Gene delivery to rat and human Schwann cells and nerve segments: a comparison of AAV 1-9 and lentiviral vectors.

Schwann cells (SCs) in an injured peripheral nerve form pathways for regenerating axons. Although these cells initially support regeneration, SCs lose their pro-regenerative properties following a prolonged period of denervation. Gene transfer to SC can enhance their therapeutic potential. In this article, we compared adeno-associated viral (AAV) vectors based on serotypes 1-9 for their capability to transduce cultured primary rat and human SCs and nerve segments. AAV1 is the best serotype to transduce rat SCs, whereas AAV2 and AAV6 performed equally well in human SCs. Transduction of monolayers of cultured rat and human SCs did not accurately predict the transduction efficiency in nerve segments. Rat nerve segments could be genetically modified equally well by a set of four AAV vectors (AAV1, AAV5, AAV7, AAV9), whereas AAV2 was superior in human nerve segments. The current experiments were undertaken as a first step towards future clinical implementation of ex vivo AAV-based gene therapy in surgical nerve repair. The transduction of rat and human SCs and nerve segments by entirely different AAV serotypes, as documented here, highlights one of the challenges of translating gene therapy from experimental animals to human patients.

Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve.

Viral vector-mediated gene transfer of neurotrophic factors is an emerging and promising strategy to promote the regeneration of injured peripheral nerves. Unfortunately, the chronic exposure to neurotrophic factors results in local trapping of regenerating axons or other unwanted side effects. Therefore, tight control of therapeutic gene expression is required. The tetracycline/doxycycline-inducible system is considered to be one of the most promising systems for regulating heterologous gene expression. However, an immune response directed against the transactivator protein rtTA hampers further translational studies. Immunogenic proteins fused with the Gly-Ala repeat of the Epstein-Barr virus Nuclear Antigen-1 protein have been shown to successfully evade the immune system. In this article, we used this strategy to demonstrate that a chimeric transactivator, created by fusing the Gly-Ala repeat with rtTA and embedded in a lentiviral vector (i) retained its transactivator function in vitro, in muscle explants, and in vivo following injection into the rat peripheral nerve, (ii) exhibited a reduced leaky expression, and (iii) had an immune-evasive advantage over rtTA as shown in a novel bioassay for human antigen presentation. The current findings are an important step toward creating a clinically applicable potentially immune-evasive tetracycline-regulatable viral vector system.

Reestablishment of the Smile after Hypoglossal-Facial Nerve Transfer: What Can We Learn?

Objective The aim of this study was to assess the ability to smile following a hypoglossal-facial nerve transfer (N12-N7). Design This is a retrospective chart review. Setting National tertiary referral center for skull base pathology. Participants Seventeen patients. Main Outcome Measures The ability to smile following an N12-N7 transfer was assessed by five medical doctors on photographs of the whole face and frontal, orbital, and oral segments. The (segmented) photographs were scored for the symmetry, asymmetry, and correct or incorrect assessment of the affected side. Results Seventeen patients were analyzed by 5 assessors providing 85 assessments. The whole face at rest was judged symmetrical in 26% of the cases and mildly asymmetrical in 56%. Frontal, orbital, and oral segments were symmetrical in 63, 20, and 35%, respectively. The affected side was correctly identified in 76%. When smiling, the whole face was symmetrical in 6% and mildly asymmetric in 59%. The affected side was correctly identified in 94%. The frontal, orbital, and oral segments during smiling were symmetrical in 67, 15, and 6%, respectively. The affected side of the frontal, orbital, and buccal facial segments during smiling was correctly identified in 89, 89, and 96%, respectively. Interobserver variability with Fleiss' kappa analysis showed that the strength of the agreement during smile of the total face was good (0.771) Conclusion Following an N12-N7 transfer, a good facial symmetry at rest can be achieved. During smiling, almost all patients showed asymmetry of the face, which was predominantly determined by the orbital and oral segments. To improve the ability to smile after an N12-N7 transfer, additional procedures are needed.

Correlating birthweight with neurological severity of obstetric brachial plexus lesions.

