Christian Witzel

Second intercostal internal mammary artery perforator (IMAP) fasciocutaneous flap as an alternative choice for the treatment of deep sternal wound infections (DSWI)

Sternal wound infections after sternotomy are associated with high morbidity, high mortality and escalating treatment costs. Repeated radical debridement - with the removal of any hardware - and wound conditioning are the prerequisites for reconstruction. Muscle and, less frequently, omentum flaps are usually used for reconstruction. However, these flaps are associated with considerable donor-site morbidity, long operation times and aesthetic impairment. Fasciocutaneous flaps seem to be an alternative. This study presents our experience of using the second intercostal mammary artery fasciocutaneous perforator flap for defect closure in nine patients (mean age: 70.2 years). Following a retrospective chart review, we assessed data on patient demographics, the type of cardiac surgery, the prevalence of deep sternal wound infection (DSWI) risk factors, identified pathogens, surgery duration, hospitalization tim patients had undergone coronary artery bypass surgery, and two had valve replacements. The mean duration of surgery (121.4 ± 39 min) was short. The patients had a mean body mass index (BMI) of 32.8 ± 4.9 kg/m(2). An average flap size of 124 ± 22 cm(2) sufficiently covered and obliterated each defect. One mediastinal haematoma required revision surgery. One wound dehiscence at the flap and two at the donor site were managed conservatively. Our experience reveals that a fasciocutaneous flap based on the second intercostal perforator of the internal mammary artery can be an alternative, quick-to-prepare flap for covering sternal defects. In adipose patients, it has sufficient bulk, and it is large enough to cover common sternal wounds. It also has low complication and morbidity rates, and it achieves an aesthetically pleasing result.

N-Propionylmannosamine stimulates axonal elongation in a murine model of sciatic nerve injury

Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-acyl side chain. N-Propionylmannosamine (ManNProp) increases neurite outgrowth and accelerates the reestablishment of functional synapses in vitro. We investigated the influence of systemic ManNProp application using a specific in vivo mouse model. Using mice expressing axonal fluorescent proteins, we quantified the extension of regenerating axons, the number of regenerating axons, the number of arborising axons and the number of branches per axon 5 days after injury. Sciatic nerves from non-expressing mice were grafted into those expressing yellow fluorescent protein. We began a twice-daily intraperitoneal application of either peracetylated ManNProp (200 mg/kg) or saline solution 5 days before injury, and continued it until nerve harvest (5 days after transection). ManNProp significantly increased the mean distance of axonal regeneration (2.49 mm vs. 1.53 mm; P < 0.005) and the number of arborizing axons (21% vs. 16%; P = 0.008) 5 days after sciatic nerve grafting. ManNProp did not affect the number of regenerating axons or the number of branches per arborizing axon. The biochemical glycoengineering of the N-acyl side chain of sialic acid might be a promising approach for improving peripheral nerve regeneration.

Fibrin glue repair leads to enhanced axonal elongation during early peripheral nerve regeneration in an in vivo mouse model

Microsurgical suturing is the gold standard of nerve coaptation. Although literature on the usefulness of fibrin glue as an alternative is becoming increasingly available, it remains contradictory. Furthermore, no data exist on how both repair methods might influence the morphological aspects (arborization; branching) of early peripheral nerve regeneration. We used the sciatic nerve transplantation model in thy-1 yellow fluorescent protein mice (YFP; n = 10). Pieces of nerve (1cm) were grafted from YFP-negative mice (n = 10) into those expressing YFP. We performed microsuture coaptations on one side and used fibrin glue for repair on the contralateral side. Seven days after grafting, the regeneration distance, the percentage of regenerating and arborizing axons, the number of branches per axon, the coaptation failure rate, the gap size at the repair site and the time needed for surgical repair were all investigated. Fibrin glue repair resulted in regenerating axons travelling further into the distal nerve. It also increased the percentage of arborizing axons. No coaptation failure was detected. Gap sizes were comparable in both groups. Fibrin glue significantly reduced surgical repair time. The increase in regeneration distance, even after the short period of time, is in line with the results of others that showed faster axonal regeneration after fibrin glue repair. The increase in arborizing axons could be another explanation for better functional and electrophysiological results after fibrin glue repair. Fibrin glue nerve coaptation seems to be a promising alternative to microsuture repair.

A self-made, low-cost infrared system for evaluating the sciatic functional index in mice

The sciatic functional index (SFI) is a popular parameter for peripheral nerve evaluation that relies on footprints obtained with ink and paper. Drawbacks include smearing artefacts and a lack of dynamic information during measurement. Modern applications use digitized systems that can deliver results with less analytical effort and fewer mice. However, the systems are expensive (€40,000). This study aimed to evaluate the applicability and precision of a self-made, low-cost infrared system for evaluating SFI in mice. Mice were subjected to unilateral sciatic nerve crush injury (crush group; n = 7) and sham operation (sham group; n = 4). They were evaluated on the day before surgery, the 2 nd , 4 th and 6 th days after injury, and then every day up to the 23 rd day after injury. We compared two SFI evaluation methods, i.e., conventional ink-and-paper SFI (C-SFI) and our infrared system (I-SFI). Our apparatus visualized footprints with totally internally reflected infrared light (950 nm) and a camera that can only detect this wavelength. Additionally we performed an analysis with the ladder beam walking test (LBWT) as a reference test. I-SFI assessment reduced the standard deviation by about 33 percent, from 11.6 to 7.8, and cut the variance around the baseline to 21 percent. The system thus requires fewer measurement repetitions and fewer animals, and cuts the cost of keeping the animals. The apparatus cost €321 to build. Our results show that the process of obtaining the SFI can be made more precise via digitization with a self-made, low-cost infrared system.

