Fatty acid amide hydrolase inhibitor URB597 may protect against kainic acid–induced damage to hippocampal neurons: Dependence on the degree of injury

Highlights

• The structure of neuronal mitochondria, ER, and nuclei altered after KA-evoked SE.

• A correlation between the severity of SE and neuronal damage was established.

• Nonconvulsive SE-induced injury was prevented or diminished by URB597.

• URB597 failed to reverse most of alterations after convulsive seizures.

Abstract

Objective

Status epilepticus (SE) provokes changes, which lead to neuronal alterations. Endocannabinoids (eCBs) can affect the neuronal survival during excitotoxicity and brain damage. Using a kainic acid (KA)-induced experimental SE model, we investigated whether cellular changes entail damage to endoplasmic reticulum (ER), mitochondria, and nuclei in hippocampal cells (CA1 field), and whether these alterations can be diminished by treatment with URB597, an inhibitor of eCB enzymatic degradation.

Material and methods

SE was induced in Wistar rats by the microinjection of KA into the lateral ventricle. URB597 or a vehicle (10% DMSO) were injected in the same way into the brain of animals 24 h after the KA infusion and then daily for the next nine days. The behavior of animals was controlled visually and recorded with a video system. The intensity of SE significantly varied in different animals. Convulsive (stages 3–5 according to the Racine scale) and nonconvulsive seizures (mainly stages 1, 2 and rarely 3, 4) were recognized.

Results

Two weeks after SE, a significant loss of hippocampal cells occurred in animals with KA injections. In survived cells, ultrastructural alterations in ER, mitochondria, and nuclei of hippocampal neurons were observed. The degree of cell injury depended on the severity of SE. Alterations evoked by moderate seizures were prevented or diminished by URB597, but strong seizures induced mostly irreversible damage.

Conclusions

The beneficial impact of the FAAH inhibitor URB597 can give impetus to the development of novel neuroprotective strategies.

Introduction

KA–induced excitotoxicity usually leads to cascades of cellular events, including activation of late cell death pathways (Macdonald and Kapur, 1999, Cock, 2002, Mikati et al., 2003). Status epilepticus (SE) is a medical emergency that can cause permanent neurologic and mental disability (Manno, 2003, Wasterlain et al., 1993). SE in humans and animal models results in considerable cerebral alterations and increases the risk of delayed repeated seizures, along with a characteristic pattern of neuronal cell loss preferentially in the hippocampus (DeGiorgio et al., 1992, Mikati et al., 2003, Wasterlain et al., 1993). As a result, the seizure focus often forms in this area, which usually leads to the development of temporal lobe epilepsy in humans (for review, see Levesque and Avoli, 2013).

To our knowledge, relatively few studies addressed the changes in the ultrastructure of hippocampal cells after SE. Evidence presented in these works suggest that the structural abnormalities and dysfunction of ER and mitochondria occur as a consequence of prolonged epileptic seizures and may play an important role in seizure-induced brain damage (Kunz et al., 1999, Kudin et al., 2002, Cock, 2002, Chuang et al., 2004, Chen et al., 2013, Kotaria et al., 2013, Zhvania et al., 2015). At the same time, the functions of ER, the nucleus, and mitochondria are crucial for cellular homeostasis, and their alterations usually lead to multiple human disorders, including neurodegenerative diseases.

One of the promising approaches to the suppression of seizure pathology may be the activation of the endocannabinoid (eCB) system as a natural homeostatic regulator (for review, see Katona and Freund, 2008, Kano et al., 2009). This system includes the cannabinoid G_i/o-coupled CB1 and CB2 receptors, their endogenous ligands (endocannabinoids, eCBs), and the associated enzymes participating in the synthesis, transport, and degradation of these ligands. CB1 and CB2 receptors are mainly expressed in the brain and immune system, respectively (Pertwee, 2008). Two primary eCB ligands have been identified: N-arachidonoyl ethanolamide (anandamide), which activates preferentially CB1 cannabinoid receptors, and 2-arachidonoyl glycerol (2-AG), which is active at both CB1 and CB2 receptors. The eCB system is abundantly expressed in the brain and regulates a plethora of physiological functions. It is interesting that CB1 receptors can be distributed in different intracellular compartments, e.g., mitochondria (for review, see Busquets Garcia et al., 2016). The synthesis of eCBs from membrane precursors is carried out “on demand” in neurons depending on spatial, temporal and cellular features of activated neural networks and in response to damaging agents (Khaspekov et al., 2004, Esch et al., 2006, Katona and Freund, 2008). eCB neuroprotection can be provided via the regulation of neuronal excitability, the control of inflammatory processes playing an important role in the neurodegeneration, the stimulation of production of neurotrophic factors, and also by means of indirect mechanisms.

It has been earlier shown that the initiation of cannabinoid receptor-dependent protection may result from toxic influence and damage (Karanian et al., 2005, Karanian et al., 2007, Khaspekov et al., 2004, Soltesz et al., 2015). We have reported previously that KA-evoked persistent seizures in limbic brain structures can be suppressed or attenuated by eCB-related drugs (Shubina et al., 2015). The present work is aimed at studying the KA-evoked subcellular damage to the hippocampus and clarifying whether the induced alterations can be diminished by the eCB-related influence after the termination of SE. We used i.c.v. injection of URB597, a selective inhibitor of fatty acid amide hydrolase (FAAH). FAAH is the primary degradation enzyme for the endocannabinoid anandamide, and, therefore, the inhibition of FAAH leads to extend the time of anandamide action in the brain and the prolongation/enhancement of CB1 receptor activity.