OBJECTIVE: To investigate the nature and extent of neurosurgically treated obstetric plexus lesions with obstetric and neonatal precedents. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Leiden, the Netherlands. POPULATION: A 9-year cohort of infants (n = 206) neurosurgically treated for obstetric brachial plexus lesion at a tertiary referral centre for nerve lesions. METHOD: Obstetric and neonatal data (parity, diabetic status, pregnancy gestation, mode of cephalic delivery and birthweight) were collected using a standardised protocol and correlated to neurological severity of the brachial plexus lesion. MAIN OUTCOME MEASURE: Neurological severity of the brachial plexus lesion. RESULTS: Nulliparous women delivered significantly lower birthweight newborns (P = 0.016), injuries in those infants were associated with the least severe injury classification. The most prominent association in ordinal logistic regression was between neurological injury severity and larger birthweight (P < 0.001). CONCLUSIONS: Birthweight is correlated with neurological severity of the injury in a group of infants experiencing brachial plexus injury resulting from cephalic vaginal delivery.

[Surgical treatment of nerve lesions]. / Operatieve behandeling van zenuwletsels.

A peripheral nerve lesion may result in loss of motor and sensory function and pain. Nerve surgery may lead to function recovery or contribute to pain reduction. Different surgical techniques are used depending on the specific type of lesion. Common techniques consist of coapting, grafting, moving or burying the nerve. The outcome depends on the interval between the time the lesion occurred and the time of repair as well as the patient's age. Here we present three case studies of patients with a different type of nerve lesion and its consequences. We describe the specific surgical intervention performed to treat the nerve lesion. Immediate repair of the nerve should be performed when the patient has an open wound. In closed lesions, the decision to repair a damaged nerve depends on the severity of the trauma, the nerve(s) involved, the location and the age of the patient. In general, repair should be performed as soon as possible.

Nerve Surgery to Treat Intractable Genitofemoral Neuropathic Pain following Laparoscopic Live Kidney Donation.

To date live laparoscopic donor nephrectomies (LLDN) are frequently performed. The most common complications entail bleeding, wound infection, and incisional hernia. Here we discuss a 50-year-old patient with a severe less known complication, namely, postoperative persistent neuropathic pain in the scrotum and left upper leg. Satisfactory pain control could not be obtained in 3 years of postoperative pain treatment which consisted of neuroleptic drugs, blocks of the L1/L2 dorsal roots with local anaesthetics, and pulsed radiofrequency lesioning. Exploratory laparoscopy was performed to assess the aspect of the genitofemoral nerve (GFN). A hemoclip used for the closure of the ureter at the time of nephrectomy was found in close relation to the GFN. The clip was removed and the GFN was subsequently cut proximal to the side of this clip. Soon after surgery the patient was completely pain-free and could return to his normal activities. Surgery should be considered in case of GFN neuropathic pain following LLDN.

Pores in synthetic nerve conduits are beneficial to regeneration.

Current opinion holds that pores in synthetic nerve guides facilitate nerve regeneration. Solid factual support for this opinion, however, is absent; most of the relevant studies assessed only morphological parameters and results have been contradictory. To evaluate the effect of pores, the rat sciatic nerve was either autografted or grafted with nonporous, macroporous (10-230 mum), and microporous (1-10 microm) biodegradable epsilon-caprolactone grafts. Twelve weeks later, the grafted nerves were resected, and the electrophysiological properties were determined in vitro. Subsequently midgraft-level sections were inspected, and peroneal nerve sections were evaluated morphometrically. Finally, the gastrocnemic and tibial muscle morphometrical properties were quantified. The microporous nerve graft performed much better than the nonporous and macroporous grafts with respect to most parameters: it was bridged by a free floating bundle that contained myelinated nerve fibers, there were more nerve fibers present distal to the graft, the electrophysiological response rate was higher, and the decrease in muscle cross-sectional area was markedly smaller. Hence, the present study demonstrates the beneficial effect of synthetic nerve guide pores on nerve regeneration, although with the caveat that not pores per se, but only small (1-10 microm) pores were effective.

Perfusion magnetic resonance imaging provides additional information as compared to anatomical imaging for decision-making in vestibular schwannoma.

OBJECTIVE: The added value of perfusion MRI for decision-making in vestibular schwannoma (VS) patients is unknown. MRI offers two perfusion methods: the first employing contrast agent (dynamic susceptibility contrast (DSC)-MRI) that provides information on cerebral blood volume (CBV) and cerebral blood flow (CBF), the second by magnetic labeling of blood (arterial spin labeling (ASL)-MRI), providing CBF-images. The goal of the current study is to investigate whether DSC and ASL perfusion MRI provides complimentary information to current anatomical imaging in treatment selection process of VS. METHODS: Nine patients with growing VS with extrameatal diameter >9 mm were included (>2 mm/year and 20% volume expansion/year) and one patient with 23 mm extrameatal VS without growth. DSC and ASL perfusion MRI were obtained on 3 T MRI. Perfusion in VS was scored as hyperintense, hypointense or isointense compared to the contralateral region. RESULTS: Seven patients showed hyperintense signal on DSC and ASL sequences. Three patients showed iso- or hypointense signal on at least one perfusion map (1 patient hypointense on both DSC-MRI and ASL; 1 patient isointense on DSC-CBF; 1 patient isointense on ASL). All patients showed enhancement on post-contrast T1 anatomical scan. CONCLUSION: Perfusion MR provides additional information compared to anatomical imaging for decision-making in VS.

