Date of Award

2009

Document type

Thesis

Degree Name

MD (Medical Doctor)

First Supervisor

Professor Arnold Hill

Second Supervisor

Dr. JM. Winograd

Keywords

Peripheral Nerves -- surgery

Abstract

Despite advances in clinical practice Sunderland's grades of nerve injury as described in 1951 remain a clinical measurement often aided by non specific diagnostic tools such as neurophysiological testing or indeed verified experimentally by destructive histological techniques. Furthermore regarding grade 5 injuries or nerve transection, microsurgical repair with sutures has remained the gold standard clinically despite a relatively poor outcome when the regenerative capability of the neural microenvironment is considered.

The objectives of this thesis are to apply novel optical technologies across the spectrum of nerve injuries as described by Sunderland. This includes evaluation of two optical diagnostic microscopes as well as a light activated neurorrhaphy addressing a grade 5 injury.

We hypothesize that Optical microscopy may have a role in analyzing injury to, and the recovery of, the peripheral nerve in vivo. Utilising Coherent Anti-stokes Raman Scattering (CARS) Microscopy to categorize peripheral nerve damage in vivo, and to monitor the recovery process of a damaged nerve and a Multifunctional Optical Coherence Tomography (OCT) system to assess quantitative and qualitative changes in optical density and polarisation of nerve tissue following injury. Regarding grade 5 injury we hypothesis that isolation of the nerve repair site using photochemical tissue bonding (PTB) in combination with human amniotic membrane can improve both functional and histological recovery.

Optical Diagnostics: A demyelinating crush injury was reproduced in the sciatic nerves of Sprague Dawley rats. Animals were randomised into groups and optical microscopy was undertaken at day one and weekly up to four weeks following completed following imaging. The uninjured nerve was used as a control.

All animals demonstrated loss of sciatic nerve function following injury. Recovery was documented with functional data approaching normal at three and four weeks. Demyelination was confirmed using CARS Microscopy in nerves up to two weeks post injury, remyelination was observed in the three week group and beyond. Imaging of normal nerve revealed structured myelin bundles. These results were consistent with histology.

OCT imaging revealed a quantifiable change in birefringence of the nerve (as measured by phase retardation graphs) following a simple crush injury. Histological markers of axonal myelination such as myelin thickness and g-ratio (axon diameter / fiber diameter) were then calculated and directly compared to the slope of the graph obtained from imaging (Birefkingence). Scatter plot analysis of the complete data set showed a statistically significant relationship with both myelin thickness and g-ratio when compared to imaging results.

Optical Therapeutics: Photochemical tissue bonding (PTB) is a sutureless tissue repair process that seals the repair site allowing prompt restoration of the normal endoneural environment and healing. In PTB, a photoactive dye is applied to tissue surfaces that are then placed in contact and irradiated with green light. This technique utilises visible light at a power that does not thermally damage the tissue. The light activates the dye leading to immediate formation of covalent crosslinks between proteins on both surfaces, thereby forming a water tight seal.

New Zealand White Rabbits (n = 24) underwent transection of the right common peroneal nerve. Standard neurorrhaphy (SN) was performed using 10/0 nylon sutures. The repair site was then wrapped in a cuff of human amniotic membrane(HAM), which was either secured with sutures (SN & HAM) or sealed using PTB (SN & HAMIPTB). Standard neurorrhaphy alone was assessed as a control group. Functional recovery was recorded at 30 day intervals post-operatively by electrophysiological assessment. At 120 days animals were sacrificed and nerves harvested for histomorphometry.

Nerves treated with amnion wraps, sealed with PTB demonstrated a statistically significant improvement across both functional and histological parameters. Functional recovery, as measured by repeated electrophysiological studies over time, revealed a 26.2% improvement over standard neurorrhaphy alone (p<0.05). Nervestreated with PTB sealed amnion wraps had significantly (p <0.001) greater axon fibre diameters as well as myelin thickness and G-ratio distal to the repair site compared to standard neurorrhaphy alone.

In conclusion, both CARS Microscopy and OCT have the ability to image the peripheral nerve revealing structural (CARS) as well as quantitative and qualitative changes (OCT) in composition which may be used to grade injury and regeneration over time and isolation of the peripheral nerve repair site using a photochemically sealed amnion wrap improves electrophysiological and histological recovery when compared to that of standard suture neurorrhaphy.

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Comments

A thesis submitted to the School of Postgraduate Studies, Royal College of Surgeons in Ireland for the degree of Doctor of Medicine from the National University of Ireland in 2009.

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