Continuous neurodegeneration and death pathway activation in neurons and glia in an experimental model of severe chronic epilepsy
Introduction
Several studies in experimental models and patients with chronic epilepsy suggested that recurring seizures could produce long-term alterations in neuronal circuits including synaptic re-organization, hippocampal mossy fiber sprouting, and neurogenesis (Buckmaster and Dudek, 1997, Cha et al., 2004, Gorter et al., 2001, Pitkänen and Sutula, 2002). Whether seizures might eventually lead to the activation of cell death pathways and whether neuronal death could play a role in epileptogenesis is still highly debated (Dingledine et al., 2014). In human patients, diffuse cell injury and death were reported in children with intractable epilepsy (Choi et al., 2009), and longitudinal or cross-sectional MRI studies suggested slowly progressive neocortical and hippocampal atrophy in drug-resistant temporal lobe epilepsy (TLE) cases (Bernhardt et al., 2009, Fuerst et al., 2003). By contrast, in experimental epilepsy models, the prevailing view considered cell death the direct consequence of the initial insult (i.e., status epilepticus or SE) and not significantly related to subsequent seizures (Gorter et al., 2003, Pitkänen et al., 2002).
Among cell death pathways, caspase-3 is the main executioner protease of the apoptotic cascade (Kajta, 2004). Data in experimental models showed that SE triggered caspase-3 activation and cell death (Ferrer et al., 2000, Henshall et al., 2000a) for a limited time-period after the initial insult (Faherty et al., 1999, Narkilahti et al., 2003a) with no direct contribution of further recurring seizures (Pitkänen et al., 2002). The application of caspase-3 inhibitors reduced SE-induced neuronal loss in the rat model based on amygdala stimulation (Narkilahti et al., 2003b). In humans, elevated levels of cleaved caspase-3 were found in the temporal cortex of patients surgically treated for drug-refractory epilepsy (Henshall et al., 2000b). All these data underscored the potential relevance of active caspase-3 in human and experimental epilepsy.
Another cell mechanism possibly leading to neuronal loss is the activation of c-Jun N-terminal kinase (JNK), which in turn activates the transcription factor c-Jun. The JNK pathway is implicated in multiple physiological processes, in particular in the response to stress stimuli (Antoniou and Borsello, 2012). Regarding cell death, c-Jun activation was associated with resistance to death in C57BL/6 mice after kainic acid (Schauwecker, 2000). However, a number of data in different experimental models pointed out that phosphorylated c-Jun (phospho-c-Jun or p-c-Jun) mediated excitotoxic cell death (Ferrer et al., 1996a, Herdegen et al., 1998, Mielke et al., 1999, Yang et al., 1997). Accordingly, inhibiting the JNK pathway with the cell-permeable peptide D-JNKI-1 provided strong neuroprotection against excitotoxicity in cerebral ischemia in vitro and in vivo (Borsello et al., 2003) and reduced excitotoxic neuronal death in kainic acid-induced SE in vivo (Spigolon et al., 2010).
Previous data in experimental epilepsy models, therefore, demonstrated that the occurrence of SE could rapidly activate pathways able to produce cell death. However, whether such activation is only temporally related to the induction of SE, whether and what pathways are activated and what cell types are involved in the cell death processes during the course of chronic seizures remain to be clarified. This is of particular relevance for brain malformations associated with severe drug-resistant epilepsy, such as focal cortical dysplasia (FCD). Apoptotic cell death may occur in FCD patients (Choi et al., 2009, Iyer et al., 2014), and we recently demonstrated reduced neuronal density in dysplastic cortical areas of FCD patients with longer vs shorter epilepsy duration, thus suggesting an ongoing, seizure-related neuronal degeneration in the malformed epileptic human brain (Finardi et al., 2013).
To address the relationship between the recurrence of seizures and the occurrence of cell death, we exploited here an experimental model of acquired FCD, the methylazoxymethanol/pilocarpine rat (MAM-PILO or MP rat, Colciaghi et al., 2011, Colciaghi et al., 2014, Marchi et al., 2006) generated in our lab. In particular, by analyzing MP rats at different time points after SE induction (from few hours up to 6 months after epilepsy onset) we verified (through Fluoro-Jade, caspase-3, and c-Jun labeling) when, where, and how cell death occurred in neurons or glia. We demonstrated the existence of an ongoing neurodegenerative process and the activation of distinct cell pathways in both neuronal and glial cells.
Section snippets
Ethical statement
Procedures were carried out with care to minimize discomfort and pain to treated rats, in compliance with national (D.L. 116 Suppl 40/1992 and D.L. 26/2014) and international guidelines and laws (2010/63/EU Legislation for the protection of animals used for scientific purposes). The experimental protocol was approved by the Ministry of Health, Italy (protocol number: BR1/2012).
MAM and pilocarpine administration
Pregnant Sprague–Dawley rats (Charles River, Calco, Italy) received two intraperitoneal (ip) doses of MAM (15 mg/kg
Seizure onset and evaluation
Four MP rats were sacrificed 18 h after SE onset and referred to as MP-acute. All the remaining 25 MP rats experienced spontaneous seizures after SE. Mean seizure onset was 8.21 ± 4.63 days after pilocarpine-induced SE. Of the 25 rats, 8 rats (referred to as MP-EC) were sacrificed 3–5 days after the onset of the first clinical seizure, 9 rats were sacrificed after 3 and 8 rats after 6 months of chronic epileptic seizures and referred to as MP-3m and MP-6m, respectively. The video-monitoring
Discussion
A bulk of evidence indicated that in experimental models of epilepsy the occurrence of SE was associated with the activation of neurodegenerative processes culminating in cell death, particularly in neurons (do Nascimiento et al., 2012, Liu et al., 2010). Most studies, however, suggested that neuronal cell death was temporally related to the acute phase following SE (Gorter et al., 2003, Pitkänen et al., 2002). In addition, some studies demonstrated that neuronal cell death, particularly in the
Conclusions
In summary, our results clearly indicate that cell death is an ongoing process active throughout all epilepsy stages in the MP model of chronic epilepsy. As already long established by other studies, and confirmed by the present data, SE is the main trigger of the cell damage. However, as clearly demonstrated here, seizure recurrence could not only contribute but also modify the diffusion and progression of cell death mechanisms in neurons as well as in glial cells. Finally, our MP rats could
Conflicts of interest
None declared.
Acknowledgments
The authors wish to thank Prof. Alessandro Vercelli for critically reviewing the manuscript, Dr. Marina Boido for support in the stereological analysis, and Dr. Andrea Legati for support in the statistical analysis. This work was supported by research grants from the Ministry of Health, Italy and AICE-FIRE to GB.
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