 |
de | fr | en print view
3R-Project 103-06
An in vitro Model of Central Nervous System Infection and Regeneration: Neuronal Stem Cells as Targets of Brain Damage & Regenerative Therapies in Bacterial Meningitis.
Stephen L. Leib
Institute for Infectious Diseases, University of Bern, CH-3010 Bern, Switzerland
stephen.leib@ifik.unibe.ch
Keywords: bacteria; rat; heart; transplantation; ischemia; cell cultures: co-cultures; slices; reduction; replacement
Duration: 3 years End of the Project: 2010
Background and Aim
Bacterial meningitis remains a devastating disease with high morbidity and mortality. Despite advances in treatment and care, bacterial meningitis holds an in-hospital mortality of approx. 25% and as a consequence of brain damage, clinically relevant neurological sequelae emerge in up to 50% of survivors. How infection leads to brain injury remains largely unresolved, but converging evidence suggests that the clinical outcome of bacterial meningitis is determined by the host’s response to the infectious agent in the brain. The research on this subject faces great challenges because of the complexity of harmful processes which involve a variety of microbial factors as well as multiple aspects of the host’s inflammatory response.
Brain injury caused by bacterial meningitis prominently affects the cortex and the hippocampus. Cortical damage is associated with areas of focal ischemic necrosis. Hippocampal injury is documented in approx. 75% of patients dying from the disease and in corresponding animal models. This form of injury is characterized by apoptotic cell death of immature neurons, e.g. neuronal stem cells and/or their progeny in the dentate gyrus (Figure 1). This form of brain damage is associated with long lasting learning deficits. Hippocampal injury is limited to the dentate gyrus, a site of continuous formation of new neurons and therefore potentially well equipped for brain repair.
Research on the pathogenesis of infections of the brain and potential therapeutic approaches supporting brain repair functions largely depends on experimental studies in animals. The grade of suffering (Belastungkategorie) according to the Swiss federal veterinary office is considered intermediate to severe (e.g. categories 2-3) for many of these studies. In terms of animal use and welfare it would be highly desirable to replace a part of this research by in vitro studies. However, due to the multi-factorial pathogenesis of meningitis involving the interplay between the susceptible brain cell type, the bacterial pathogen and the host’s inflammatory reaction, the majority of the current research activities on meningitis are performed in vivo. With this project we plan to establish an in vitro model that reproduces important patho-physiological processes of damage and tissue regeneration in infectious diseases of the brain. In the framework of the project we propose:
AIM 1: To reduce and replace studies in animals by developing an in vitro system for the study of neuronal brain damage due to bacterial infection. This will be done by differentiating neuronal stem cells into defined developmental stages susceptible for injury, and subsequently challenging them with infectious pathogens and/or inflammatory mediators.
AIM 2: To reduce and replace studies in animals by establishing a co-culture model of neuronal stem cells and organotypic hippocampal cultures for the evaluation of the integrative potential of early neuronal cell lines as transplants into the targeted brain tissue.
Method and Results
in progress (present status)
We have developed an organotypic cell culture system which allows to culture hippocampal or cortical brain tissue in its original architecture and distribution of cell types (Figure 2). This system is a cornerstone of the proposed co-culture system of organotypic brain tissue with stem or progenitor cells isolated from different origins. This co-culture system will allow us to screen stem cells from different sources e.g. embryonic stem cells from the fetal brain or liver; stem cells of placental origin or stem cells of adult origin e.g. from neuronal or haematopoetic origin for a potential therapeutic application of neuronal stem cells. Furthermore, this approach will allow us to assess the stage of differentiation that is best suited for integration into the host tissue. This system may lead to a substantial reduction in the number of animals used, since only approaches that prove successful in the vitro system would be considered for further evaluation in vivo.
Conclusions and Relevance for 3R
The availability of an in vitro system would lead to a substantial reduction of animal use by the possibility to screen pathogenetic mechanisms for their relevance and therapeutic approaches for their potential effects prior to conducting studies in vivo. The proposed experimental in vitro system would allow the reduction or replacement of the following in vivo investigations:
i) Testing of pathogenic hypothesis by in vitro screening of potential bacteria-derived mediators (e.g. bacterial cell wall components) and potential host factors (e.g. host inflammatory mediators).
ii) Assessment of the intrinsic properties of the differentiated cells, which contribute to their selective vulnerability.
iii) Evaluation of therapeutic approaches able to counteract the selective vulnerability investigated under ii)
iv) Evaluation of therapeutic approaches that involve the grafting of stem cells / neuronal precursors into brain tissue.
Thus, once this system is established, studies screening for pathogenic factors and therapeutic feasibility studies in animals can be substantially replaced by in vitro studies.
Figures
Figure 1: Brain histology of infant rats with bacterial meningitis 24 h after infection.
A) Hippocampal histology of infant rats with pneumococcal meningitis 24 h after infection. Neurons forming apoptotic bodies (arrowhead), a morphological feature characteristic for programmed cell death, are scattered along the inner rim of the dentate gyrus and evenly distributed over the lower and the upper blade (Cresyl violet, x200). In the cortex neuronal injury consists of areas with markedly reduced neuronal density in a wedge-shaped distribution (arrowheads) suggestive of ischemic damage (x10).
B) Neurons with apoptotic bodies consisting of condensed and fragmented nuclei (arrowheads, x400).
C) Neurons in the subgranular zone of the dentate gyrus labeled with BrdU to identify multiplying cells (brown) and cells undergoing apoptosis by staining for activated caspase-3 (green).
D) Close up of apototic cells double positive for BrdU and caspase 3, indicating that they are multiplying progenitor cells or their progeny undergoing apoptosis.
Figure 2: Architecture and cell-type characterization and documentation of apoptosis in organotypic slices.
A) The cellular organization of the hippocampal slice culture system is shown in an overview stained with cresyl violet. Hippocampi are explanted from seven-day-old rats and cut into 400µm-thick slices. Experiments are started on day 11.
B) After in vitro stimulation with pneumococci, apoptosis with formation of characteristic apoptotic bodies (arrows) occurs in neurons of the dentate gyrus.
C and D) The cellular distribution pattern is assessed by cell-type specific markers. Shown here is a horizontal © and a vertical (D) section of organotypic hippocampal cultures. Cytoarchtectonic and cell type specific composition of organotypic cultures is shown by the immunohistochemical documentation of astrocytes by GFAP (green) and neurons by NeuN (red). Cellular nuclei are stained by DAPI (blue).
| Last modified 2010/08/29 |  |