DEVELOPMENT AND REGENERATION LABORATORY

David F. Davey

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 reconstruct-ion, and electrophysiology.

RESEARCH in 1993

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, 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 1 million Schwann cells. In 1993 the Laboratory extended these observations to animals allowed to survive over a much longer time. Initial results from these long-term animals suggested that regeneration has continued, and quantitative analysis of the numbers of axons reaching the lower leg is in progress.

Culture of Schwann cells from adult humans

It is 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. The Laboratory's success in culturing cells from adult animals is based upon the use of a conditioning nerve lesion. Schwann cells can 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. As a part of an on-going collaborative study between members of the Development & Regeneration laboratory and John Pollard in the Dept of Medicine, the Laboratory has continued to study human Schwann cells. The idea behind the conditioning lesion has been adapted to the nerve biopsies by maintaining the latter in organ culture for about one week prior to cell harvesting. This allowed the mobilization and proliferation of Schwann cells to begin while the biopsy was untouched, and the Schwann cells became much easier to extract. The Laboratory can now establish Schwann cell cultures from small adult human nerve biopsies on a regular basis and have cells in culture 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. To investigate the possibility that Schwann cells may respond to activity-dependent signals from axons, a study was begun in 1993 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 are sensitive to a variety of molecules at very low concentrations. For example some neurotransmitters applied to Schwann cells bring about changes in the intracellular concentration of calcium in a short time. The cells are 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.

Schwann cell ion channels

Preliminary experiments were begun to test the feasibility of examining ion channels in the cultured adult Schwann cells. These served as a basis for a successful grant proposal which will see a new study begin in 1994.

RESEARCH PLANNED for 1994

The present nerve guide assessments will be completed in 1994. Discussions have been conducted 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 collaboration with Annik Ansselin, a patch clamp facility is being established in her laboratory at the University of New South Wales. The channels expressed in adult Schwann cells will be investigated with a view to assisting in the interpretation of the calcium imaging experiments which will be continued by the 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.

PERSONNEL in 1993 and 1994

Dr David F. Davey	Associate Professor (in-charge)	University	1974-
Dr Annick D. Ansselin 	Research Affiliate		Microsearch Fdn	1985-92
			Postdoctoral Fellow		Medical Fdn	1993
			Lecturer			Univ. of N.S.W.	1994-
Thomas Fink 		PhD student (co-supervised with Dr A. Ansselin) 1993-

COLLABORATIONS

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

In vitro assessment of Schwann cell pathology: Dr John D. Pollard, Dept of Neurology (1992-present).

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

FACILITIES

This 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, still, two refrigerators, oven, incubator, routine light microscope and three dissecting microscopes.

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

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. Uncertainty over the future of this instrument early in 1993 was resolved with its repair late in the year. A Leitz Ortho-plan photomicroscope, 4nd 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.

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).

FUNDING

Private donations	Nerve regeneration	Davey DF	1993	$2,500
Total for 1993: $2,500

Total for 1993: NIL

TEACHING in 1993

Medicine 2

Lectures: 15, on excitable cell physiology.

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

Tutorials: 12: 2, of 1 h, repeated 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 X h, repeated 4 times, on the compound action potential.

Tutorials: 12: 2, of 1 h, on practical work and 1 on lecture material, each repeated 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 (10 student-exam h).

Neuroscience 3 (BMedSc)

Course supervisor for the Physiology contribution

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

Examinations: 0.4 h of examination time consisting of short essay questions (10 student-exam h).

Honours: BSc(Hons), BSc(Med)(Hons)

General supervisor for the Honours Sessions.

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

Tutorials: The main component of this program consists of the students presenting their own work. Each student made 4 presentations (on background, methods and two on results), with 2 students presenting in each session. Sessions averaged 1.5 h. There were 7 students in 1992? Physiology. Thus there were 14 sessions.