Electrical Nerve Stimulation Enhances Perilesional Branching after Nerve Grafting but Fails to Increase Regeneration Speed in a Murine Model.

Background Electrical stimulation immediately following nerve lesion helps regenerating axons cross the subsequently grafted nerve repair site. However, the results and the mechanisms remain open to debate. Some findings show that stimulation after crush injury increases axonal crossing of the repair site without affecting regeneration speed. Others show that stimulation after transection and fibrin glue repair doubles regeneration distance. Methods Using a sciatic-nerve-transection-graft in vivo model, we investigated the morphological behavior of regenerating axons around the repair site after unilateral nerve stimulation (20 Hz, 1 hour). With mice expressing axonal fluorescent proteins (thy1-YFP), we were able to calculate the following at 5 and 7 days: percentage of regenerating axons and arborizing axons, branches per axon, and regeneration distance and speed. Results Brief stimulation significantly increases the percentage of regenerating axons (5 days: 35.5 vs. 27.3% nonstimulated, p < 0.05; 7 days: 43.3 vs. 33.9% nonstimulated, p < 0.05), mainly by increasing arborizing axons (5 days: 49.3 [4.4] vs. 33.9 [4.1]% [p < 0.001]; 7 days: 42.2 [5.6] vs. 33.2 [3.1]% [p < 0.001]). Neither branches per arborizing axon nor regeneration speed were affected. Conclusion Our morphological data analysis revealed that electrical stimulation in this model increases axonal crossing of the repair site and promotes homogeneous perilesional branching, but does not affect regeneration speed.

N-Propionylmannosamine：using biochemical glycoengineering to promote peripheral nerve regeneration