Reduced functional connectivity within the primary motor cortex of patients with brachial plexus injury.

This study aims at the effects of traumatic brachial plexus lesion with root avulsions (BPA) upon the organization of the primary motor cortex (M1). Nine right-handed patients with a right BPA in whom an intercostal to musculocutaneous (ICN-MC) nerve transfer was performed had post-operative resting state fMRI scanning. The analysis of empirical functional correlations between neighboring voxels revealed faster correlation decay as a function of distance in the M1 region corresponding to the arm in BPA patients as compared to the control group. No differences between the two groups were found in the face area. We also investigated whether such larger decay in patients could be attributed to a gray matter diminution in M1. Structural imaging analysis showed no difference in gray matter density between groups. Our findings suggest that the faster decay in neighboring functional correlations without significant gray matter diminution in BPA patients could be related to a reduced activity in intrinsic horizontal connections in M1 responsible for upper limb motor synergies.

Intercostal and pectoral nerve transfers to re-innervate the biceps muscle in obstetric brachial plexus lesions.

In obstetric brachial plexus lesions with avulsion injury, nerve grafting for biceps muscle re-innervation may not be possible owing to the unavailability of a proximal stump. In such cases, the intercostal nerves or medial pectoral nerve can serve as donor nerves in an end-to-end transfer to the musculocutaneous nerve. The present study reports the results of both techniques from a single institution in a consecutive series of 42 patients between 1995 and 2008. From 1995 to 2000 we always used the intercostal nerve transfer, and from 2001 to 2008 both techniques were used. Biceps muscle force ≥ Medical Research Council Grade 3 was achieved in 37 of 42 patients after a mean follow-up of 44 months. There was no statistical difference in the results in the medial pectoral nerve transfer group (n = 25) and the intercostal nerve transfer group (n = 17).

Neonatal brachial plexus palsy with neurotmesis of C5 and avulsion of C6: supraclavicular reconstruction strategies and outcome.

BACKGROUND: Nerve reconstruction strategies for restoration of elbow flexion and shoulder function in patients with neonatal brachial plexus palsy with neurotmesis of C5 and avulsion of C6 are not well defined and the outcomes are unclear. METHODS: From 1990 to 2008, nerve surgery was performed in 421 patients with neonatal brachial plexus palsy. This study focused on thirty-four infants who had a neurotmetic lesion of C5 and avulsion or intraforaminal neurotmesis of C6, irrespective of C7. The C8 and T1 functions were intact. Intraplexal transfer of C6 to C5 with direct coaptation was preferred for restoration of elbow flexion. The suprascapular nerve was reconnected either by extra-intraplexal transfer of the accessory nerve or by grafting from C5 to restore shoulder function. Additional grafts were attached from C5 to the C5 contribution of the posterior division of the superior trunk when technically possible. RESULTS: Transfer of either the C6 anterior root filaments or the entire C6 nerve to C5 was performed in seventeen patients (group A) with direct coaptation in fifteen of them. Grafting from C5 to the anterior division of the superior trunk was performed in the remaining seventeen infants (group B). An accessory-to-suprascapular nerve transfer was applied in twenty-nine infants. The suprascapular nerve was reconnected in five patients by grafting from C5. It was possible to attach one, two, or three additional grafts from C5 to the posterior division of the superior trunk in twenty-one patients. All infants had biceps muscle recovery to a Medical Research Council (MRC) grade of ≥4, twenty-two (65%) of the thirty-four patients obtained Mallet grade-IV abduction, and eleven (32%) of the thirty-four obtained Mallet grade-IV external rotation. CONCLUSIONS: In patients with neonatal brachial plexus palsy who have neurotmesis of C5 and avulsion of C6, elbow flexion can be successfully restored with supraclavicular intraplexal reconstruction with use of C5 as the proximal outlet. However, shoulder function recovery following suprascapular nerve reinnervation and additional grafting from C5 to the posterior division of the superior trunk is less successful. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Developing core sets for patients with obstetricbrachial plexus injury based on the International Classificationof Functioning, Disability and Health.