Total distribution (hours of formal teachning)

				Med2	MedSci2	MedSci3	Sci3	Honours	Total
Lectures			15	11	5	(5*)	2	33
Prac classes (no.)		32(8)	16(4)	-	-	-	48
Tutorial hours			12	10	-	-	21	43
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			-	-	-	-	6	6
Other (as course supervisor)	-	-	16	32	32	80
(*Given concurrently with MedSc3)
Total teaching time = 324 h.

OTHER ACTIVITIES in 1993

Refereeing

Grant and fellowship applications: for the National Health and Medical Research Council and for the Australian Research Council.

Official of scientific society

• Local Secretary for the Feb 1994 meeting of the Australian Neuroscience Society (ANS) held at the Univ. of Sydney.

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

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 committee’s to award clinical academic titles.

Member of a number of appointment committees.

Member, Anderson Stuart Building Committee.

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

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 1993.

Coordinator for library purchases.

Supervisor of Computing staff.

University administration

Member of two of the committees participating in the University’s information technology planning process (ITPC): the committee for High Performance Computing and Visualisation; the Communications Committee. This process resulted in a very large commitment for expenditure on computer networking beginning in 1994, which includes network links to Faculty of Medicine staff in the teaching hospitals.

5-YEAR RESEARCH PUBLICATIONS

JOURNAL ARTICLES

1990

Ansselin AD, Pettigrew AG (1990) Development of innervation patterns in auditory nuclei of the chick brainstem following unilateral otocyst removal. Developmental Brain Research, 54, 177-186

Ansselin AD, Pollard JD (1990) Immunopathological factors in peripheral nerve allograft rejection: quantification of lymphocyte invasion and major histocompatibility complex expression. Journal of the Neurological Sciences, 96, 75-88

Ansselin AD, Westland K, Pollard JD (1990) Low dose, short term Cyclosporin A does not protect the Schwann cells of allogeneic nerve grafts. Neuroscience Letters, 119, 219-222

1991

Davey DF, Ansselin AD (1991) Labelling of restricted numbers of axons by solid rhodamine implantation into nerve trunks. Neuroscience Letters, 121, 83-87

1992

Ansselin AD, Pollard JD, Davey DF (1992) Immunosuppression in nerve grafting: Is it desirable? Journal of the Neurological Sciences, 112, 160-169

1993

Ansselin AD, Davey DF (1993) The regeneration of axons through normal and reversed peripheral nerve grafts. Restorative Neurology and Neuroscience, 5, 225-240

PUBLICATIONS IN UNREFEREED JOURNALS

Davey DF (1989) Getting started and debugging guide for SUNIII. Australian UNIX systems User Group Newsletter, 10 (1), 17-38

CONFERENCE ABSTRACTS AND PRESENTATIONS in 1993

Ansselin AD, Davey DF, Fink T (1993) The use of a biosynthetic nerve guide seeded with cultured adult Schwann cells to repair an 18mm gap in the sciatic nerve of the rat. Proceedings of the Australian Physiological and Pharmacological Society, 24, 80P. Melbourne (Feb)

Brown CS, Davey DF, Nichol KA, Schulz M, Bennett MR (1993) Molecules bearing the HNK-1 epitope having an in vitro survival action on purified chick motoneurons. Proceedings of the Australian Neuroscience Society, 4, 72. Melbourne, (Feb)

Ansselin AD, Davey DF, Allen DG (1993) ATP evokes a rise in intracellular calcium in cultured adult rat Schwann cells. International Journal of Neuroscience (in press), Australasian Winter Conference on Brain Research, Queenstown, (Aug)

Davey DF, Janod-Groves FMM (1993) Hodgkin-Huxley simulations in science courses. International Journal of Neuroscience (in press), Australasian Winter Conference on Brain Research, Queenstown (Aug)

Fink T, Ansselin AD, Davey DF (1993) Nerve regeneration through 18mm biosynthetic nerve guides is improved by seeding with cultured adult Schwann cells. International Journal of Neuroscience (in press), Australasian Winter Conference on Brain Research, Queenstown (Aug)