Makwana, M.; (2008) Intracellular mediators of axonal sprouting in vivo. Doctoral thesis , University of London.

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Abstract

Generation of new axonal sprouts and the process of axonal elongation play a vital role in neural regeneration and repair. The facial nerve axotomy model is a well-established, prototypical experimental paradigm for the systematic study of nerve regeneration and degeneration, providing insights into molecular signals that determine axonal regeneration and neuronal cell death. Interestingly, this model of peripheral injury induces a delayed appearance of galanin+ and calcitonin gene-related peptide+ (CGRP) neuropeptide-immunoreactive growth cones in the facial nucleus which peak at 14 days following axotomy and surprisingly, increase in number if recut within this time window. Furthermore, application of the retrograde tracer mini-ruby to the distal portion of the cut nerve demonstrates the motoneuron origin of these sprouting neurites. To gain an insight into the molecular mechanisms inducing the sprouting response we examined how neuronal cell death and the inflammatory response of various transgenics affected sprouting and regeneration. Deletion of the a7 integrin, which demonstrated a moderate reduction in regeneration, showed enhanced sprouting neural c-jun blocked regeneration, abolished regeneration-associated neuronal proteins and neuronal cell death, also completely eliminated central axonal sprouting. Absence of TNFR1&2 which displayed reduced neuronal cell death and inflammation, showed a tendency toward enhanced sprouting TGF(31 deletion, which showed an elevated inflammatory response and a 4-fold increase in neuronal cell death, resulted in decreased central sprouting. Similarly, enhanced neural inflammation following systemic injection of E.coli lipopolysaccharide (LPS) also reduced central sprouting. Finally, neuronally expressed constitutively active Ras (Ras+), dominant-negative MEK1 (MEKIdn) and Ras+xMEK1dn double mutant (DM) all demonstrated reduced neuronal cell death as well as substantially enhanced central sprouting, particularly in the MEKIdn mutant, suggesting that the sprouting response in these mutants may be beneficial for improving regeneration in the CNS. Sprouting and regeneration studies in Ras+ and MEKIdn mutants were therefore extended to the injured corticospinal tract (CST) and rubrospinal tract. These mutants showed extensive collateral sprouting of corticospinal tract (CST) axons, in the grey and white matter on the ipsilateral side in Ras+, MEKIdn and DM animals compared with wild-type (WT) controls when the injury spared the dorsolateral CST, enhanced green fluorescent protein (EGFP) labelled rubrospinal axons showed increased sprouting below the site of injury following a C4 injury in Ras+, MEKIdn and DM mice but this was not statistically significant compared with wild-type controls. To determine functional recovery rearing and grid-walk tests over 28 days following a unilateral left dorsal hemisection (DH) at C4 were used. Ras+, MEKIdn and DM groups performed significantly better in left forepaw use than WT in the rearing test at day 28 (25.0% 3.0% 32% 1% 50.0% 9.0%) compared with WT (13.0% 5.0%). Similarly, Ras, MEKIdn and DM animals showed significantly less footslips on the left forepaw compared with WT at day 28 (11.0% 2.0% 10.0% 1.0%+/- 12.0% 1.0% 19.0% 2.0% respectively). Overall, data from facial nucleus studies suggest central axonal sprouting is an injury but not a reinnervation-driven response that it is not directly connected to neuronal cell death, that excessive inflammation is detrimental, and that jun-, Ras-, and MEK1-mediated changes in regeneration-associated gene and protein expression play a vital part in shaping the growth cone response. Following spinal injury, expression of MEKIdn enhanced CST sprouting below the injury site. Furthermore, the combination with Ras+ also enhanced functional recovery following C4 DH. These data suggest that neuronal expression of active Ras and MEKIdn might serve as a promising biochemical strategy for regrowth in the injured spinal cord.

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