Previous workshops

Neurodegenerative diseases, Uppsala, 11-13 September 2009

Workshop title: Harmonization of neuropathological diagnostics of neurodegenerative disorders, a practical approach

Location:
Uppsala University, Uppsala Sweden
Department of Genetics and Pathology,
Rudbeck Laboratory (Dag Hammarskölds street 20)
Building C11 / Rudbecks sal

Organizers: BrainNet-Europe and EURO-CNS

Programme:

More information can be obtained from:
Irina Alafuzoff

The workshop is fully booked.


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Laser-Capture-Microdissection (2nd), May 2009, Lyon


Date: 13-15 May 2009

Place: Centre de Pathologie et Neuropathologie Est, Laboratoire de Biochimie, Hospices Civils de Lyon

Organised by: Dr. David Meyronet and Dr. Aline Dorey (Lyon)

Summary:
Hospices Civils de Lyon has been responsible for laser capture microdissection (LCM) within the BNE II network. We have optimised a protocol to cut tissue, stain slides, capture cells and extract RNA from human post-mortem brain tissue for further molecular biological analysis. We have also built protocols to better handle high trough-put and highly sensitive protein analysis with mass spectrometry (SELDI TOF)

This course was intended to explain how to use LCM in the research environment, to learn best practice procedures in LCM in order to preserve the quality of yields and understand implications of using LCM in a research project from the sample to the management of the project.
The two days course was divided in four workshops an 8 keynotes attended alternatively by small groups of four participants in order to have a real “hands on“ experience. These workshop covered tissue sampling, safety procedures, pre-treatments, quality control of RNA yields quality, presentation of different LCM systems.

Sixteen participants from different BNE II centres were trained by nine persons. An evaluation form was filled in by all attendees at the end of the course. All of them gave enthusiastic comments and agreed that the course was highly relevant to their research on brain tissue with LCM.


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Stereology Workshop (2nd), May 2009, Aarhus


Date: 1-3 May 2009

Place: Stereology and EM Research Lab., University of Aarhus

Organised by: Dr. Nenad Bogdanovic (Huddinge) and Dr. Jens Randel Nyengaard (Aarhus)

Summary:
Seventeen scientists had signed up for this BrainNet Europe course in stereology and only three were BNE members. The scientists came from Denmark, Sweden, Germany, Turkey, Australia, England, Greece and Poland. We were five instructors (Nenad Bogdanovic, Karl-Anton Dorph-Petersen, Johnnie Bremholm Andersen, Gorm Bennedsen and Jens Randel Nyengaard), who were present all three course days, except Gorm Bennedsen who was present only the first day.

The course was taking place in the new building for the Danish Neuroscience Center at Aarhus University Hospital, Noerrebrogade 44, 8000 Aarhus C, Denmark. We used a new lecture hall with all modern electronic equipment for visualization of slides and movies. The general design of the course was that lectures on each topic would be followed by hands-on exercises to bring theory to practice. The focus was on description of heterogeneous structure and morphology for applications in neuroscience. This included cells (neurons and glia), subcellular structures, axons, dendrites, synapses, nerve fibres, blood vessels etc.

On day 1, the following topics were discussed: sampling, probes and basic stastistics, estimation of total volume with point counting and the principle of Cavalieri, as well as number estimation with the fractionator and disector. At the end of the day we had a practical session in the new Stereology Laboratory at the 3rd floor in the Danish Neuroscience Center, where practical demonstrations of agar embedding, slicing and sampling of brains where performed. Furthermore, a brightfield microscope equipped with a fluorescent system and modified for stereology with a fast digital camera, a computer-controlled motorized stage and a microcator was presented for the participants. Finally, we went to get-together-dinner at Restaurant “Olive” in Aarhus.

On day 2 followed lectures and exercises about estimation of surface area and length using isotropic section planes. Sessions continued with multilevel sampling designs, ratio estimators including the reference trap, and how to design a stereological study with number of subjects/animals, number of blocks, number of fields of view and number of counts. The afternoon included demonstration of software for computer-assisted stereology, a lecture and exercise about connectivity and various theoretical aspects of tissue deformation, slicing, sectioning, stereological tools and gadgets. The day was rounded off with dinner at Restaurant “Le Regal” in Aarhus.

Day 3 included a lecture about vertical section planes and two practical exercises with estimation of surface area using thin vertical sections and cycloids and length estimation using total vertical projection. Local volume estimators were discussed in a lecture after lunch and a practical exercise with the 3D nucleator on dorsal root ganglion neurons was finishing the course.

