Erik T. Walbeehm

A distal Schwann cell‐specific enhancer mediates axonal regulation of the Oct‐6 transcription factor during peripheral nerve development and regeneration

The POU domain transcription factor Oct‐6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct‐6 is under the control of axonal signals. Identification of the cis‐acting elements necessary for Oct‐6 gene regulation is an important step in deciphering the complex signalling between Schwann cells and axons governing myelination. Here we show that a fragment distal to the Oct‐6 gene, containing two DNase I‐hypersensitive sites, acts as the Oct‐6 Schwann cell‐specific enhancer (SCE). The SCE is sufficient to drive spatially and temporally correct expression, during both normal peripheral nerve development and regeneration. We further demonstrate that a tagged version of Oct‐6, driven by the SCE, rescues the peripheral nerve phenotype of Oct‐6‐deficient mice. Thus, our isolation and characterization of the Oct‐6 SCE provides the first description of a cis‐acting genetic element that responds to converging signalling pathways to drive myelination in the peripheral nervous system.

INFLUENCE OF CORE SUTURE GEOMETRY ON TENDON DEFORMATION AND GAP FORMATION IN PORCINE FLEXOR TENDONS

The effects of core suture geometry on the mechanics of failure in flexor tendon surgery are investigated. Forty porcine flexor tendons were repaired using a Kessler; a Kessler-Pennington; a double Kessler; a continuous Kessler; and a cruciate repair. At maximum breaking strength, the cruciate repair gapped more then the double Kessler (12.8 mm vs 9.1 mm), but the double Kessler was less strong (37N vs 45 N). Transverse narrowing was 22% and 24% for the Kessler and the Kessler-Pennington, 11% for the double Kessler, and 0% for the continuous Kessler and the cruciate repair. Kessler-type sutures failed by suture breakage and the cruciate repair by pull-out. Under load, the transverse part of the Kessler sutures narrows, allowing longitudinal parts to lengthen, leading to gapping. The double Kessler shortened transverse segment decreases gapping. Eliminating a transverse component (the cruciate repair) decreased gapping, but the cruciate failed at higher loads by suture pull-out.

Natural conduits for bridging a 15-mm nerve defect: Comparison of the vein supported by muscle and bone marrow stromal cells with a nerve autograft

OBJECT The gold standard for reconstructing large nerve defects, the nerve autograft, results in donor-site morbidity. This detrimental consequence drives the search for alternatives. We used a vein filled with a small piece of fresh muscle to prevent the vein from collapsing and with bone marrow stromal cells (BMSCs) to enhance regeneration. METHODS In 60 rats, a 15-mm sciatic nerve defect was bridged with a nerve autograft, a vein filled with muscle or a vein filled with muscle and BMSCs. Toe spread and pinprick were used to evaluate motor and sensory function. Compound muscle action potentials (CMAPs) and the gastrocnemius muscle index (GMI) were recorded to assess conduction properties and denervation atrophy. Extensive histology was performed to confirm presence of BMSCs and to evaluate regeneration by staining neural tissue for Schwann cells and neural growth factor. RESULTS After 12 weeks, all animals responded with toe spread and pinprick reaction; significant differences were found between groups. Six weeks post grafting no difference was found comparing the GMI between the groups. Group I had a significant increase in GMI at 12 weeks compared to group II and group III. The CMAP measurements showed comparable results at 6 weeks post grafting. Twelve weeks after reconstruction, group I had significantly better results compared to group II and group III. Group III showed a tendency to outperform group II at 12 weeks postoperatively. Immunofluorescence analysis showed an increased number of Schwann cells in group III compared to group II. The BMSCs were visible 6 and 12 weeks postoperatively. CONCLUSIONS This study is a step forward in the search for an alternative to the nerve autograft because it demonstrates the beneficial effect of BMSCs to a conduit. However, our data do not demonstrate sufficient benefit to warrant clinical implementation at this stage.

Re-innervation patterns by peptidergic Substance-P, non-peptidergic P2X3, and myelinated NF-200 nerve fibers in epidermis and dermis of rats with neuropathic pain.

