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1.  In vitro and In vivo Release of Nerve Growth Factor from Biodegradable Poly-Lactic-Co-Glycolic-Acid Microspheres 
Regeneration of peripheral nerves after injury is suboptimal. We now report the long term delivery of nerve growth factor (NGF) by biodegradable poly-lactic-co-glycolic acid (PLGA) microspheres in vitro and in vivo. Lactic to glycolic acid ratios of 50:50 and 85:15 were fabricated using the double emulsion solvent, evaporation technique. Three different inherent viscosities (0.1dL/g: 1A, 0.4dL/g: 4A, 0.7dL/g: 7A) were analyzed. In vitro, release of NGF for 23 days was measured. Electron microscopy demonstrated intact spheres for at least 7 days (50:50 1A), 14 days (50:50 4A) or 35 days (50:50 7A and 85:15 7A). In vitro release kinetics were characterized by burst release, followed by release of NGF at a rate of 0.6%-1.6% a day. Release curves for 50:50 1A and 85:15 7A differed significantly from other compositions (p<0.01). In vivo, release was characterized by a novel radionuclide tracking assay. Release rates varied from 0.9%-2.2% per day with linear kinetics. All but the 85:15 type of spheres showed different release profiles in vivo compared to in vitro conditions. Based on the surface morphology and release profiles we found microspheres fabricated from 50:50 4A PLGA to be best suited for the use in a rat sciatic nerve injury model.
PMCID: PMC2989534  PMID: 20878933
Nerve Growth Factor; Microspheres; Peripheral Nerve; Poly-lactic-co-glycolic-acid; Dorsal root ganglia
2.  Homodimerization of the Wnt Receptor DERAILED Recruits the Src Family Kinase SRC64B 
Molecular and Cellular Biology  2013;33(20):4116-4127.
Ryk pseudokinase receptors act as important transducers of Wnt signals, particularly in the nervous system. Little is known, however, of their interactions at the cell surface. Here, we show that a Drosophila Ryk family member, DERAILED (DRL), forms cell surface homodimers and can also heterodimerize with the two other fly Ryks, DERAILED-2 and DOUGHNUT ON 2. DERAILED homodimerization levels increase significantly in the presence of its ligand, WNT5. In addition, DERAILED displays ligand-independent dimerization mediated by a motif in its transmembrane domain. Increased dimerization of DRL upon WNT5 binding or upon the replacement of DERAILED's extracellular domain with the immunoglobulin Fc domain results in an increased recruitment of the Src family kinase SRC64B, a previously identified downstream pathway effector. Formation of the SRC64B/DERAILED complex requires SRC64B's SH2 domain and DERAILED's PDZ-binding motif. Mutations in DERAILED's inactive tyrosine kinase-homologous domain also disrupt the formation of DERAILED/SRC64B complexes, indicating that its conformation is likely important in facilitating its interaction with SRC64B. Finally, we show that DERAILED's function during embryonic axon guidance requires its Wnt-binding domain, a putative juxtamembrane extracellular tetrabasic cleavage site, and the PDZ-binding domain, indicating that DERAILED's activation involves a complex set of events including both dimerization and proteolytic processing.
PMCID: PMC3811672  PMID: 23979591
3.  The Evidence for Nerve Repair in Obstetric Brachial Plexus Palsy Revisited 
BioMed Research International  2014;2014:434619.
Strong scientific validation for nerve reconstructive surgery in infants with Obstetric Brachial Plexus Palsy is lacking, as no randomized trial comparing surgical reconstruction versus conservative treatment has been performed. A systematic review of the literature was performed to identify studies that compare nerve reconstruction to conservative treatment, including neurolysis. Nine papers were identified that directly compared the two treatment modalities. Eight of these were classified as level 4 evidence and one as level 5 evidence. All nine papers were evaluated in detail to describe strong and weak points in the methodology, and the outcomes from all studies were presented. Pooling of data was not possible due to differences in patient selection for surgery and outcome measures. The general consensus is that nerve reconstruction is indicated when the result of nerve surgery is assumedly better than the expected natural recovery, when spontaneous recovery is absent or severely delayed. The papers differed in methodology on how the cut-off point to select infants for nerve reconstructive surgical therapy should be determined. The justification for nerve reconstruction is further discussed.
PMCID: PMC3914347  PMID: 24551845
4.  Lentiviral Vector-Mediated Gradients of GDNF in the Injured Peripheral Nerve: Effects on Nerve Coil Formation, Schwann Cell Maturation and Myelination 
PLoS ONE  2013;8(8):e71076.
Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the “candy store” effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.
PMCID: PMC3741360  PMID: 23951085
5.  Outcome following Nerve Repair of High Isolated Clean Sharp Injuries of the Ulnar Nerve 
PLoS ONE  2012;7(10):e47928.
The detailed outcome of surgical repair of high isolated clean sharp (HICS) ulnar nerve lesions has become relevant in view of the recent development of distal nerve transfer. Our goal was to determine the outcome of HICS ulnar nerve repair in order to create a basis for the optimal management of these lesions.
High ulnar nerve lesions are defined as localized in the area ranging from the proximal forearm to the axilla just distal to the branching of the medial cord of the brachial plexus. A meta-analysis of the literature concerning high ulnar nerve injuries was performed. Additionally, a retrospective study of the outcome of nerve repair of HICS ulnar nerve injuries at our institution was performed. The Rotterdam Intrinsic Hand Myometer and the Rosén-Lundborg protocol were used.
The literature review identified 46 papers. Many articles presented outcomes of mixed lesion groups consisting of combined ulnar and median nerves, or the outcome of high and low level injuries was pooled. In addition, outcome was expressed using different scoring systems. 40 patients with HICS ulnar nerve lesions were found with sufficient data for further analysis. In our institution, 15 patients had nerve repair with a median interval between trauma and reconstruction of 17 days (range 0–516). The mean score of the motor and sensory domain of the Rosen's Scale instrument was 58% and 38% of the unaffected arm, respectively. Two-point discrimination never reached less then 12 mm.
From the literature, it was not possible to draw a definitive conclusion on outcome of surgical repair of HICS ulnar nerve lesions. Detailed neurological function assessment of our own patients showed that some ulnar nerve function returned. Intrinsic muscle strength recovery was generally poor. Based on this study, one might cautiously argue that repair strategies of HICS ulnar nerve lesions need to be improved.
PMCID: PMC3474788  PMID: 23082230
6.  Accuracy of Motor Axon Regeneration Across Autograft, Single Lumen, and Multichannel Poly(lactic-co-glycolic Acid) (PLGA) Nerve Tubes 
Neurosurgery  2008;63(1):144-155.
Accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons.
Simultaneous tracing of the tibial and peroneal nerves with fast blue (FB) and diamidino yellow (DY), 8 weeks after repair of a 1-cm nerve gap in the rat sciatic nerve, was performed to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with DY 1 week before and FB 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons.
In the cases in which there was successful regeneration across single lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair.
Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis in the development of a nerve tube for motor nerve repair.
PMCID: PMC3463233  PMID: 18728579
misdirection; axon targeting; double labeling; peripheral nerve regeneration; rat sciatic nerve model; retrograde tracing
7.  Intratumoral hemorrhage, vessel density, and the inflammatory reaction contribute to volume increase of sporadic vestibular schwannomas 
Virchows Archiv  2012;460(6):629-636.
Vestibular schwannomas show a large variation in growth rate, making prediction and anticipation of tumor growth difficult. More accurate prediction of clinical behavior requires better understanding of tumor biological factors influencing tumor progression. Biological processes like intratumoral hemorrhage, cell proliferation, microvessel density, and inflammation were analyzed in order to determine their role in vestibular schwannoma development. Tumor specimens of 67 patients surgically treated for a histologically proven unilateral vestibular schwannoma were studied. Preoperative magnetic resonance imaging (MRI) scans were used to determine tumor size and to classify tumors as homogeneous, inhomogeneous, and cystic. Immunohistochemical studies evaluated cell proliferation (histone H3 and Ki-67), microvessel density (CD31), and inflammation (CD45 and CD68). Intratumoral hemorrhage was assessed by hemosiderin deposition. The expression patterns of these markers were compared with tumor size, tumor growth index, MRI appearance, patients’ age, and duration of symptoms. No relation between cell proliferation and clinical signs of tumor volume increase or MRI appearance was found. Intratumoral hemosiderin, microvessel density, and inflammation were significantly positively correlated with tumor size and the tumor growth index. Cystic and inhomogeneous tumors showed significantly more hemosiderin deposition than homogeneous tumors. The microvessel density was significantly higher in tumors with a high number of CD68-positive cells. The volume increase of vestibular schwannomas is not based on cell proliferation alone. Factors like intratumoral bleeding, (neo)vascularization, and intensity of the inflammatory reaction also influence tumor volume.
Electronic supplementary material
The online version of this article (doi:10.1007/s00428-012-1236-9) contains supplementary material, which is available to authorized users.
