| Summary: | Background & Objectives: The potential benefits of adjuvant pharmacotherapy with N-acetylcysteine and acetyl-L-carnitine to reduce the amount of primary sensory neurons in the dorsal root ganglion (DRG) which die by apoptosis after peripheral nerve injury has been previously established. However, the duration of NAC therapy sufficient to achieve neuroprotection is unknown. Establishment of clinical trials of NAC/ALCAR therapy is hindered by relatively poor existing methods of assessment of neuronal populations and nerve regeneration in human subjects. Magnetic Resonance Imaging (MRI), a non-invasive modality, has been demonstrated in a pilot study to provide volumetric analysis of rat L4 DRG after sciatic nerve transection; detecting DRG volume reductions which reflect neuronal loss. Further, MRI has been reported to detect changes occurring in peripheral nerves distal to the site of injury, corresponding to axonal degeneration and subsequent regeneration. High Frequency Ultrasound (HFUS) is a new, high resolution imaging modality, not previously applied to the study of animal or human peripheral nervous system. Experiments were designed to test the duration of NAC treatment needed to achieve a neuroprotectant effect; to further evaluate the MRI DRG imaging protocol under interventional experimental conditions and in vivo; to investigate the ability of MRI and HFUS to visualise murine sciatic nerve and its response to injury and to assess the ability of HFUS to visualise human digital nerves in the hand. Further, a pilot study investigating the effect of NAC and ALCAR on primary sensory neurons in tissue culture was carried out to assess the neurotrophic effect of these agents in vitro. Methods: 1. Groups of rats underwent sciatic nerve transection, and NAC treatment for 7, 14, 30 or 60 days. At 60 days post injury neuron counts from L4 and L5 DRG were assessed histologically using a stereological optical fractionator analysis, and compared to the contralateral noN-axotomised DRG. 2. Animals from treatment groups were MRI scanned after two months to measure the L4 DRG volume bilaterally. A single animal was also scanned under anæsthesia and after death. 3. Numerous MRI techniques were used to scan rats after sciatic nerve crush, division and intraneural injection of contrast, attempting to identify the nerve and its response to injury. 4. Normal and injured rat sciatic nerve and intact human digital nerve were assessed by HFUS. 5. Cultured DRG neurons were exposed to a range of ALCAR/NAC doses or NGF, with assessment of cell survival and neurite formation at 24h. Results: 1. Neuronal death was related to duration of NAC treatment; with losses of 20%, 9%, 4% & 0% after treatment for 7, 14, 30 and 60 days respectively. 2. MR measured L4 DRG volume reduction correlated well with neuron loss. In vivo MRI scanning is feasible; however images are subject to motion artefact distortion, precluding accurate quantification. 3. The rat sciatic nerve could not reliably and consistently be identified by MRI in this model. 4. High frequency ultrasound can image sciatic nerve and its branches and human digital nerve with good resolution and can detect neurotmesis and axonotmesis grade injurys. 5. Non-toxic in vitro NAC/ALCAR dosage regimens have been defined, but do not affect neuron survival or neuritogenesis in the absence of NGF. Conclusions: 1. One month treatment with NAC post neurotomy prevents death of the majority of axotomised neurons. Apoptosis is prevented rather than delayed for some neuronal populations by NAC treatment. 2. MRI is a valuable objective tool, and DRG volume serves as a proxy measure of neuronal loss, fit for translation to human studies. 3. In vivo MRI DRG imaging requires motion compensation techniques such as respiratory gaiting. 4. Further investigation is needed to facilitate murine sciatic nerve imaging with MRI in this model, but HFUS easily demonstrates two grades of peripheral nerve injury, and is proposed for investigation of human digital nerve trauma. 5. The neuroprotective and regenerative effects of NAC/ALCAR on primary sensory neurons in vivo can not be replicated by direct treatment of isolated neurons in culture.
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