DEVELOPMENT & REGENERATION LABORATORY

David F. Davey

  

  

RESEARCH

PERSONNEL in 1994 and 1995

Dr David F. Davey Associate Professor (in-charge) University 1974-
Dr Annick D. Ansselin Research Affiliate
Lecturer

Univ. of N.S.W.
1985-
1994-
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.
  

PROJECTS in 1994

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.
  

RESEARCH PLANNED for 1995

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.
  

COLLABORATIONS

Axonal regeneration through biosynthetic nerve guides:
Bio Nova Neo Technics Pty Ltd (1989-present).

In vitro assessment of Schwann cell pathology:
Assoc. Prof. John Pollard, Dept of Medicine (1992-present).

Calcium imaging of Schwann cells from hyperalgesic rats:
Assoc. Prof. David Tracey, Dept of Anatomy, Univ. of N.S.W. (1994-present).
  

FACILITIES

The Laboratory occupies rooms 281-283 and 257-259 of the Anderson Stuart building. Dr Davey's office is in room 280.

Histology: Main laboratory has fume cupboard, two balances, pH meter, stirrer, hotplate, two refrigerators, oven, incubator, routine light microscope and three dissecting microscopes.

Microtomy: Cambridge-Huxley microtome and LKB Ultratome V, LKB knifemaker.

Tissue culture: CO2-incubator, autoclave, oven, centrifuge, Olympus inverted microscope, Zeiss upright microscope, Laminar flow.

Microscopy: The E.M. suite sits directly on Sydney's sandstone for vibration free conditions, is electromagnetically shielded and air conditioned. It houses a Philips EM 201c with plate camera and goniometric stage. A Leitz Orthoplan photomicroscope, and a Zeiss Axiovert 35 inverted microscope, both equipped with incident light and fluorescence optics, are housed in a microscope room with a graphics workstation coupled to the Departmental MicroVax. A CCD camera or a high-sensitivity image intensified camera can be used with either microscope and connected to the computer's frame grabber for image analysis and/or ion-imaging.

Photography: A small darkroom contains EM plate developing and printing equipment.

Computing: The laboratory's computer is a MicroVax II. It is housed in the Departmental computer room and supports two graphics setups (graphics terminals, digitizers, plotter, printer) and three X terminals. It uses the Unix operating system and is connected to the main Departmental computers and thence to the world network (node name: phoenix.physiol.su.oz.au).
  

OTHER RESEARCH ACTIVITIES in 1994

Refereeing

Grant and fellowship applications: National Health and Medical Research Council (4); Australian Research Council (1).

Official of scientific societies

Local Secretary for Feb 1994 meeting of the Australian Neuroscience Society (ANS) held at Univ. of Sydney. This meeting had a record registration of more than 500 neuroscientists.

Member, organizing committee for the Glial Workshop run in conjunction with the ANS meeting.

Invited speaker

Australian Association of Neurologists Symposium on Nerve Regeneration, Canberra (May).

Seminar

Faculty of Health Sciences, Univ. of Sydney (Sep).

Appointment

D.F. Davey was appointed a Visiting Fellow in the Dept of Anatomy, Univ. of N.S.W.
  

5-YEAR RESEARCH PUBLICATIONS

MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): 16
  

FUNDING in 1994 and 1995

Ramaciotti Foundation Membrane channels in
adult Schwann cells
(*administered by UNSW)
Ansselin AD* 1994 $20,000
Davey DF

Private Donations Nerve regeneration Davey DF 1994 $2,000
1995 $3,000

Total for 1994: $25,000 + share of confocal equipment grants

Total for 1995: $3,000

  

  

TEACHING

COURSES TAUGHT

Medicine 2

Lectures: 15, on excitable cell physiology.

Practical classes: 1, of 3 h, presented 4 times, on the compound action potential.

Tutorials: 12: 2, of 1 h, presented 4 times, on practical work, and 4, of 1 h, on lecture material.

Examinations: 0.9 h of examination time consisting of short answer and multiple-true false questions (216 student-exam h).

Medical Science 2

Lectures: 11 on excitable cell physiology.

Practical classes: 1, of 3 h, presented 4 times, on the compound action potential.

Tutorials: 12: 2, of 1 h, on practical work and 1 on lecture material, each presented 4 times.

Question periods: 2, on lecture material.

Examinations: 0.7 h of examination time consisting of short answer questions (84 student-exam h).

Science 3

Course supervisor: for the Neuroscience semester.

Lectures: 5 lectures on peripheral nerve physiology, development and regeneration.

Examinations: 0.4 h of examination time consisting of short essay questions (8 student-exam h).

Medical Science 3: Neuroscience 3

Course supervisor: for the Physiology contribution.

Lectures: 5 lectures on peripheral nerve physiology, development and regeneration. (These lectures were in common with the Science 3 ones listed above).

Examinations: 0.4 h of examination time consisting of short essay questions (12 student-exam h).

Medical Science 3: Neuroscience Advanced

Lectures: 4 on neuron-Schwann cell interactions.

Honours

General supervisor: for the Honours Sessions.

Lectures: 2 formal sessions on the honours program and on computing.

Tutorials: The main component of this program consisted of the students presenting their own work. Each student made 4 presentations (on background, methods and 2 on results), with 4 students presenting in each session. Sessions averaged 1.5 h. There were 15 students in 1994 Physiology. Thus there were 16 sessions.
  

TEACHING HOURS in 1994

Med 2 MedSc 2 MedSc 3 Sc 3 Honours Total
Lectures 15 11 9 * 9 * 2 37
Practical classes (no.) 32 (8) 16 (4) - - - 48
Tutorial hours 12 10 - - 24 46
Student consultations 8 3 2 2 3 18
Preparing lectures 17 10 - 10 1 38
Setting exams 4 3 - 1 - 8
Marking exams 10 6 3 3 - 22
Marking essays - 16 5 5 2 28
Marking theses - - - - 12 12
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

  

  

ADMINISTRATION

(see also OTHER RESEARCH ACTIVITIES in 1994)

University committees

Elected member of the Academic Board (from 1990, re-elected 1992 and 1994).

Member, Standing Committee of the Academic Board (1990-present).

Academic Board representative on a number of appointment committees.

Member of a number of appointment committees considering appointments in cognate departments.

Faculty of Medicine

Member, Senior Lecturer promotions core committee (1991-94).

Member, Burkitt Library Users' Committee (1987-present).

Member of a number of committees to award clinical academic titles.

Member of a number of appointment committees.

Chairman, Faculty Computer Policy Committee (1992-94).

Faculty of Science

Member, Library Committee (1987-present).

Member, Timetable Committee (1982-present).

Faculty adviser during enrolment period.

Departmental administration

Several periods as Acting Head of Department in 1994.

Co-ordinator for library purchases.

Supervisor of Computing staff.