Elsevier

Neurobiology of Disease

Volume 113, May 2018, Pages 70-81
Neurobiology of Disease

Seizure progression and inflammatory mediators promote pericytosis and pericyte-microglia clustering at the cerebrovasculature

https://doi.org/10.1016/j.nbd.2018.02.002Get rights and content

Highlights

  • Pericyte-microglia pathology is a mechanism of BBB damage in epilepsy.

  • Pericyte-microglia clusters form at the hippocampal capillaries post-SE and during epileptogenesis.

  • Pericytosis is provoked by pro-inflammatory cytokines.

  • Human TLE-HS and FCD type IIb display perivascular microglia aggregates in the lesional areas.

Abstract

Background

Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications.

Methods

In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1β, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly.

Results

A disarray of NG2DsRed+ pericyte soma and ramifications was found 72 h post-SE and 1 week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72 h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1β elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes.

Conclusions

These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.

Introduction

Blood-brain barrier (BBB) damage and inflammation occur during the progression of central nervous system (CNS) diseases, including epilepsy (Marchi et al., 2014, Marchi et al., 2012; Vezzani et al., 2011; van Vliet et al., 2014). Recent evidence has introduced pericytes as a possible contributor to neuro-inflammation (Rustenhoven et al., 2017), with pericyte damage facilitating BBB failure (Sweeney et al., 2016). Pericytes are located on the abluminal side of brain arterioles and capillaries and are characterized by a round soma and ramifications enveloping the endothelium. Pericytes communicate with astrocytic end-feet and participate in structural and homeostatic BBB functions (Armulik et al., 2005, Armulik et al., 2011). Pericytosis, a phenomenon where pericytes redistribute and participate in immune changes, may occur in pathological conditions (Sweeney et al., 2016; Armulik et al., 2011, Armulik et al., 2010; Bell et al., 2010; Rustenhoven et al., 2017; Zehendner et al., 2015). Recent findings have demonstrated that pericytes can acquire a myo-fibroblast phenotype in response to pathological insults, (Rustenhoven et al., 2017; Goritz et al., 2011).

Although BBB damage and inflammation are well accepted to occur in epilepsy, limited information exists on whether pericytes are involved in the epileptic pathophysiology (Marchi et al., 2014, Marchi et al., 2007; Marchi and Lerner-Natoli, 2013). This constitutes a knowledge-gap considering that, based on their anatomical position at the abluminal side of capillaries, pericytes could mediate a pathological crosstalk between activated parenchymal glia and the BBB endothelium during seizures (Librizzi et al., 2017; Abbott et al., 2010). Initial evidence has indicated pericyte reorganization at the BBB 24 h after experimental status epilepticus (Librizzi et al., 2017; Milesi et al., 2014). Pericyte hypertrophy and detachment from the capillary basal lamina were found in human brain specimens obtained from temporal lobe or focal cortical epilepsies (Garbelli et al., 2015; Liwnicz et al., 1990).

Here we tested the hypothesis that pericyte reactivity, or pericytosis, occurs during experimental epileptogenesis and human epilepsy, interplaying with activated microglia around the capillaries. We tested the corollary hypothesis that pro-inflammatory cytokines, present in the brain parenchyma during seizure activity, initiate pericyte modifications. These hypotheses were investigated using an in vivo model of SE-induced epileptogenesis and spontaneous recurrent seizures (Heinrich et al., 2011, Heinrich et al., 2006; Meier et al., 2007; Pallud et al., 2011; Sierra et al., 2015), in vitro hippocampal slices derived from NG2DsRed mice, and human-derived pericytes culture. To further study pericytes and microglial cells at the capillaries, we analyzed human brain resections obtained from patients affected by drug-resistant epilepsy. Cases of epilepsy where an involvement of inflammatory pathways has been described, e.g., hippocampal sclerosis and focal cortical dysplasia (FCD) type II, were examined (Boer et al., 2006; Iyer et al., 2010; Ravizza et al., 2008).

Section snippets

Patient selection and neuropathological evaluation

We used brain tissues previously obtained from patients who underwent surgery for intractable epilepsy (C. Besta Neurological Institute and C. Munari Epilepsy Surgery Centre, Niguarda Hospital, Milan, Italy). The surgical specimens were removed for therapeutic reasons and after informed consent. A total of 13 cortices and five hippocampi (temporal lobe epilepsy, n. 6, 7, 8 and 12, 13; Table 1) were available and evaluated for histology. Specifically: i) five patients with a neuropathological

Pericyte modification and cerebrovascular microglia aggregates during epileptogenesis

In the hippocampus of control mice, NG2DsRed+ pericytes presented with a typical bump-on-a-log soma and thin ramifications lining the capillary surface. IBA1+ microglial cells were uniformly distributed in the parenchyma and proximal to the capillaries (Fig. 1A, and Supplemental Movie 1). At one week post-SE (epileptogenesis), pericyte showed disorganized soma and ramifications, especially in the CA3 and DG areas where IBA1+ microglial cells accumulated around the pathological pericytes (Fig. 1

Discussion

We here report pericytosis topographically coinciding with cerebrovascular clusters of microglial cells in experimental epileptogenesis and in human epilepsy. We have identified pericyte engulfment by the accumulating microglia, a potentially harmful event to the BBB during seizures. Our results suggest that IL-1β promotes pericyte modifications and pericyte-microglia clustering. A spatio-temporal proliferation of pericytes in the hippocampus is consistent with a pericytosis phenotype occurring

Acknowledgement

This work was supported by ANR-Epicyte, Fédération pour la Recherche sur le Cerveau (FRC-Marchi) and Cure-Innovator Award (CURE) to NM. ERANET-NEURON JTC2014 BriE ANR14 Neur0004, EU grant FP7 (DESIRE) No. 602531.

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