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Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4(pos) T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350-1362., (© 2016 Wiley Periodicals, Inc.)

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45 ± 0.07 mm(2) in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P<0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

Status epilepticus (SE) induced by pilocarpine or kainate is associated with yet not systemically investigated astrocytic and vascular injuries. To investigate their possible association with neuronal damage, the changes in glial fibrillary acidic protein (GFAP), laminin and neuron-specific nuclear protein (NeuN) immunoreactivities were analyzed in rats treated with pilocarpine (380 mg/kg) or kainate (15 mg/kg), and receiving diazepam (20mg/kg) after 10 min of SE. A different group of rats was injected with endothelin-1 (ET-1) in the caudate putamen to reproduce the changes in GFAP and laminin immunoreactivities associated with ischemia. Focal loss of GFAP immunostaining was accompanied by increased laminin immunoreactivity in blood vessels, in all the examined groups. Regression analysis revealed a significant (P<0.01) relationship between astrocytic lesion and increased laminin immunoreactivity in the piriform cortex (Pir) of both pilocarpine (R(2)=0.88) and kainate (R(2)=0.94) groups of treatment. A significant relationship (P<0.01; R(2)=0.81) was also present in the cornu Ammonis 3 (CA3) hippocampal region of pilocarpine-treated rats. At variance, neuronal and glial lesions were significantly related (P<0.05, R(2)=0.74) only in the substantia nigra of pilocarpine-treated rats. The ratio between areas of GFAP and laminin changes of immunoreactivity in the ET-1 group was similar to those found in pilocarpine- and kainate-treated rats in specific brain regions, such as the hippocampal CA3 subfield, Pir and the anterior olfactory nucleus. The amygdala and submedius thalamic nucleus in the pilocarpine group, and the perirhinal and entorhinal cortices in the kainate group, also presented ischemic-like changes. These results indicate that laminin immunoreactivity is upregulated in the basal lamina of blood vessels after SE induced by pilocarpine or kainate. This phenomenon is significantly associated with lesions involving more glial than neuronal cells, in specific cerebral regions., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Understanding the pathophysiogenesis of temporal lobe epilepsy (TLE) largely rests on the use of models of status epilepticus (SE), as in the case of the pilocarpine model. The main features of TLE are: (i) epileptic foci in the limbic system; (ii) an "initial precipitating injury"; (iii) the so-called "latent period"; and (iv) the presence of hippocampal sclerosis leading to reorganization of neuronal networks. Many of these characteristics can be reproduced in rodents by systemic injection of pilocarpine; in this animal model, SE is followed by a latent period and later by the appearance of spontaneous recurrent seizures (SRSs). These processes are, however, influenced by experimental conditions such as rodent species, strain, gender, age, doses and routes of pilocarpine administration, as well as combinations with other drugs administered before and/or after SE. In the attempt to limit these sources of variability, we evaluated the methodological procedures used by several investigators in the pilocarpine model; in particular, we have focused on the behavioural, electrophysiological and histopathological findings obtained with different protocols. We addressed the various experimental approaches published to date, by comparing mortality rates, onset of SRSs, neuronal damage, and network reorganization. Based on the evidence reviewed here, we propose that the pilocarpine model can be a valuable tool to investigate the mechanisms involved in TLE, and even more so when standardized to reduce mortality at the time of pilocarpine injection, differences in latent period duration, variability in the lesion extent, and SRS frequency.