PMCID: PMC3371334  PMID: 22555941
Vestibular schwannoma; Neuropathology; Tumor biology
8.  Severe Obstetric Brachial Plexus Palsies Can Be Identified at One Month of Age 
PLoS ONE  2011;6(10):e26193.
To establish whether severe obstetric brachial plexus palsy (OBPP) can be identified reliably at or before three months of age.
Severe OBPP was defined as neurotmesis or avulsion of spinal nerves C5 and C6 irrespective of additional C7-T1 lesions, assessed during surgery and confirmed by histopathological examination. We first prospectively studied a derivation group of 48 infants with OBPP with a minimal follow-up of two years. Ten dichotomous items concerning active clinical joint movement and needle electromyography of the deltoid, biceps and triceps muscles were gathered at one week, one month and three months of age. Predictors for a severe lesion were identified using a two-step forward logistic regression analysis. The results were validated in two independent cohorts of OBPP infants of 60 and 13 infants.
Prediction of severe OBPP at one month of age was better than at one week and at three months. The presence of elbow extension, elbow flexion and of motor unit potentials in the biceps muscle correctly predicted whether lesions were mild or severe in 93.6% of infants in the derivation group (sensitivity 1.0, specificity 0.88), in 88.3% in the first validation group (sensitivity 0.97, specificity 0.76) and in 84.6% in the second group (sensitivity of 1.0, specificity 0.66).
Infants with OBPP with severe lesions can be identified at one month of age by testing elbow extension, elbow flexion and recording motor unit potentials (MUPs) in the biceps muscle. The decision rule implies that children without active elbow extension at one month should be referred to a specialized center, while children with active elbow extension as well as active flexion should not. When there is active elbow extension, but no active elbow flexion an EMG is needed; absence of MUPs in the biceps muscle is an indication for referral.
PMCID: PMC3197138  PMID: 22043309
9.  Designing ideal conduits for peripheral nerve repair 
Neurosurgical focus  2009;26(2):E5.
Nerve tubes, guides, or conduits are a promising alternative for autologous nerve graft repair. The first biodegradable empty single lumen or hollow nerve tubes are currently available for clinical use and are being used mostly in the repair of small-diameter nerves with nerve defects of < 3 cm. These nerve tubes are made of different biomaterials using various fabrication techniques. As a result these tubes also differ in physical properties. In addition, several modifications to the common hollow nerve tube (for example, the addition of Schwann cells, growth factors, and internal frameworks) are being investigated that may increase the gap that can be bridged. This combination of chemical, physical, and biological factors has made the design of a nerve conduit into a complex process that demands close collaboration of bioengineers, neuroscientists, and peripheral nerve surgeons. In this article the authors discuss the different steps that are involved in the process of the design of an ideal nerve conduit for peripheral nerve repair.
PMCID: PMC2978041  PMID: 19435445
biomaterial; growth factor; nerve conduit; nerve guide; nerve tube; polymer; Schwann cell
10.  Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model 
Experimental neurology  2008;211(2):339-350.
Misdirection of regenerating axons is one of the factors that can explain the poor results often found after nerve injury and repair. In this study, we quantified the degree of misdirection and the effect on recovery of function after different types of nerve injury and repair in the rat sciatic nerve model; crush injury, direct coaptation, and autograft repair. Sequential tracing with retrograde labeling of the peroneal nerve before and 8 weeks after nerve injury and repair was performed to quantify the accuracy of motor axon regeneration. Digital video analysis of ankle motion was used to investigate the recovery of function. In addition, serial compound action potential recordings and nerve and muscle morphometry were performed. In our study, accuracy of motor axon regeneration was found to be limited; only 71% (±4.9%) of the peroneal motoneurons were correctly directed 2 months after sciatic crush injury, 42% (±4.2%) after direct coaptation, and 25% (±6.6%) after autograft repair. Recovery of ankle motion was incomplete after all types of nerve injury and repair and demonstrated a disturbed balance of ankle plantar and dorsiflexion. The number of motoneurons from which axons had regenerated was not significantly different from normal. The number of myelinated axons was significantly increased distal to the site of injury. Misdirection of regenerating motor axons is a major factor in the poor recovery of nerves that innervate different muscles. The results of this study can be used as basis for developing new nerve repair techniques that may improve the accuracy of regeneration.
PMCID: PMC2967197  PMID: 18448099
Aberrant reinnervation; Accuracy of regeneration; Ankle motion analysis; Double labeling; Sequential retrograde tracing

Results 1-10 (10)