We had both an oral and a written evaluation (enclosed) of the course and in general the participants were very happy and satisfied with the content and design of the course. The instructors were also given credit for the success of the course and several collaborations and visits between the laboratories have been planned. Some participants wanted a longer course to get more experience with stereological software, which we would support.

 

Training Material:
Estimation of volume by the Cavalieri method (2MB)
More disector sampling and connectivity (6MB)
The fractionator (2MB)

 


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Tryptophan measurement, April 2009, Würzburg


Date: 20-21 April 2009

Place: TDM-Labor, Department of Clinical Neurochemistry, Füchsleinstraße 15, 97080 Würzburg.

Organised by: Prof. Dr. Peter Riederer (Würzburg)

Summary:
This workshop gave insight into the method of High Pressure Liquid Chromatography (HPLC) as tool for quality control of human postmortem brain tissue. Next to a theoretical introduction for the HPLC equipment the participants had the opportunity during the practical part to prepare a tissue sample for the determination of the essential aminoacid tryptophan. The tryptophan level of a tissue sample provides an easy applicable indicator for protein degradation. The results gained within this workshop were discussed and interpreted based on previous results on this topic. This method is next to pH measurement useful for the estimation of tissue quality.


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DNA/protein preservation, April 2009, Barcelona


Date: 01 - 03 April 2009

Place: Universidad de Barcelona, campus de Bellvitge, calle Feixa LLarga sn, 08907 Hospitalet de LLobregat

Organised by: Prof Isidro Ferrer, Dr. Gabriel Santpere (Barcelona)

Summary:
The workshop on proteomics was carried out in Barcelona the days 1, 2 and 3 of April. Six persons attended the workshop. Attendants were from Wurzburg, Lyon, Vienna and London. The course included a tutorial practical part with two persons per "teacher", and several talks on different topics: gele electrophoresis and western blotting, bi-dimensional gel electrophoresis, mass spectrometry, redox proteomics, mass chromatography, and three lectures focused on brain banks and application of protemics on the sdtudy diseases of the nervous system. The evaluation resuts of the course show that it was successfull albeit the background was very variable among participants.


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Laser-Capture-Microdissection (1st), March 2007, Lyon


Date: 26 – 27 March 2007

Place: Department of Pathology and Neuropathology, Hospices Civils de Lyon, France

Organised by: Dr David Meyronet (Lyon)

Summary:
Pathological lesions in tissues are often a heterogeneous process in which many different cell types are encountered. Laser capture microdissection techniques give researchers the ability to select a specific population of cells based on its morphological characteristics. The success of gene or pro-teins expression applications will depend on the isolation and purification of intact cellular mRNA or proteins from laser captured cells from human post-mortem brain tissue. The integrity of RNA and protein yields may be affected by each steps from the cryostat sectioning to the extraction. Therefore it is important to use protocols that prevent further degradation of the cellular RNA/proteins by endogenous and exogenous RNases or proteases. Extraction protocols have also to be adapted to very small quantity of material in order to avoid dilution of information.

Hospices Civils de Lyon has been responsible for laser capture microdissection (LCM) within the BNE II network. We have optimised a protocol to cut tissue, stain slides, capture cells and extract RNA from human post-mortem brain tissue for further molecular biological analysis.

This course was intended to explain how to use LCM in the research environment, to learn best practice procedures in LCM in order to preserve the quality of yields and understand implications of using LCM in a research project from the sample to the management of the project.

The two days course was divided in four workshops an 8 keynotes attended alternatively by small groups of four participants in order to have a real “hands on“ experience. These workshop covered tissue sampling, safety procedures, pre-treatments, quality control of RNA yields quality, presenta-tion of different LCM systems.

The equipment of our research platform, Neurobiotec services, was kindly completed for the train-ing purpose by Arcturus (Alphelys) and Leica microsystems. Trainees had the opportunity to train themselves on 2 cryostats, 1 optical microscope, 3 Laser Capture/Microdissection Microscopes, 1 Agilent Bioanalyser and 1 Nanodrop spectrophotometer.

Sixteen participants from different BNE II centres were trained by nine persons : 2 pathology techni-cians (E. Alix, E. Gros) (Centre for Pathology and Neuropathology - HCL), 1 Neuropathologist (D. Meyronet) (Centre for Pathology and Neuropathology - HCL), 2 researchers for keynotes (IN-SERM, CNRS), 1 research engineer (C. REY) (Neurobiotec Services - INSERM / IFR 19), 3 engi-neers from LEICA (A. Caroff, A. Galland, L. Bompard) and 1 from ALPHELYS (ARCTURUS) (P. Defrenaix). They received a manual with a CD ROM version containing all protocols used during the session, alternative published protocols, full text references, website links and contacts needed.

