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Basic Research on Multiple Sclerosis: animal and cellular models
Spain (IFMSA-SPAIN) - University of Oviedo, Oviedo
Morphology and Cell Biology Department. Faculty of Medicine. C/Julián Clavería, 8. 33006. Oviedo. Principado de Asturias. Spain. (Head of the Department: Santos Suárez Garnacho)
Ana María Navarro Incio, Eva Martínez Pinilla, Eva María del Valle Suárez
Ana María Navarro Incio, Eva Martínez Pinilla, Eva María del Valle Suárez
English, Spanish, Italian, Portuguese
Type of Research Project
- Basic science
What is the background of the project?
This project is part of a bigger one that focuses on the role of Apolipoprotein D in the demyelination and remyelination processes. Multiple Sclerosis (MS) is a central nervous system inflammatory and demyelinating disease, mainly of autoimmune aetiology. Most of the approved treatments for MS are immunologic but those focused on direct protection of oligodendrocytes, the myelin producer cells, are scarcely used. Apolipoprotein D (Apo D) is an oligodendrocyte- synthesized lipocalin that stands out for being one of the most overexpressed molecules during nervous repair process, being necessary for a correct remyelination. For these reasons, it is among our aims to know the role of Apo D in a mouse MS experimental model (wild type, KI and KO for Apo D) by cuprizone treatment as a demyelinating agent. On the other hand, we propose the intervention in the remyelination process by clozapine treatment (this drug is a well-known antipsychotic, which causes Apo D overexpression in the central nervous system) in animals with induced MS to prevent axonal damage. At the same IFMSA International Secretariat, c/o IMCC, Nørre Allé 14, 2200 København N., Denmark time, we will address the study of the functions and interactions of Apo D, cuprizone and clozapine in cell lines and mouse oligodendrocyte primary cultures. The study will be completed with the analysis of Apo D expression and its relation with glial and inflammatory cells in samples from MS patients.
What is the aim of the project?
To deepen in the role of Apo D in the demyelination and remyelination processes in cellular and animals models of Multiple Sclerosis
What techniques and methods are used?
The study will be addressed at two levels: in vitro studies, in oligodendrocytes and neuronal cultures, and in vivo in an animal model of MS already established and based on cuprizone as a demyelinating agent. The in vitro studies will be performed at the cell culture facility of Department. The student will be working with HOG (human oligodendroglioma) and SH-SY5Y (human neuroblastoma) cells, performing protocols for the maintenance of the cell lines as well as different treatments with chemical and biological substances. Analysis of the viability of the cells will be performed by MTT and trypan blue assays. The in vivo studies will be performed at the animal house of the University and imply day to day routines as feeding and weighting of the animals every two days (the treatment with cuprizone and clozapine is provided with the food and water respectively) and neurological tests once a week. At the end of the treatment animals will be euthanized by intracardiac perfusion with fixative or by CO2 inhalation. After that, tissues will be collected for paraffin inclusion or frozen in nitrogen. With the first group, histochemical and immunohistechemical techniques will be performed to detected myelin levels and the presence of different protein; the second group of tissues will be used to DNA, RNA and total protein extraction, the samples obtained will be analyzed by Slot-blot, Western Blot and real time quantitative PCR (Polymerase Chain Reaction). This will allow us to measure proteins levels as well as the possible differences in gen expression due to the treatments. We use photographic techniques to document the experiments (in vivo and in vitro) and perform quantification analysis of the results (for a detailed explanation of the quantification technique see Tolivia Jet al. Anal Quant Cytol Histol. 2006 Feb;28(1):43-53). As the statistical analysis is the final state of the project is not likely that the student would be involved in that part.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Every task performed by the student will be under supervision at the beginning but, depending on the skills of the student, eventually he/she will perform the task on his/her own. 1) Animal handling: change the food and water of the animals and weight them every two days. 2) Neurological test (rota-rod): the student will collaborate in this task by taking record of the times and speed of the animals. 3) Mice treatments: the student will prepare the amount of cuprizone and clozapine necessary for the treatment of the animals.4) Histochemistry: the student will perform the Luxol Fast Blue technique to visualize the myelin in tissue samples. 5) Cell culture: the student will help to maintain the cell lines in the lab (changing media and passing cells). 6) Help with the day to day routine in the lab: as the student will be fully integrated in our lab he/she will help with any routine task as, for example, preparation of buffers.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
As part of the induction program, the student will attend to a short seminar about the previous development of the project; specific literature in English will be provided as well.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic lab skills; willing to learn; teamwork; independent thinking
Are there any legal limitations in the student’s involvement
Unless the student had an animal handling certificate, the tasks performed with living animals must be always performed under supervision.
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields
- del Valle E; Navarro A; Martinez-Pinilla E; Torices S; Tolivia J. Apo J and Apo D: Complementary or Antagonistic Roles in Alzheimer's Disease? J Alzheimers Dis. 2016 May 17;53(2):639-50. doi: 10.3233/JAD-160032.
- Martinez-Pinilla E; Navarro A; Ordóñez C; del Valle E; Tolivia J. Apolipoprotein D subcellular distribution pattern in neuronal cells during oxidative stress. Acta Histochem. 2015 Jul;117(6):536-44. doi: 10.1016/j.acthis.2015.04.003. Epub 2015 May 3.- Navarro A; Méndez E; Diaz C; Del Valle E; Martinez-Pinilla E; Ordóñez C; Tolivia J. Lifelong expression of apolipoprotein d in the human brainstem: correlation with reduced age-related neurodegeneration. PLoS One. 2013 Oct 22;8(10):e77852.
- Martinez E; Navarro A; Ordóñez C; Del Valle E; Tolivia J. Amyloid-β25-35 induces apolipoprotein D Synthesis and growth arrest in HT22 hippocampal cells.J Alzheimers Dis. 2012;30(2):233-44
- Rioseras B; Rodríguez A;. Torices S; González C; del Valle E; Navarro A; Tolivia J. Apoliporptoein D decrease indemyelination plaques in multiple sclerosis. TERMIS- EU;26:424(2011).ISSN:0213-3911.
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