Nerve endings in the epidermis, termed nociceptors, conduct information on noxious stimuli to the central nervous system. The precise role of epidermal nerve fibers in neuropathic pain is however still controversial. Here, we have investigated the re-innervation patterns of epidermal and dermal nerve fibers in a rat neuropathic pain model. After applying the spared nerve injury (SNI) model, we determined the mechanical and thermal withdrawal thresholds in the uninjured lateral (sural) and medial (saphenous) areas of the affected hind paw and investigated the innervations patterns of Substance P (SubP), Neurofilament-200 (NF-200) and P2X3-immunoreactive (IR) nerve fibers in the epidermis and dermis. We found a significant loss in the density of peptidergic (Sub P and NF-200) and non-peptidergic (P2X3) nerve fibers in the center area of the foot sole at 2 weeks postoperatively (PO). The densities of Sub P-IR fibers in the epidermis and upper dermis, and the density of P2X3-IR fibers in the upper dermis were significantly increased at 10 weeks PO as compared to 2 weeks PO, but were still significantly lower than the densities in controls. However, the density of NF-200-IR fibers in the center area reached control levels at 10 weeks PO. No changes were found in the densities of any of the fibers in the medial and lateral parts of the foot sole. The present results suggest that after peripheral nerve injury, specific nerve fibers have different re-innervation patterns in the epidermis and dermis and that they might be involved in the development of neuropathic pain.

Adipose-derived stem cells inhibit the contractile myofibroblast in Dupuytren's disease.

Background: In an attempt to provide minimally invasive treatment for Dupuytrens disease, percutaneous disruption of the affected tissue followed by lipografting is being tested. Contractile myofibroblasts drive this fibroproliferative disorder, whereas stem cells have recently been implicated in preventing fibrosis. Therefore, the authors tested the role of stem cells in modulating myofibroblast activity in Dupuytrens disease. Methods: The authors compared the effect of co-culturing Dupuytrens myofibroblasts with either adipose-derived or bone-marrow–derived stem cells on isometric force contraction and associated levels of &agr;-smooth muscle actin mRNA and protein expression. The authors also tested the effect of these stem cells on Dupuytrens myofibroblast proliferation and assessed whether this was mediated by cell-to-cell contact or by a paracrine mechanism. Results: Addition of adipose-derived stem cells to Dupuytrens myofibroblasts reduced the contraction of the latter, with a corresponding reduction of &agr;-smooth muscle actin protein expression, probably through a dilution effect. In contrast, bone marrow–derived stem cells increased myofibroblast contractility. In addition, adipose-derived stem cells inhibit myofibroblast proliferation and mediate these effects by soluble factors, influenced by cell-to-cell contact–dependent signaling. Conclusion: Adipose-derived stem cells inhibit the contractile myofibroblast in Dupuytrens disease, and these findings lend support to the potential benefit of lipografting in conjunction with aponeurotomy as a novel strategy for the treatment of Dupuytrens disease.

Cost analysis of surgically treated pressure sores stage III and IV.

Health-care costs associated with pressure sores are significant and their financial burden is likely to increase even further. The aim of this study was to analyse the direct medical costs of hospital care for surgical treatment of pressure sores stage III and IV. We performed a retrospective chart study of patients who were surgically treated for stage III and IV pressure sores between 2007 and 2010. Volumes of health-care use were obtained for all patients and direct medical costs were subsequently calculated. In addition, we evaluated the effect of location and number of pressure sores on total costs. A total of 52 cases were identified. Average direct medical costs in hospital were €20,957 for the surgical treatment of pressure sores stage III or IV; average direct medical costs for patients with one pressure sore on an extremity (group 1, n = 5) were €30,286, €10,113 for patients with one pressure sore on the trunk (group 2, n = 32) and €40,882 for patients with multiple pressure sores (group 3, n = 15). The additional costs for patients in group 1 and group 3 compared to group 2 were primarily due to longer hospitalisation. The average direct medical costs for surgical treatment of pressure sores stage III and IV were high. Large differences in costs were related to the location and number of pressure sores. Insight into the distribution of these costs allows identification of high-risk patients and enables the development of specific cost-reducing measures.

Ultrasound imaging of the rabbit peroneal nerve

Abstract  Ultrasound imaging of peripheral nerves is increasingly used in the clinic for a wide range of applications. Although yet unapplied for experimental neuroscience, it also has potential value in this research area. This study explores the feasibility, possibilities and limitations of this technique in rabbits, with special focus on peripheral nerve regeneration after trauma. The peroneal nerve of 25 New Zealand White rabbits was imaged at varying time intervals after a crush lesion. The ultrasonic appearance of the nerve was determined, and recordings were validated with in vivo anatomy. Nerve swelling at the lesion site was estimated from ultrasound images and compared with anatomical parameters. The peroneal nerve could reliably be identified in all animals, and its course and anatomical variations agreed perfectly with anatomy. Nerve diameters from ultrasound were related to in vivo diameters (p < 0.001, R2 = 77%), although the prediction interval was rather wide. Nerve thickenings could be visualized and preliminary results indicate that ultrasound can differentiate between neuroma formation and external nerve thickening. The value of the technique for experimental neuroscience is discussed. We conclude that ultrasound imaging of the rabbit peroneal nerve is feasible and that it is a promising tool for different research areas within the field of experimental neuroscience.

