Accuracy of sampling methods in morphometric studies of the sural nerve in man

A variety of sampling methods are used in quantitative studies of myelinated sural nerve fibres, however there is no consensus as to which method is most accurate. This study compares whole fascicular sampling and systematic sampling of myelinated fibres with evaluation of the total myelinated nerve fibre population. Two control and eighteen pathological sural nerves showing varying degrees of demyelination/remyelination and axonal degeneration were examined. The fascicular area, number of myelinated fibres, myelinated fibre density, fibre diameter and axonal diameter were measured in each fascicle of all the nerves using I micron plastic cross sections stained with osmium tetroxide. Each fascicle was divided into measuring frames, and the number and size of myelinated fibres in each frame (field) counted using the Quantimet 500MC computer-assisted image analysis system (Leica-Cambridge, UK). Frequency distributions of myelinated fibre density and size were calculated. The mean values and frequency distributions of fibre density, fibre diameter and axonal diameter of each sample were compared to the whole population by the Wilcoxon Rank-Sum test and Kolmogorov- Smirnov goodness-of-fit test. Fascicular sampling of the two control sural nerves (14 fascicles) showed that 8 fascicles had different myelinated fibre density (P<0.05), and 8 fascicles had different fibre diameter and/or axonal diameter (P<0.05) when compared to the whole population. In the 18 pathological nerves there were 168 fascicles. When compared to the whole population, 61 fascicles had different myelinated fibre density (P<0,05), and 90 fascicles had different fibre diameter and/or axonal diameter (P<0.05). There was no relationship between the inated fibre density of each fascicle and the fascicle diameter or area in either control and pathological sural nerves. It is concluded that morphometric study of myelinated fibres of one or part of a fascicle cannot accurately represent the whole myelinated fibre population in the sural nerve. Systematic sampling of one third to half of the total transverse fascicular area in control and pathological sural nerves did not accurately depict the fibre diameter or axonal diameter of the whole myelinated fibre population. The myelinated fibre density derived from systematic sampling was more accurate than that derived from fascicular sampling. The spatial distribution of the number and size of myelinated fibres within and between fascicles is heterogeneous in the sural nerve. It is necessary to quantitate more than half the area of every fascicle to acquire accurate data about myelinated fibres that is representative of the whole myelinated fibre population.Thesis (MMSc)--University of Adelaide, Dept. of Medicine, [1998

Digital electron microscopic examination of human sural nerve biopsies

Diabetic peripheral polyneuropathy is characterized by axonal degeneration and regeneration as well as by Schwann cell and microvascular changes. These changes have been described at both the light (LM) and the electron microscopic (EM) levels; however, EM has not been applied to large clinical trials. Our goal was to adapt the rigorous techniques used for quantifying human biopsies with LM image analysis to accommodate ultrastructural analyses. We applied digital image capture and analysis to the ultrastructural examination of axons in sural nerve biopsies from diabetic patients enrolled in a multicenter clinical trial. The selection of sural nerve biopsies was based on the quality of specimen fixation, absence of physical distortion, and nerve fascicle size (≥100 000; ≤425 000 µm 2 ). Thin sections were collected on formvar-coated slot grids, stabilized with carbon and scanned on a Phillips CM100 transmission electron microscope. Digital images were captured with a Kodak Megaplus 1.6 camera. A montage was constructed using software derived from aerial mapping applications, and this virtual image was viewed by EM readers. Computer-assisted analyses included identification and labeling of individual axons and axons within regenerating clusters. The average density of regenerating myelinated axon clusters per mm 2 was 65.8 ± 5.1, range of 0–412 ( n  = 193). These techniques increase the number of samples that may be analyzed by EM and extend the use of this technique to clinical trials using tissue biopsies as a primary endpoint.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72529/1/j.1085-9489.2003.03030.x.pd

Imaging fascicular organisation in mammalian vagus nerve for selective VNS

Nerves contain a large number of nerve fibres, or axons, organised into bundles known as fascicles. Despite the somatic nervous system being well understood, the organisation of the fascicles within the nerves of the autonomic nervous system remains almost completely unknown. The new field of bioelectronics medicine, Electroceuticals, involves the electrical stimulation of nerves to treat diseases instead of administering drugs or performing complex surgical procedures. Of particular interest is the vagus nerve, a prime target for intervention due to its afferent and efferent innervation to the heart, lungs and majority of the visceral organs. Vagus nerve stimulation (VNS) is a promising therapy for treatment of various conditions resistant to standard therapeutics. However, due to the unknown anatomy, the whole nerve is stimulated which leads to unwanted off-target effects. Electrical Impedance Tomography (EIT) is a non-invasive medical imaging technique in which the impedance of a part of the body is inferred from electrode measurements and used to form a tomographic image of that part. Micro-computed tomography (microCT) is an ex vivo method that has the potential to allow for imaging and tracing of fascicles within experimental models and facilitate the development of a fascicular map. Additionally, it could validate the in vivo technique of EIT. The aim of this thesis was to develop and optimise the microCT imaging method for imaging the fascicles within the nerve and to determine the fascicular organisation of the vagus nerve, ultimately allowing for selective VNS. Understanding and imaging the fascicular anatomy of nerves will not only allow for selective VNS and the improvement of its therapeutic efficacy but could also be integrated into the study on all peripheral nerves for peripheral nerve repair, microsurgery and improving the implementation of nerve guidance conduits. Chapter 1 provides an introduction to vagus nerve anatomy and the principles of microCT, neuronal tracing and EIT. Chapter 2 describes the optimisation of microCT for imaging the fascicular anatomy of peripheral nerves in the experimental rat sciatic and pig vagus nerve models, including the development of pre-processing methods and scanning parameters. Cross-validation of this optimised microCT method, neuronal tracing and EIT in the rat sciatic nerve was detailed in Chapter 3. Chapter 4 describes the study with microCT with tracing, EIT and selective stimulation in pigs, a model for human nerves. The microCT tracing approach was then extended into the subdiaphragmatic branches of the vagus nerves, detailed in Chapter 5. The ultimate goal of human vagus nerve tracing was preliminarily performed and described in Chapter 6. Chapter 7 concludes the work and describes future work. Lastly, Appendix 1 (Chapter 8) is a mini review on the application of selective vagus nerve stimulation to treat acute respiratory distress syndrome and Appendix 2 is morphological data corresponding to Chapter 4

