GSBS Dissertations and Theses

Approval Date

December 1997

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Cell Biology

Subjects

Hypothalamo-Hypophyseal System; Vasopressins; Academic Dissertations

Abstract

The hypothalamo-neurohypophysial model has been studied for many years. Of note, when the axons of the magnocellular, peptidergic neurons of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) are transected or crushed, varying degrees of polydipsia and polyuria ensue as the result of measurable losses of vasopressin (AVP) within the organism's circulation. Following insult, these hypothalamic cells show a remarkable capacity to reorganize themselves within the proximal areas of the infundibular stalk and median eminence and form what has come to be known as a new 'mini neural lobe' . While the surviving neurons sprout new projections toward the level of the external zone, vascular hypertrophy is marked throughout the new neurohypophysis and new neurohemal contacts have been identified (at the ultrastructural level) associated with these vessels. In parallel with this vascular hypertrophy is a measurable re-release of vasopressin into the circulation. This new 'mini neural lobe' now has the morphological and physiological appearance of an intact neural lobe and is capable of releasing AVP in response to changes in water balance.

While the ability of these axons to reorganize is more characteristic of the peripheral nervous system (PNS), this model system provides an unique opportunity to study axonal regeneration of the central nervous system (CNS). Not only the mechanisms underlying the restoration of AVP function following axotomy but the extent to which various magnocellular neuron populations are involved in the regenerative process may also be analyzed. Before attempting to identify putative markers associated with this regenerative process, it was necessary to carefully characterize the system following axonal injury. Using Sprague Dawley rats, we repeated previous physiological studies which had examined the intake of water and output of urine following hypophysectomy. In addition, we also correlated the restoration of water balance with the return of AVP release, as measured by radioimmunoassay. These data defined a temporal framework in which magnocellular AVP regeneration occurs.

As a result of repeating these physiological studies, we noted several inconsistencies between other previously published work. First, the time course of AVP recovery did not agree with other published results, nor did the first appearance of AVP immunoreactivity . We did not observe a complete recovery of water balance as previously reported and the degree of magnocellular death was inconsistent with other reports. In light of these many conflicting observations between several historical reports and our own results, we did a basic physiological re-characterization of the hypothalamo-neurohypohysial system following hypophysectomy. By means of immunohistochemistry, we also demonstrated the re-appearance of AVP within the new the 'mini neural lobe ' concomitant with the increased appearance of synapsin I, a marker associated with the presence of mature and presumably functioning synapses to be no sooner than 28 days following surgical removal of the hypophysis.

Immunocytochemistry was also used in conjunction with retrograde fluorescent labeling to extend the previous studies and include a 2-D analysis of cell survival throughout the PVN and SON following hypophysectomy or neurohypophysectomy. As reported previously, magnocellular neuronal loss is greater within the SON, particularly the hypophysectomized subject, and less so within the PVN; again with the greater loss in the PVN of the hypophysectomized animal. Based upon our observations and other recent reports, we suggest the possibility that some cells of the hypothalamo-neurohypophysial system or some other extrahypothalamic cell population may be capable of expressing vasopressin in response to neurohypophysectomy. We provide initial evidence that glial cells of the third ventricle may indeed be involved.

Finally, one of the ultimate goals of using this as a model system of CNS regeneration is to understand the underlying mechanisms and components essential to central nervous tissue regeneration. Toward that end I have been involved with the initial studies to optimize an adenovirus delivery system which will be capable of incorporating various putative neurotransmitter and/or peptide anti-sense messages, being injected into the neurohypophysis and transported back into the cells of the hypothalamo-neurohypophysial system. Once these antisense sequences are expressed by the cells following axotomy, the sequence of expression of various proteins in response to injury may be elucidated.

Comments

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