Nerve compound action current (NCAC) measurements and morphometric analysis in the proximal segment after nerve transection and repair in a rabbit model

Abstract  In the evaluation of nerve regeneration using magneto‐neurography (MNG), the proximal segment showed a reproducible decrease in peak–peak amplitude of the nerve compound action currents (NCAC) of 60%. To explain these changes, morphometry of myelinated axons in the proximal segment is compared to the MNG signals. A standardised nerve transection and reconstruction was performed in rabbits. NCACs were measured approximately 5 cm proximal to the lesion from operated and control nerves after 12 weeks. Histological samples were taken from the same area of the nerve where the NCACs were obtained. Results showed a decrease of the peak–peak amplitude of the NCAC of 57% compared to the control. Conduction velocity decreased 15%(not significant). Morphometry elicited a decrease in larger (10–15 μm) axons (284 ± 134 vs 82 ± 55) and an increase in smaller (2–5 μm) axons (1445 ± 360 vs 1921 ± 393). A strong correlation existed between the decrease in amplitude and the decrease in larger axons (0.85). Peak–peak amplitude varies approximately with the square of the diameter axon. Therefore, because peak–peak amplitude is mainly dependent on the larger‐diameter axons, the decrease in peak–peak amplitude of the NCACs may be explained by a decrease in numbers of 10–15‐μm axons.

An anatomical study of the ECRL and ECRB: feasibility of developing a preoperative test for evaluating the strength of the individual wrist extensors

BACKGROUND Tendon transfers are essential for reconstruction of hand function in tetraplegic patients. To transfer the extensor carpi radialis longus (ECRL), the extensor carpi radialis brevis (ECRB) has to be sufficiently strong. However, there is currently no reliable clinical test to individually analyse both muscles. In order to develop a reliable preoperative clinical test, the anatomy of the muscle (innervation) areas of ECRB, ECRL and brachio-radialis (BR) was examined. METHODS In 20 arms, the ECRB, ECRL and BR were dissected and localised. Subsequently, muscle-innervation points were mapped and categorised. A novel method, computer-assisted surgical anatomy mapping (CASAM), was used to visualise muscle areas and innervation points in a computed arm with average dimensions. RESULTS For both ECRL and ECRB a 100% area could be identified, a specific area in the computed average arm in which the muscle was present for all 20 arms. For the ECRL, this area was situated at 16% of the distance between the lateral epicondyle and the deltoid muscle insertion. The ECRB 100% area was 5 times bigger than that of the ECRL and was located at 40% of the distance between the lateral epicondyle and the radial styloid process. The ECRL and BR showed one to three innervation points, the ECRB one to four. In 47% of the cases, there was a combined nerve branch innervating both the ECRL and the ECRB. CONCLUSIONS It is feasible to develop a preoperative test; the 100% areas can be used for needle electromyography (EMG) or local anaesthetic muscle injections.

An anatomical study of the length of the neural pedicle after the bifurcation of the thoracodorsal nerve: implications for innervated free partial latissimus dorsi flaps.

Background: For innervated functional muscle transplant procedures, it is essential to have knowledge about the length of nerve pedicles available for nerve anastomosis. For the latissimus dorsi muscle, the thoracodorsal nerve divides into two funicles that separately innervate the medial and lateral portions of the muscle. This suggests the possibility of a multiple, segmentally innervated latissimus dorsi muscle transfer. The branching and length of the thoracodorsal nerve distal to the bifurcation have not been described. This surgical-anatomical study presents anatomical data on these practical/clinical issues. Methods: Eleven latissimus dorsi muscles were dissected in eight adult embalmed human specimens. The thoracodorsal neurovascular bundle was dissected from insertion to proximal of the bifurcation. Measurements were taken indirectly from standardized photographic images and analyzed with ImageJ and standard spreadsheet software. Results: The mean age of the specimens was 66 years. The median pedicle length of the lateral part of the muscle was 3.8 cm (range, 2.41 to 5.93 cm). The median length of the medial branch was 3.49 cm (range, 1.7 to 5.12 cm). Proximal branching of the medial pedicle of the bifurcation was identified in approximately two-thirds of the specimens and had a median of 3.55 cm (range, 2.54 to 4.68 cm). The veins and arteries showed a similar pattern, with a median length slightly less than that of the thoracodorsal nerve. Conclusion: The separate neurovascular branches and its minimal pedicle length make the latissimus dorsi muscle very suitable for single functional free muscle transfer, using only the lateral part of the latissimus dorsi muscle, and double functional free muscle transfer using only one vascular pedicle.