Hart, Andrew McKay (2001) Peripheral nerve injury: primary sensory neuronal death & regeneration after chronic nerve injury. MD thesis http://theses.gla.ac.uk/4472/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Peripheral Nerve Injury: Primary Sensory Neuronal Death & Regeneration After Chronic Nerve Injury Thesis Submittedfor Doctor of Medicine University ofGlasgow June 2001 Mr. Andrew MCKay Hart BSc. M.R.C.S. A.F.R.C.S. Blond-lvflndoe Centre, Royal Free University College Medical School, London, u.K. Department ofSurgical & Perioperative Science, Section for Hand & Plastic Surgery, Umea University, Sweden Abstract Peripheral nerve trauma remains a major cause of morbidity, healthcare expenditure, and social disruption, largely because the death of up to 50% of primary sensory neurons ensures that sensory outcome remains overwhelmingly poor despite major advances in surgical technique. Hence the principal aim of this project was to identifY novel, clinically applicable strategies for the prevention of sensory neuronal death after peripheral nerve injury. After a defined unilateral sciatic nerve transection in the rat, a novel triple staining technique was employed in order to enable the detection of neuronal death in L4 & L5 dorsal root ganglia by light microscopic morphology, and TdT Uptake Nick-End Labelling (TUNEL). Optical disection was then used to quantifY neuronal loss from statistically unbiased estimates of the number of surviving neurons. Neuronal death was demonstrated to begin within 24 hours of injury and to peak 2 weeks later, while neuronal loss plateaued 2 months after axotomy, and 39.2% of neurons died overall. Thus the most relevent experimental timepoints at which to examine the effects of putative neuroprotective strategies are 2 weeks and 2 months after axotomy, until which time a window of opportunity exists for therapeutic intervention. The principal that sensory outcome might be related to the delay between injury and nerve repair was confirmed by the fact that although surgical nerve repair reduced neuronal death 2 weeks after axotomy, the neuroprotective benefit depended upon how soon after injury the nerve was repaired. Even immediate repair did not entirely eliminate neuronal loss, confirming the need for an adjuvant therapy. Hence the effect of two promising agents with established clinical safety records was examined. N-acetyl-cysteine (NAC) is a clinically proven glutathione substrate with antioxidant, and anti-mitotic properties. Systemic treatment caused a dose-dependent improvement in neuronal morphology, a significant reduction in the number of TUNEL positive neurons 2 weeks after axotomy (p<0.05), and 2 months after axotomy it was found to have reduced neuronal loss from 35% to only 3% (p<0.001). L-acetyl-carnitine (LAC) is a physiological peptide integral to mitochondrial aerobic glycolysis that was found to be even more neuroprotective than NAC, since after LAC treatment no neuronal loss was detected 2 months after axotomy (no treatment 35% loss; high­ dose LAC -4% loss, p<O.OOl). LAC was also found to promote peripheral nerve regeneration (p<0.05) independently of its effect upon neuronal survival, suggesting an additional role in the treatment of chronic peripheral nerve pathologies. This was confmned by a small clinical trial using immunohistochemical quantification of cutaneous innervation in nucleoside analogue related distal symmetrical polyneuropathy (DSP) in HN disease. Innervation was found to be reduced in established DSP, but after 6 months of oral LAC treatment (l500mg b.d.) substantial increases were found (up to 599% for some fibre types). Small sensory (C, A8) fibres showed the greatest response to treatment, and innervation with some fibre types returned to the same level as that found in asymptomatic HN negative controls. Given its demonstrated neuroprotective potential, and ability to promote peripheral nerve regeneration in both experimental and clinical settings, LAC is proposed as the first agent clinically suitable for the adjuvant pharmacotherapy of peripheral nerve trauma. If current clinical trials are successful, then LAC may also have potential in spinal cord and traumatic brain injury, and in a wider range of neurodegenerative disorders with dysfunctional mitochondrial bioenergetic aetiologies. 2 Table of Contents ABSTRACT ....................................................................................................................................................... 1 TABLE OF CONTENTS ................................................................................................................................. 2 LIST OF FIGURES .......................................................................................................................................... 5 LIST O}"" T ABLES ............................................................................................................................................ 6 ACKNOWLEDGEMENTS ...................................................................... , ....................................................... 7 AUTHOR DECLARATION ............................................................................................................................ 8 CHAPTER 1. INTRODUCTION .................................................................................................................... 9 1.1 THE CLINICAL PROBLEM: SENSORY OUTCOME AFTER PERIPHERAL NERVE TRAUMA ............................. 9 1.2 PERIPHERAL NERVE INJURY, REPAIR & REGENERATION ....................................................................... 10 1.3 CELLULAR EVENTS AFTER NERVE INJURY ............................................................................................. 12 1.4 AxOTOMY INDUCED CELL DEATH WITIllN THE DORSAL ROOT GANGLIA ............................................. 14 1.5 MECHANISM OF DRG NEURONAL DEATH .............................................................................................. 18 1.6 POTENTIAL INTERVENTIONS TO REDUCE PRIMARY SENSORY NEURONAL DEATH ................................. 22 1.7 AlMs ....................................................................................................................................................... 24 CHAPTER 2. METHODS............................................................................................................................. 26 2.1 PRE-OPERATIVE PREPARATION OF MATERIALS ...................................................................................... 26 2.2 OPERATIVE PROCEDURES: ...................................................................................................................... 26 2.2.1 Anaesthesia.................................................................................................................................. ... 26 2.2.1 a Inhalational Anaesthesia .............................................................................................................. 28 2.2.1b Terminal Anaesthesia ................................................................................................................... 28 2.2.1 c CO2 Narcosis ................................................................................................................................ 28 2.2.2 Sciatic Nerve Division & Capping ................................................................................................. 28 2.2.3 L-Acetyl-Carnitine (LAC) Treatment after Sciatic Nerve Division & Capping ............................. 29 2.2.4 N-Acetylcysteine (NAC) Treatment After Sciatic Nerve Division & Capping ............................... 30 2.2.5 Sciatic Nerve Division & Primary Repair ...................................................................................... 30 2.2.6 Sciatic Nerve Division & Early Secondary Repair ........................................................................ 32 2.2.7 Sciatic Nerve Division & Late Secondary Nerve Graft Repair ...................................................... 32 2.3 TISSUE HARVESTING PROCEDURES ..............................................•....................................................•..... 33 2.3.1 Schedule 1 Termination ...... ............................................................................................................ 33 2.3.2 Dorsal Root Ganglia ...................................................................................................................... 33 2.3.3 Late Secondary Nerve Graft Repairs ............................................................................................. 35 2.3.4 Human Skin Biopsies ...................................................................................................................... 36 2.4 TIssUE SECTIONING & STAINING ........................................................................................................... 37 2.4.1 a Preparation ofFrozen Blocks .....................................................................................................