Groves, Michael J.; (1997) An immunocytochemical, morphological and morphometric study of the axotomy-induced degeneration in dorsal root ganglia and the effect of administered neurotrophic factors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Following a lesion of their peripheral processes, dorsal root ganglion (DRG) neurons undergo a series of regressive and regenerative changes during which their morphology as well as the content and distribution of their components vary considerably. It was also recognised that some neurons die as a result of peripheral axotomy. This degeneration and death occurs in other types of peripheral and central neuron after de-afferentation, but the mechanism of this reaction remains obscure. Any insight into the factors affecting the progress of this process may enable the development of therapeutic strategies aimed at reducing it. The effects of peripheral axotomy upon the morphology and neuropeptide content of sensory neurons have been described in extensive studies, some of which are nearly a century old. However some of the morphometric methods used have had doubt cast on their reliability, and accuracy. In the last decade, antibodies have become available that are directed against cell surface oligosaccharide antigens that characterise subsets of dorsal root ganglion neurous projecting to different laminae of the dorsal horn of the spinal cord, providing an alternative biochemical classification to that of neuropeptide or neurofilament content. The effect of axotomy upon the expression of oligosaccharides in neurons has not been examined. Other recent advances of relevance include the re-evaluation of the phenomenon known as apoplosis which now appears to be a fundamental physiological process, particularly important in development and disease; and the recent insights into the roles of the ever-growing family of neurotrophins and neurotrophic factors which have important roles during development, but whose function in adults is less well understood. This thesis tests the hypothesis that the sensory neuron response to axotomy depends upon the type of neuron axotomised, and that this response may be ameliorated by the administration of neurotrophins or neurotrophic factors. During the course of the study, the 4th and 5th lumbar dorsal root ganglia and the corresponding levels of the spinal cord of 120 adult rats of either sex were examined up to 18 months after unilateral (permanent and transient) lesions to the sciatic nerve. The monoclonal antibodies (for oligosaccharides) LA4, LD2 and MC-813-70 (anti-SSEA4) were used to characterise DRG neurons, in addition to calcitonin gene-related peptide and GAP43 antisera. Other histochemical and conventional morphological techniques were used to estimate and characterise the incidence of neuronal death and vacuolation up to six months following axotomy. A stereological neuronal counting technique was also employed to assess the incidence of neuronal loss in ipsilateral ganglia when compared with the contralateral ganglia. The effect of administration of the neurotrophins nerve growth factor, brain derived neurotrophic factor and neurotrophin-3, and the neurotrophic factor ciliary derived neurotrophic factor upon these parameters was also assessed at one month after axotomy. The results obtained suggest that: 1) at least some of the neuronal loss following axotomy occurs via an apoptotic pathway and proceeds for longer periods than previously thought. Previous confusion concerning neuronal loss probably arose through comparing axotomised ganglia with the contralateral, unaxotomised ganglia, a method that is probably unable to detect small losses of neurons reliably. 2) The tempo and rate of reaction to peripheral axotomy varies between different subsets of DRG neurons. 3) A subset of small sensory neurons is particularly susceptible to trauma when a transient model of axotomy is examined (p[less-than]0.05, students t-test). 4) Vacuolation is a specific reaction to axotomy by some of the large, neurofilament-containing sensory neuron population. 5) At the time of observation, neurotrophin-3 administration reduced the incidence of neuronal apoptosis and vacuolation whereas nerve growth factor probably only reduced neuronal loss (p[less-than]0.05, AVOVA with post hoc Newman-Keuls range test). Nerve growth factor administration actually increased the amount of vacuolation, whereas ciliary neurotrophic factor and brain derived neurotrophic factor administration did not seem to affect any of the parameters studied. In conclusion, it appears from my results that a subset of small diameter sensory neurons is particularly vulnerable to axotomy-induced damage, and that some neurons belonging to this subset probably undergo apoptosis as a result, although this will be very difficult to prove conclusively. Furthermore, some large diameter sensory neurons (with myelinated axons) which appear to be relatively resistant to the effects of axotomy, form grossly distorting intracytoplasmic vacuoles as a response, and this does not appear to lead to the loss of the affected neurons. Finally, the local administration of neurotrophin-3 can reduce the incidence of all of the degenerative changes studied, whilst nerve growth factor administration only reduced the incidence of neuronal loss, and actually increased the amount of vacuolation seen. Brain derived neurotrophic factor and ciliary neurotrophic factor administration had no effect on these parameters. The working hypothesis that can be drawn from these results would state that the phenotype of neurons may determine their response to axotomy, and that this in turn may depend upon their trophic requirements. It is therefore likely that different populations of neuron may require different neurotrophic factors to resist the effects of axotomy. This hypothesis has implications for the use of neurotrophic factors in neuropathies, and further work to examine whether this is true in other neuron populations is needed.

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