Investigating human Schwann cell phenotypes and outcome measures of muscle reinnervation

Peripheral Nerve Injury (PNI) often causes partial or complete paralysis and/or loss of sensation of the segment of the body involved. Traumatic PNI is a global problem and can result in significant disability and socio-economic impacts. Clinical translation of new therapeutics for the treatment of PNI is challenged by the little information that is known about the cellular and molecular features that underpin human nerve regeneration. Moreover, clinical models and measurements that can quantify the efficacy of new treatments for PNI are not well established. Therefore, this PhD explored injured and healthy human nerve samples liberated from reconstructive nerve procedures to characterise the cellular and molecular features of human peripheral nerve degeneration. Associated with this theme of characterisation of human nerve injury, the recovery of motor units in reinnervated elbow flexor muscles following nerve transfer was quantified using Motor Unit Number Estimation (MUNE). In order to better understand the relationship of MUNE with the biological process of nerve regeneration, an animal model of nerve injury was used to investigate the association between MUNE and histological markers of regeneration. MUNE was found to be a sensitive marker of muscle reinnervation in human and animal models of nerve regeneration. Moreover, MUNE demonstrated a correlation with histological markers of muscle reinnervation. It is known that these changes in the number of motor units are accompanied by changes in muscle volume. Therefore, using the same surgical scenario of nerve transfer to reanimate elbow flexor muscles, this PhD measured the recovery of muscle volume following nerve transfer to reanimate elbow flexor muscles using quantitative Magnetic Resonance Imaging (MRI) techniques. It was found that MRI assessment of muscle volume is a measure that is sensitive to the biological process of nerve regeneration. With further data, this has the capacity to determine the efficacy of new therapeutics for the treatment of PNI and predict the likely functional recovery following PNI. In summary, the findings represent an important step towards understanding the in vivo cellular and molecular events in human nerve degeneration. In addition, MUNE and quantitative MRI techniques were found to represent sensitive and responsive measures of nerve regeneration. With further data, the findings presented here will help new therapeutic options for human nerve injury advance

Functional assessment after peripheral nerve injury : kinematic model of the hindlimb of the rat

Doutoramento em Motricidade Humana na especialidade de FisioterapiaGait analysis is increasingly used on research methodology to assess dynamics aspects of functional recovery after peripheral nerve injury in the rat model, which ultimately is the goal of treatment and rehabilitation. In this thesis we studied nerve regeneration using techniques of molecular and cellular biology. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and hindlimb kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. From our results, hybrid chitosan membranes after sciatic nerve crush, either alone or enriched with N1E-115 neural cells, may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery. Collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT. Extending the kinematic analysis during walking to the hip joint improved sensitivity of this functional test. For motor rehabilitation, either active or passive exercises positively affect sciatic nerve regeneration after a crush injury, possibly mediated by a direct mechanical effect onto the regenerating nerve.FCT- Fundação para a Ciência e Tecnologi

The Physiological Changes That Occur Post Endovascular Renal Denervation in Dialysis Patients

Sympathetic neural activation is markedly increased in end-stage kidney disease (ESKD). Catheter-based renal denervation (RDN) reduces sympathetic over-activity and blood pressure in resistant hypertension. The effect of renal denervation on sympathetic neural activation and left ventricular mass was investigated in patients with ESKD. Nine ESKD (six haemodialysis and three peritoneal dialysis) patients with a dialysis vintage of ≥11 months were treated with RDN (EnligHTN system). Data were obtained on a non-dialysis day; at baseline, one (1M), three (3M) and twelve months (12M) post-RDN. At baseline, sympathetic neural activation measured by muscle sympathetic nervous activity (MSNA) and plasma norepinephrine concentrations were markedly elevated. Left ventricular hypertrophy (LVH) was evident in eight of the nine patients. At 12M post-RDN, blind analysis revealed that MSNA frequency (-12.2 bursts·min-1, 95% CI [-13.6, -10.7]) and LV mass (-27 g·m-2, 95% CI [-47, -8]) were reduced. Mean ambulatory BP (systolic: -24 mmHg, 95% CI [-42, -5] and diastolic: -13 mmHg, 95% CI [-22, -4]) was also reduced at 12M. Office BP was reduced as early as 1M (systolic: -25 mmHg, 95% CI [-45, -5] and diastolic: -13 mmHg, 95% CI [-24, - 1]). Both ambulatory and office BP had clinically significant reductions in at least 50% of patients out to 12M. Catheter-based RDN significantly reduced MSNA and LV mass as well as systemic BP in this group of patients with ESKD

GBS100: Celebrating a Century of Progress in Guillain-Barré Syndrome

No abstract available