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Role of Microglia Cells & Application to Clinics

Role of microglia cells is studied in two contexts, the first one is related to nerve cord regeneration of the Medical Leech (H. medicinalis) and the second one to spinal cord injury of vertebrates (rat model)

Hirudo medicinalis
Microglia are intrinsic components of the central nervous system (CNS). During pathological events in mammals, inflammatory processes implicate the resident microglia and the infiltration of peripheral immune cells of which macrophages. Because they are implicated as sensors of pathological changes in CNS tissue, the cytokines produced by microglial cells represent interesting markers of cell activation and contribute to reactive processes. Functions of microglia appear to be complex as they exhibit both neuroprotective and neurotoxic effects. For a long time, some authors have focused on the understanding of mechanisms of microglial activation during neuropathological conditions in vivo and in vitro. But the limiting question is to discriminate the activated microglial cells from the peripheral infiltrating macrophages, a step which is a necessary prerequisite to understand successive steps occurring in CNS pathological situations.

Highlight the recruitment process of leech microglia In our project, the medicinal leech Hirudo medicinalis is used to highlight the activation processes of resident microglial cells. In the laboratory for many years, the leech was studied due to its ability to naturally repair its central nervous system (CNS) following injury such as a crush or a complete section of the nerve cord. This experimental model offers original opportunities to study the molecular and cellular basis of the CNS repair processes leading to functional recovering. Of interest, when the leech CNS is injured, resident microglial cells migrate and accumulate at the site of lesion. The microglial activation can be studied using similar markers to mammalian microglia. Importantly, this phenomenon is known to be essential for the usual sprouting of injured axons and does not require any infiltrating blood cells as we will describe in the proposal. The group activities focus on (i) the complete characterization of leech chemotactic factors responsible of the microglial recruitment, (ii) the identification of nerve cells producing these molecules, (iii) the action kinetics of the different chemoattractants on microglia, and (iv) the study of different microglial subsets by identifying the specific receptors for the respective cytokines. In any case, the activation of such microglial sub-populations is studied to evaluate their respective effect on the neuritic growth. This topic aims to better understand the mechanisms of microglial recruitment in order to explore in parallel the crosstalk between damaged neurons and the different microglia subsets. Indeed, this dialog is necessary to understand the balance of inflammation leading to the leech CNS repair.

Specify the functions of activated microglia in the crosstalk with damaged neurons The leech microglial cells can be activated in vitro or ex vivo by recombinant cytokines in order to study the molecular response to the activation. We focused the analysis on the secreted molecules to identify important proteins involved in the dialog with neurons. This topic uses our proteomic platform by combining nano-LC trap and Mass spectrometry. First results have been obtained from mouse BV2 microglial cells and showed the possibility to characterize the differential secretome of cells following the specific activation by only one type of cytokine. The study can be reproduced by using different cytokines exerting pro- or anti-inflammatory functions. In fine the aim is the identification of the different functional microglial sub-populations which are involved in the crosstalk with neurons. In the medicinal leech, this interaction is considered as an efficient mechanism since scientists have observed a complete, innate and functional nerve repair.

Spinal cord injury (SCI) belongs to serious, currently incurable disorders of CNS, which is often accompanied by permanent health consequences-disability. Many patients remain paralyzed with complete or partial loss of neurological function below the injury site. The clinical symptoms are consequences of degenerative changes manifested by cell loss, demyelization and the formation of central cystic cavity that disrupts the ascending and descending axonal pathway. Treatment of patients with SCI is one of the major challenges for modern medicine and for the clinician in addition to restore motor functions, sensation and relieve accompanying complications, which may partially improve the quality of life for patients. Regenesis project is based on the knowledge of nervous system regeneration of the leech to better improve the limited regeneration of spinal cord (SC) in higher vertebrates (mammals). Indeed, we previously demonstrated that regeneration is tightly related to 1) inflammation control through at least microglial cells and 2) neurite outgrowth, recognition and reconnection through trophic factors and gap junction molecules. The basic concept underlying the Regenesis project is to control inflammation especially by switching microglia through M2 polarization (Figure 1), use leech secretome in conjunction of secretome of Mesenchymal stem cells (MSCs) (Figure 2) and apply them on rodent models in order to improve SC regeneration after injury in mammals. For this purpose, MALDI-MSI, and on tissue proteomic technologies as well as shot-gun proteomic analyses on different collected secretomes will be undertaken. In vitro, ex vivo and in vivo experiments (Figure 3) on rat will be also the key technologies used to realize this project.