicle from the cross-facial nerve graft to the masseteric nerve. This method allows for leaving the fixation sutures of the muscle at the oral commissure in place. All patients showed muscle contraction after 3 months and a smile with open mouth after 6 months. No significant difference in the range of commissure excursion was observed between the healthy and operated sides. CONCLUSIONS AND RELEVANCE: Recoaptation of the neural pedicle from the cross-facial nerve graft to the masseteric nerve, leaving the muscle transplant in place, is a suitable salvage procedure after unsuccessful reconstruction with a cross-facial nerve graft, avoiding a second free muscle transfer. LEVEL OF EVIDENCE: 4.

Dissociation of pentameric to monomeric C-reactive protein localizes and aggravates inflammation: in vivo proof of a powerful proinflammatory mechanism and a new anti-inflammatory strategy.

BACKGROUND: The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. METHODS AND RESULTS: We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. CONCLUSIONS: These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2-dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy.

A conformational change of C-reactive protein in burn wounds unmasks its proinflammatory properties.

Tissue damage in burn injury leads to a rapid increase of leukocytes and acute phase reactants. Plasma levels of C-reactive protein (CRP) rise within hours after the insult. No deficiency of this protein has been reported in humans, suggesting it plays a pivotal role in innate immunity. CRP in circulation is composed of five identical subunits [pentameric CRP (pCRP)]. Recently, deposits of structurally modified CRP (mCRP) have been found in inflammatory diseases. Little is known about this structural change and how it affects CRP functions. We analyzed CRP deposits in burn wounds and serum by immunohistochemistry, western blot and dot blot analysis. CRP was deposited in necrotic and inflamed tissue, but not in adjacent healthy tissue. Tissue deposited CRP was detected by mCRP-specific antibodies and structurally different from serum pCRP. mCRP but not pCRP induced reactive oxygen species production by monocytes and facilitated uptake of necrotic Jurkat cells by macrophages. In addition, it accelerated migration of keratinocytes in a scratch wound assay. The structural changes that occur in pCRP upon localization to damaged and inflamed tissue in burn wounds result in a functionally altered protein with distinct functions. mCRP exhibits opsonic, proinflammatory and promigratory properties which modulate wound healing.

Pentameric CRP attenuates inflammatory effects of mmLDL by inhibiting mmLDL--monocyte interactions.

Previous studies have reported that C-reactive protein (CRP) interacting with low-density lipoproteins (LDL) affects macrophage activation and LDL uptake. However, the physiological relevance of CRP-LDL interaction with circulating monocytes remains elusive. Moreover, recent studies have shown that CRP exists in two isoforms with partly opposing characteristics pentameric (pCRP) and monomeric CRP (mCRP). Here we investigated the effects of CRP interacting with minimally modified low-density lipoprotein (mmLDL) interaction in regard to events involved in formation of atherosclerotic plaque. We analyzed the effect of mmLDL on human monocytes and found a substantial increase in monocyte activation as evaluated by CD11b/CD18 expression and increased monocyte adhesion under static and under shear flow conditions to human endothelial cells. Monocyte adhesion and activation was attenuated by pCRP via the prevention of mmLDL binding to monocytes. These anti-inflammatory properties of pCRP were lost when it dissociates to the monomeric form. Our results elucidate the physiological relevance of the CRP-mmLDL interaction and furthermore confirm the importance of the previously described pCRP dissociation to mCRP as a localized inflammatory "activation" mechanism.

Real-time digital imaging of leukocyte-endothelial interaction in ischemia-reperfusion injury (IRI) of the rat cremaster muscle.

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery. Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion. Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor. While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM). Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968. Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality. We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method. In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

C-reactive protein: how conformational changes influence inflammatory properties.

Recent evidence suggests that the prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP belongs to the family of pentraxins and as such consists of five identical non-covalently linked subunits. Contradictory data on the characteristics of CRP as either being pro- or anti-inflammatory may be explained by the existence of two conformations of the protein: the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. In vitro both isoforms exhibit a very distinct inflammatory profile. We recently identified a localized, physiologically relevant pCRP dissociation mechanism by activated platelets and apoptotic cells and showed the deposition of mCRP in inflamed tissue. Here we review the literature on the causal role of pCRP and mCRP in the light of our findings and critically analyze the current controversies around CRP. The novel understanding of the localized dissociation of circulating pentameric CRP to the distinctively pro-inflammatory monomeric CRP allows for a new view on CRP in inflammatory reactions and further highlights mCRP and the pCRP dissociation process as a potential therapeutic target.