An evaluation form was filled in by all attendees at the end of the course. All of them gave enthusiastic comments and agreed that the course was highly relevant to their research on brain tissue with LCM. The course organisers were complimented on the organisation, the practical aspects of presentations and on the adequate time given for interaction and questions. Although the course may be improved by increasing the level of English of some trainers, the participants found this course will  enable the use of LCM for their own research projects.

 


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Basic RNA, February 2007, London


Date: 18 – 19 February 2007

Place: Dept. of Cellular and Molecular Neuroscience, Charing Cross Campus, Imperial College, London, UK

Organised by: Dr. Shama Fernando and Professor Richard Reynolds (London Imperial)

Summary:
This course was intended to transfer RNA isolation and quality assessment skills to other groups within the BNE Network. The aim was to show researchers who are new to the technique, that it is important to follow certain guidelines in experimental procedures in order to improve the quality of results. Especially for RNA, it is not only the protocol that matters, but also the precautions that have to be taken to maintain the quality of the samples.

The one-day course had three main parts and there was time for questions and discussion during all three parts.

  1. Introductory talk – The covered topics included information about the importance to isolate good quality RNA, factors affecting quality of RNA, tissue collection, handling and storage procedures, dissecting tissue for RNA, outline of the isolation protocol and various methods of quality assessment, and use of RNA for molecular biology
  2. RNA isolation procedure using the Qiagen Lipid Tissue Mini Kit– The method was first demonstrated and then the attendees were given hands-on experience
  3. Quality assessment – Detailed explanation of Nanodrop spectrophotometer and Bioanalyser methods with demonstration and hands on experience

There were eight applicants from several BNE member laboratories and the course was run on two consecutive days, accommodating four attendees on each day.  Attendees were drawn from the following BNE centres:.
February19th
1. Bologna, Italy
2. Budapest, Hungary
3. Lyon, France
4. Stockholm, Sweden

February 20th
1. Institute of Psychiatry, London, UK
2. Goettingen, Germany
3. Paris, France
4. Imperial College, London, UK

An evaluation form was filled in by all attendees at the end of the course.  All of them enjoyed the course very much and agreed that the course was highly relevant to brain banking practice and a very useful addition to their skills.  The course organisers were complimented on the clarity and precision of the presentations and on the level of interaction provided for the attendees.  By taking part in this practical, lab based course, the participants emerged with confidence that they could undertake the procedures themselves.

Conclusion:

This course provided tuition with hands-on experience in a technique which should become part of 21st century best practice for brain banking.

 


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Mystery Case Workshop, December 2006, Vienna


Date: 1 December 2006

Place: Medical University of Vienna, Austria

Organised by: Prof. Herbert Budka, Dr. Ellen Gelpi and Ms Irene Leisser (Vienna)

Summary:
The main objective of the Medical University of Vienna is to reach a consensus diagnosis in each “mystery case“ (case with difficult, atypical, rare or combined neuropathological changes). This requires contribution by the most experienced neuropathologists and should improve diagnostic security, contribute to quality control for neuropathological diagnoses and train neuropathologists on appropriate diagnostic set-up for difficult or atypical neuropathological conditions. Eventual description of new disease (sub-)types and eventual development of harmonised diagnostic criteria for new disease (sub-) types.

Therefore, a one-day workshop on “Mystery cases” and “Teaching cases” was organized in Vienna. All BNE partners were invited to participate in the workshop and asked whether they would contribute with some case of their bank.

The workshop had two main parts:

  1. Microscope session: each participant had four hours to look at the sections for its own
  2. Case presentations and discussion. Cases were presented by each contributing partner and actively discussed.

A summary of neuropathological features of each disease type, (possible) pathogenesis and differential diagnosis was presented. Boxes containing sections of the cases discussed have been circulated to the partners.

 


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Synuclein Pathology, May 2006, Edinburgh


Date: May 2006

Place: Edinburgh University, UK

Organised by: Dr Irina Alafuzoff (Kuopio) and Professor Jeanne Bell (Edinburgh)

Summary:
This closed workshop drew together 3 BNE members together with 3 external experts who served as referees to assist us in devising an assessment and grading programme for synuclein pathology. In sitting at a multihead discussion microscope with the 3 experts, the BNE members derived considerable training benefit and the experts themselves were made aware of the opportunities for training in viewing at one sitting a large collection of affected cases with multiple brain areas from each case. Neither the BNE members nor the experts had truly investigated grading systems in this way before and this proved a useful exercise.