The peripheral nervous system, in contrast to the central nervous system, is capable of spontaneous regeneration. However, nerve reconstruction in the peripheral nervous system remains a major challenge, as the functional outcomes following nerve repair are variable. Quantitative parameters such as the number of regenerating axons and degree of myelination are crucial, but correct axon target organ allocation, time to regeneration and target organ quality are also equally important. After nerve transection, the distal part of the nerve undergoes Wallerian degeneration, while the Schwann cells change from a myelinating to a regenerating phenotype and form bands of Bungner. In doing so, they determine the conditions for the elongating axons to reach a target organ. On the other side, the injured nerve also goes into regeneration mode by activating regeneration-associated genes and elongating the transected axon until it reaches a target organ. This is a complex, time-consuming process that presents the regenerating axon with several obstacles. Crossing the coaptation site is one of the first. Once it has done this, the regenerating axon has to decide which Schwann cell tube it will enter to reach a target organ. Several different behavioral patterns have been observed here. Some axons penetrate the distal nerve segment as a single axon, mainly via a lateral movement. Others arborize into several branches that enter separate Schwann cell tubes and elongate towards the target organ. In our laboratory, we found that single axons crossing the site can reach as many as 142 Schwann cell tubes after 7 days, whereas arborizing axons can reach up to 68 Schwann cell tubes in the same period (Witzel et al., 2005). It is of note that axon branches are internal competitors for the supply of energy and structural materials, and external competitors for growth factors and Schwann cell tubes. According to our unpublished data, the mean number of branches per arborizing axon is limited to an average of one to two. This internal and external competition might be a regulation process designed to single out the stable branches (Smalheiser and Crain, 1984). Thus, misdirected branches are eventually pruned back so as to achieve specificity of regeneration (Brushart et al., 1998). Sialic acid of glycoproteins and gangliosides plays an integral role in the development and regeneration of the nervous system, as well as in neural plasticity. Polysialylation of the neural cell adhesion molecule (NCAM) is an important posttranslational modification that is crucial to the development of the nervous system. Polysialylation of NCAM decreases during adulthood, but increases again after nerve injury and thus contributes to regeneration success. Several therapeutic strategies show promise for promoting the quantity, quality, and duration of peripheral nerve regeneration. The newly described metabolic glycoengineering (MGE) seems capable of stimulating nerve regeneration. MGE involves the metabolic modification of the N-acetyl side chain of N-acetylneuraminic acid (= sialic acid, Sia). The modification of Sia is achieved via a simple biochemical procedure using new unphysiological precursors of Sia. Whereas the N-acetyl group of N-acetylneuraminic acid originates from the physiological precursor N-acetylmannosamine, slight modifications of N-acetylneuraminic acid are obtained via the use of analogs of N-acetylmannosamine with elongated N-acyl side chains. In the simplest manner, it is elongated by one or more methylene groups, leading to N-propionyl derivatives, N-butanoyl derivatives or an azido group leading to N-azido Sia. The promiscuity of the enzymes that catalyze the biosynthesis of sialic acid is essential to the success of this new biochemical method; most modifications of the N-acyl side chain are tolerated (Keppler et al., 2001) (Figure 1). The biosynthetic pathway of polysialic acid and posttranscriptional modification of proteins and lipids to glycoproteins and glycolipids continues from the cytosol to the nucleus and then to the Golgi apparatus, as shown in Figure 2. The enzymes that perform polysialylation of specific glycoproteins are the two polysialyltransferases ST8SIA2 and ST8SIA4. Figure 1 Biosynthesis of N-acetylneuraminic acid. Figure 2 Neuraminic acid and nerve regeneration. The enzymes’ ability to incorporate unnatural precursors during Sia biosynthesis creates new scope for engineering N-acetylneuraminic acid to N-acylneuraminic acid in vitro and in vivo (Buttner et al., 2002). This biochemical glycoengineering has revealed unexpected biological characteristics of Sia. N-Propionylmannosamine (ManNProp), one unnatural precursor, is metabolized to the new N-propionylneuraminic acid (Neu5Prop) and replaces partially physiological N-acetylneuraminic acid (Keppler et al., 2001). Recent in vitro results showed that feeding PC12 cells with ManNProp resulted in increased neurite outgrowth, and that feeding organotypical in vitro co-cultures with ManNProp accelerated the reestablishment of functional synapses (Buttner et al., 2002). We recently found that a systemic application of ManNProp increased the distance of axonal elongation and the number of arborizing axons in a murine injury model (Witzel et al., 2015 in press). We also showed that the polysialyltransferase ST8SiaII is substantially involved in stimulating axonal elongation and arborization. Using nerve grafts from ST8SiaII–/– knockout mice in a sciatic nerve transection and transplantation mouse model, we showed that the absence of the polysialyltransferase ST8SiaII significantly reduced the regeneration distance and number of arborizing axons during early peripheral nerve regeneration (Koulaxouzidis et al., 2015). The presence of ST8SiaII in the distal nerve graft appears to be a pivotal factor in axonal extension and arborization induced by ManNProp. Others have shown that a deficiency of ST8SiaII reduced the number and size of regenerating fibers without impairing remyelination. PolySia was up-regulated by the polysialyltransferases ST8SiaII and ST8SiaIV. The cellular localization of polySia seems to be crucial for peripheral nerve regeneration (Jungnickel et al., 2009). However, several mechanisms appear to be involved in the beneficial effect that ManNProp has on nerve regeneration. Firstly, the incorporation of N-propionylneuraminic acid (Neu5Prop) and partial replacement of N-acetylneuraminic acid modulates the glycan structure of glycoproteins (such as NCAM) and gangliosides (Buttner et al., 2002; Franz et al., 2005). This is followed by differentiation of the extracellular environment and might be a further interface between regenerating axons and their environment (Brushart et al., 1998). Secondly, the resulting unnatural sialic acids lead to expression genes involved in cell differentiation, such as the transcription factors c-Jun and TOAD-64/Ulip/CRMP. These stimulate the phosphorylation of erk1/2 within the nucleus, a process that enhances the activation of regeneration-associated genes (Kontou et al., 2009; Horstkorte et al., 2010). Thirdly, ManNProp stimulates the secretion of thioredoxin, a small protein that promotes neurite outgrowth and has neuroprotective and neurotrophic effects (Horstkorte et al., 2010) (Figure 2).

In Situ Deactivation of Interleukin-6 Enhances Early Peripheral Nerve Regeneration in a Murine Injury Model

BACKGROUND Systemic alteration of interleukin-6 (IL-6) influences peripheral nerve regeneration. We investigated the potential influences of in situ (at the coaptation site) IL-6 modulation in a peripheral-nerve-transection/sciatic-nerve-graft in vivo model. METHODS We quantified the elongation of regenerating axons, the number of arborizing axons, and the number of branches per arborizing axon 7 days after the injury in mice expressing axonal fluorescent proteins (thy-1-YFP mice). Sciatic nerves from nonexpressing mice (C57Bl6 or IL-6(-/-) mice) were grafted into those expressing yellow fluorescent protein. We altered the in situ IL-6 concentration by loading a topical gelatin sponge with an inhibiting IL-6 receptor antibody or IL-6 combined with a soluble IL-6 receptor. Sciatic nerves from IL-6(-/-) mice were grafted into an additional group. The contralateral sham-operated side served as control in all the groups. RESULTS Axonal elongation increased significantly with the in situ application of the IL-6 receptor antibody, while topical IL-6 significantly reduced the regeneration distance. The number of arborizing axons increased significantly in nerves grafted from IL-6(-/-) mice, whereas branches per arborizing axons remained stable. CONCLUSION In situ IL-6 receptor inhibition and IL-6(-/-) nerve grafting enhance early peripheral nerve regeneration in an acute murine injury model.