|Dr David F. Davey||Associate Professor (in-charge)||University||1974-|
|Dr Annick D. Ansselin||Research Affiliate
Univ. of N.S.W.
|Thomas Fink||PhD student (Assoc. Supervisor: A. Ansselin)||1993-|
Current effective full-time personnel = 3.0
This Laboratory is concerned with the embryological development of motor nerves, the innervation of skeletal muscle, and the regeneration of nerves following injury, with a particular emphasis on the role of Schwann cells. The Laboratory's technical specialty is structure-function correlations using a combination of light- and electron-microscopy, computer-aided three dimensional reconstruction, and electrophysiology.
Biosynthetic nerve guides seeded with cultured Schwann cells
The Laboratory has been examining the use of biosynthetic nerve guides as an alternative to grafts. These guides are manufactured by Bio Nova Neo Technics Pty Ltd (Melbourne) as vascular prostheses. They have a collagen surface potentially well suited to axon guidance. Peripheral nerve regenerates through these guides in a manner different to the regeneration in plastic tubes; the tissue fills the guides, suggesting the initial growth is along the internal surface of the guide. To assist axons to regenerate through biosynthetic nerve guides, the guide lumen has been seeded with Schwann cells proliferated in tissue culture. In 1992-93, using 22mm long guides to bridge a gap in the sciatic nerve much longer than has been repaired before using guides, initial phases of regeneration were observed when the guides were seeded with more than one million Schwann cells. In 1994 regeneration was quantified in animals allowed to survive over a much longer time (12-15 months). Results from these long-term animals suggest that regeneration through the guides accelerates once some axons have navigated through the guide, and that seeding the guides with Schwann cells has two important aspects: it greatly enhances the probability that any regeneration will occur through long guides; it shortens the time required for a particular degree of regeneration to be achieved in those cases where regeneration occurs.
Culture of Schwann cells from adult humans
It has been the aim of the nerve guide study to assist in human nerve repair. This would involve the use of a patient's own Schwann cell line. Success in culturing cells from adult animals was based upon the use of a conditioning nerve lesion. Schwann cells could be extracted from the nerve distal to lesion in large numbers about a week later. This approach cannot be used in humans, so a method to obtain Schwann cells from human nerve biopsies is needed. This has been pursued as part of an on-going collaboration with members of the Development & Regeneration Laboratory and John Pollard in the Dept of Medicine. The conditioning lesion approach has been adapted by maintaining nerve biopsies in organ culture for about one week prior to cell harvesting. This has allowed the mobilization and proliferation of Schwann cells to begin while the biopsy is untouched, and the Schwann cells have become much easier to extract. Schwann cell cultures can now be established from small adult human nerve biopsies on a regular basis, and cells in culture have been obtained from individuals with a variety of peripheral neuropathies.
Schwann cell responses to neuroligands
The Laboratory is interested in the role of Schwann cells in the nutritional support of peripheral nerve axons. In 1993, to investigate the possibility that Schwann cells may respond to activity dependent signals from axons, studies were begun of cultured Schwann cells using fluorescence microscopy and intracellular calcium indicators to assess their responsiveness to molecules that might be released by active neurons. This has proven to be a fascinating study, for the Schwann cells were found to be sensitive to a variety of molecules at very low concentrations. For example, some neurotransmitters applied to Schwann cells brought about changes in intracellular concentration of calcium in a short time. The cells were also able to propagate calcium waves though dense cultures indicating there is Schwann cell-to-Schwann cell communication of some kind, that might be indicative of such coupling in vivo.
Studies of Schwann cells derived from peripheral nerve of hyperalgesic rats were then begun in collaboration with David Tracey at the Univ. of N.S.W. These cells showed abnormal responsiveness to noradrenaline, raising the possibility that Schwann cells might be involved in the raised sensitivity in peripheral pathways.
Schwann cell coupling
Prompted by the observations of calcium waves, the nature of the coupling between Schwann cells in culture was examined. The mechanism whereby Schwann cells align when in proximity in culture is also of interest. Confocal microscopy has displayed tiny cytoplasmic processes from the long (usually) biopolar processes that give Schwann cells their characteristic shape in culture. These small lateral processes frequently contact adjacent cells, and may subserve coupling and cell-cell recognition. (See figure) Dye injection studies confirmed cytoplasmic continuity between contacting cells, and scanning electron microscopy on fixed cultures established the validity of the small lateral projections seen with confocal microscopy of living cells.
Schwann cell ion channels
In the latter half of 1993, Annick Ansselin, then a Postdoctoral Medical Foundation Fellow in this Laboratory, took up a position in the Dept of Anatomy, Univ. of N.S.W. Since she continues to be an active member of the Laboratory, advantage was taken of her new Laboratory at Univ. of N.S.W. to establish, with the help of a Ramaciotti Foundation grant, a patch clamping facility. Studies were begun of ion channels in cultured adult Schwann cells. These experiments were driven by a need to understand the mechanisms of neuroligand responses first observed with ion imaging.
The analysis of nerve guide regeneration will be completed in 1995. Discussions have been entered into with Bio-Nova, with a view to developing a permeable guide. If these guides can be developed, they will be tested later in the year. In Annick Ansselin's patch clamp facility at the Univ. of N.S.W., the channels expressed in adult Schwann cells will be investigated. This is intended to assist in the interpretation of the calcium imaging experiments which will be continued in this Laboratory. The axon-Schwann cell signalling hypothesis will also be investigated in intact axons examined using the confocal microscope. This instrument should enable separation of the axon and Schwann cell signals.
|Ramaciotti Foundation||Membrane channels in
adult Schwann cells
(*administered by UNSW)
|Private Donations||Nerve regeneration||Davey DF||1994||$2,000|
Total for 1994: $25,000 + share of confocal equipment grants
Total for 1995: $3,000
|Med 2||MedSc 2||MedSc 3||Sc 3||Honours||Total|
|Practical classes (no.)||32||(8)||16||(4)||-||-||-||48|
|Other (as course supervisor)||-||-||16||32||32||80|
|* 9 lectures given concurrently to MedSc 3 and Sc 3|
Total formal contact teaching time = 131 h
Total time = 337 h
(see also OTHER RESEARCH ACTIVITIES in 1994)