BACKGROUND: Symptoms of obstetric brachial plexus injury (OBPI) vary widely over the course of time and from individual to individual and can include various degrees of denervation, muscle weakness, contractures, bone deformities and functional limitations. To date, no universally accepted overall framework is available to assess the outcome of patients with OBPI. The objective of this paper is to outline the proposed process for the development of International Classification of Functioning, Disability and Health (ICF) Core Sets for patients with an OBPI. METHODS: The first step is to conduct four preparatory studies to identify ICF categories important for OBPI: a) a systematic literature review to identify outcome measures, b) a qualitative study using focus groups, c) an expert survey and d) a cross-sectional, multicentre study. A first version of ICF Core Sets will be defined at a consensus conference, which will integrate the evidence from the preparatory studies. In a second step, field-testing among patients will validate this first version of Core Sets for OBPI. DISCUSSION: The proposed method to develop ICF Core Sets for OBPI yields a practical tool for multiple purposes: for clinicians to systematically assess and evaluate the individual's functioning, for researchers to design and compare studies, and for patients to get more insight into their health problems and their management.

Nerve surgery and gene therapy: a neurobiological and clinical perspective.

Despite major microsurgical improvements the clinical outcome of peripheral nerve surgery is still regarded as suboptimal. Over the past decade several innovative techniques have been developed to extend the armamentarium of the nerve surgeon. This review evaluates the potential of gene therapy in the context of peripheral nerve repair. First the main challenges impeding peripheral nerve regeneration are presented. This is followed by a short introduction to gene therapy and an overview of its most important advantages over the classical delivery of therapeutic proteins. Next, this review focuses on the most promising viral vectors capable of targeting the peripheral nervous system and their first application in animal models. In addition, the challenges of translating these experimental results to the clinic, the limitations of current vectors and the further developments needed, are discussed. Finally, four strategies are presented on how gene therapy could help patients that have to undergo reconstructive nerve surgery in the future.

Establishing a method to predict the outcome of vestibular schwannoma surgery based on one's own results.

OBJECTIVES: To establish a prognostication method based on our own results of vestibular schwannoma surgery. DESIGN: Retrospective data analysis. SETTING: Tertiary referral centre. PARTICIPANTS: 141 ambulatory patients operated for unilateral vestibular schwannoma by the translabyrinthine approach in the period 1996--2003. MAIN OUTCOME MEASURES: Facial impairment defined by House Brackmann grade III-VI, and the relation with tumor size. RESULTS: For our institution we found that in a range of tumor sizes the tumor size of 17.5 mm was the cut-off point associated with highest sensitivity and specificity values available concerning the prediction of facial impairment, these were 0.86 and 0.61. Compared to a random cut-off point (11 mm), this leads to 36% more accurate predictions. CONCLUSIONS: By establishing sensitivity and specificity values of predictions, one is aware of the rate of false predictions. By Receiver Operating Curve analysis the rate of false predictions can be minimised. In vestibular schwannoma surgery this leads to more precise predictions concerning outcome, as we have demonstrated for the facial function.

Type grouping in skeletal muscles after experimental reinnervation: another explanation.

Type grouping signifies clustering of muscle fibres of the same metabolic type, and is a frequent finding in reinnervated muscles. To elucidate the mechanism behind it, the rat sciatic nerve was either autografted or grafted with hollow synthetic nerve grafts. Twelve weeks later the number and fibre area of the type I and type II muscle fibres in the gastrocnemic and anterior tibial muscles were determined after ATP-ase staining. The number and diameter of peroneal nerve fibres distal to the grafts were measured, and the number of Aalpha-nerve fibres was derived. Nearly all nerve and muscle morphometrical parameters changed equally in both experimental groups. However, type grouping occurred frequently only after autografting, whereas the number of nerve fibres and the number of Aalpha-nerve fibres increased in this group. Hence type grouping cannot be explained by increased intramuscular sprouting subsequent to a decrease in the number of innervating nerve fibres, as previously presumed. Regenerating axons branch along their course through the peripheral nerve. We propose that the probability of the occurrence of type grouping is related to the dispersion of sibling branches in the nerve. In the autograft, emerging branches are kept together by Schwann cell basal lamina scaffolds, in contrast to the hollow synthetic nerve grafts where the emerging branches become dispersed. Thus, in muscles reinnervated after autografting, the probability that nerve branches that arrive at a specific muscle territory are sibling branches is greater than after hollow tube grafting. Consequently, the probability that type grouping will occur is greater.