Final Report Summary - MICROGLIA-EPILEPSY (Microglia phenotype in temporal lobe epilepsy)
Mesial temporal lobe epilepsy (TLE) is the most common form of epilepsy in adult humans, where the epileptic focus is most frequently found in the hippocampal formation. This type of epilepsy is generally triggered by an insult, which includes trauma, infection, stroke, febrile convulsions and status epilepticus (SE). This is followed by a period called epileptogenesis where several cellular and neuronal circuit alterations occur, accompanied by prominent structural changes, which collectively configure the so-called mesial sclerosis, which include neuronal loss, connectivity abnormalities and gliosis.
A growing body of evidence is now supporting a relationship between inflammation and epilepsy. Indeed, activated microglia, reactive astrocytes, local expression of pro-inflammatory cytokines, blood brain barrier leakage and peripheral immune cell infiltration have all been observed in TLE animal models as well as in humans. Accordingly, inflammatory mechanisms are thought to play a central role in the initiation and maintenance of seizures, starting in the acute phase represented by status epilepticus (SE) induction. Microglia activation has been correlated with the expression of several pro-inflammatory cytokines which are thought to contribute to the neuronal cell death occurring after SE. Data point towards a pro-inflammatory phenotype of microglia that precedes neuronal injury and cell death. Because of this, microglia are generally considered to play a pro-epileptogenic role. However, infiltration of peripheral immune cells during epileptogenesis such as leukocytes, granulocytes and monocytes/macrophages might also contribute to the development of chronic epilepsy and recurrent seizures. Uncertainty on the role of these different inflammatory cells depended on technical limitations in the discrimination of microglia from macrophages. For this reason, it is possible that the detrimental function that is currently attributed to microglia might be incorrect and should be ascribed to infiltrating macrophages.
The project objectives were:
1- Determine the gene and protein expression profile of phenotype markers in microglia vs macrophages during epileptogenesis.
2- Examine of the localization of the most prominent markers identified in objective 1 and perform a correlation with the phases of epileptogenesis.
Both microglia and macrophages were acutely isolated from the hippocampi of control and pilocarpine-treated CD1 mice (24h and 96h after SE) and FACS sorted. Microglia were defined as CD11b+ CD45int Ly-6Cneg and infiltrated macrophages as CD11b+ CD45hi Ly-6Cpos. After sorting, qPCR analyses were performed. Some animals were sacrificed at 24h and 96h after SE
RESULTS. During epileptogenesis, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecule CD40 and pro-inflammatory gene IL-1beta. In contrast, infiltrated macrophages were strongly immune activated. Both cell types expressed high levels of the phagocytosis marker AXL.
CONCLUSIONS. These data suggest that macrophages might be more detrimental than microglia during epileptogenesis.
SOCIO-ECONOMIC IMPACTS. Based on results described in the previous section, a patent application is currently under development.
A growing body of evidence is now supporting a relationship between inflammation and epilepsy. Indeed, activated microglia, reactive astrocytes, local expression of pro-inflammatory cytokines, blood brain barrier leakage and peripheral immune cell infiltration have all been observed in TLE animal models as well as in humans. Accordingly, inflammatory mechanisms are thought to play a central role in the initiation and maintenance of seizures, starting in the acute phase represented by status epilepticus (SE) induction. Microglia activation has been correlated with the expression of several pro-inflammatory cytokines which are thought to contribute to the neuronal cell death occurring after SE. Data point towards a pro-inflammatory phenotype of microglia that precedes neuronal injury and cell death. Because of this, microglia are generally considered to play a pro-epileptogenic role. However, infiltration of peripheral immune cells during epileptogenesis such as leukocytes, granulocytes and monocytes/macrophages might also contribute to the development of chronic epilepsy and recurrent seizures. Uncertainty on the role of these different inflammatory cells depended on technical limitations in the discrimination of microglia from macrophages. For this reason, it is possible that the detrimental function that is currently attributed to microglia might be incorrect and should be ascribed to infiltrating macrophages.
The project objectives were:
1- Determine the gene and protein expression profile of phenotype markers in microglia vs macrophages during epileptogenesis.
2- Examine of the localization of the most prominent markers identified in objective 1 and perform a correlation with the phases of epileptogenesis.
Both microglia and macrophages were acutely isolated from the hippocampi of control and pilocarpine-treated CD1 mice (24h and 96h after SE) and FACS sorted. Microglia were defined as CD11b+ CD45int Ly-6Cneg and infiltrated macrophages as CD11b+ CD45hi Ly-6Cpos. After sorting, qPCR analyses were performed. Some animals were sacrificed at 24h and 96h after SE
RESULTS. During epileptogenesis, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecule CD40 and pro-inflammatory gene IL-1beta. In contrast, infiltrated macrophages were strongly immune activated. Both cell types expressed high levels of the phagocytosis marker AXL.
CONCLUSIONS. These data suggest that macrophages might be more detrimental than microglia during epileptogenesis.
SOCIO-ECONOMIC IMPACTS. Based on results described in the previous section, a patent application is currently under development.