 


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Dementia pathology and immunohistochemical practice, March 2006, Munich


Date: 6-8 March 2006

Place: Munich Centre for Neuropathology and Prion Research, Germany

Organised by: Dr Irina Alafuzoff (Kuopio) and Dr Thomas Arzberger (Munich), Ms Kauppinen (Kuopio) and Mrs Henn (Munich)

Summary:
This 2.5 day course allowed parallel session activities for neuropathologists and biomedical scientists with a final joint session at a multihead discussion microscope. The neuropathologists took part in joint assessment of alpha synuclein and Tau stained slides with the opportunity to interact with external experts including Professor H Braak. Participants were again reminded of the criteria for assessment and were able to approach some consensus on grading. At the same time, the biomedical scientists were participating in wet lab exercises using different immunohistochemical protocols for antibodies used in the assessment of neurodegeneration. A final joint session was held during the last morning of the workshop where pathologists and biomedical scientists undertook joint review of the staining results at a multihead discussion microscope. Agreement was reached on the best protocols for staining, which has implications for best laboratory practice in the different BNE centres and elsewhere after publication.

Programme:


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Database development, September 2005, Munich


Date: September 2005

Place: Munich Centre for Neuropathology and Prion Research, Germany

Organised by: Mr Peer Schmitz (Munich) and Professor Jeanne Bell (Edinburgh)

Summary:
This course covered the essentials of data protection and steps which must be taken to protect confidentiality in databases that contain patient information. The principles of access to database, password policies, antivirus software, network management and back-up polices were all covered.
This course was run in conjunction with a laboratory and microscope based course in the comfortable accommodation of the Munich Centre for Neuropathology and Prion Research


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Stereology Workshop (1), September 2005, Aarhus


Date: September 2005

Place: Aarhus University, Denmark

Organised by: Prof N Bogdanovic (Huddinge)

Attended by: 16 neuroscientists and neuropathologists
Advertised among BNE members

Summary of Course

This 3 day course commenced with a general introduction to the principles of stereology and provided seminars on basic statistics and the definition of probes, volume estimation, Cavalieri and fractionator principles, dissector and 3D sampling, and factors which might influence the practice of stereology such as tissue deformation.  Practical exercises were made available for the participants.

During the evenings, the participants had the opportunity of attending a concert in the Musikhuset in Aarhus, followed by dinner with the time to get to know each other.  They were also well looked after on the second evening with dinner in a local restaurant – an opportunity to relax after two intensive working days.

Course Evaluation

13 responses were received.  All but 2 had limited or very limited previous experience in stereology whereas 2 participants rated their knowledge as satisfactory or good.  The overall assessment of seminars was good to very good by 11 participants and the remaining two considered most of the content good to very good.  The number of participants and the duration of the course was rated just right by 11 participants; 2 participants thought that the course should have been longer.

Individual comments commended the exercises and the availability of lecturers for help and dealing with queries.  The course opened the eyes of participants to the potential pitfalls in methodology and allowed participants to take a more rigorous approach to these problems in their further work.  Requests were made for hand-outs of the actual presentations, a glossary of definitions and some pre-course reading material.  All felt that this provided a good foundation for further work and it is notable that both the participants with some previous experience of stereology rated the course to be very good and would definitely recommend it to other people as did all the other participants.
Summary

This was a well organised course meeting a particular need of BNE members.  Useful training was delivered and was much appreciated.  Future courses in stereology should not seek to have greater numbers of participants and, on balance, should be about the same duration.  Some pre-course preparation for those who would welcome it might be valuable and copies of all the presentations should be made available to maximise the further benefit.

Conclusion

A well run course meeting a particular need and which should be repeated.

 


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Macro- and Microdissection, June 2005, Budapest


Date: 8 June 2005

Place: Semmelweis University, Budapest, Hungary

Organised by: Prof Miklos Palkovits & Team

Summary:
This half day workshop provided an opportunity for neuroscientists and neuropathologists to observe the microdissection technique developed in the Anatomy Department of Semmelweis University. Using a punch biopsy applied to slices of fresh brain tissue, the individual nuclei of the brain can be targeted for separate storage and subsequent analysis. This course provided an update in the technique and an overview of neuroanatomy of the human brain. An opportunity was provided for participants to have hands on practice while observing appropriate safety techniques. This technique is particularly suitable for researchers wishing to undertake neurochemical analsyses and proteomics. A training video has been prepared to provide details of microdissection and this was made available to members of BNE.


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