The Characteristic Pattern And Related Mechanisms Of Acute And Delayed Neuronal Degeneration In Experimental Models Of Temporal Lobe Epilepsy

Temporal lobe epilepsy presents a serious neurological disorder in human beings and its long-term pathological events largely remain an obscure and severe obstacle in clinical treatment of patients. The epileptic process usually consists of three phases: initial insult→latency period (epileptogenesis)→recurrent seizures (symptomatic epilepsy). The epileptogenesis and recurrent seizure mechanisms in humans are poorly understood, but growing evidences have been obtained from animal models of epilepsy. The rodent animal models of status epilepticus (SE) have been extensively utilized in experimental studies of chronic epilepsy by injection of pilocarpine. Systemic administration of pilocarpine can induce neuronal loss in central nervous system, which shows striking similarities to human temporal lobe epilepsy in rats or mice.There are two major mechanisms of neuronal death in models of temporal lobe epilepsy. One mechanism is acute oedemic or ischemic death of the effected neurons. The other mechanism is delayed, and is likely to be mediated by a transient but abnormal rise in intracellular free calcium concentration. This may overactivate various enzyme systems normally involved in synaptic plasticity, e.g. calpain and protein kinases. Caspase-mediated programmed cell death (PCD) can contribute to delayed damage. Neuronal loss is one of the major components of circuitry reorganisation during epileptogenesis. Thus,the injury extent and time-course of neuronal death induced by SE attack still remain crucial question to answer, which may encase key neural basis of long-term changes and recurrent seizure in chronic epilepsy. Evidences have showed that neuronal loss in the chronic epilepsy could partially result from cell apoptosis, which is a programmed physiological event but also occurred in various toxic insults and neurological diseases through multiple ways of death signaling receptors, mitochondrial dysfunction, activation of caspase enzymes, and DNA damage. It is still important to further elucidate the contribution of an apoptotic mechanism to the pathological process of chronic epilepsy.Purpose: In the present study, we are interested in elucidating long-term brain injury and related mechanisms that may occur in the temporal lobe epilepsy. Methods: The regional distribution and time-course of degenerative neurons were examined in a mouse pilocarpine model of chronic epilepsy by Fluoro-Jade C (FJC) dye that can specifically stain degenerating neurons in the central nervous system. The type of affected neurons and mechanisms involved in cellular damage were also elucidated by combining with immunofluorescence technique. FJC is a new-developed fluorescent Fluoro-Jade (FJ) dye which has high affinity for degenerating neurons including cell body, dendrites, axons and axon terminals. Results: (1) The FJC stain combined with neuronal nuclear specific protein (NeuN) immunofluorescence revealed that pilocarpine-induced SE resulted in massive degenerative death of neuronal cells in many brain regions from olfactory bulb to midbrain. (2) Moreover, cellular degeneration including acute and delayed neuronal death revealed by FJC stain was time-dependent. The FJC-positive degenerating neurons occurred at 4h, increased into peak levels at 12h–3d, and then gradually went down, even resolved to baseline or disappeared at 7d–14d after onset of SE. (3) Double-labeling data revealed that cellular co-localization of FJC and Hoechst was abundantly observed and most of FJC-positive degenerating neurons also expressed apoptosis signaling molecules such as cytochrome C, caspase-9, and activated caspase-3, indicating that these FJC-positive cells maybe were undergoing apoptotic processes and were in an early phase of apoptosis. These may also imply that caspase-mediated PCD plays an important role in the delayed damage. (4) More interestingly, a large percentage (about 88%) of FJC-positive degenerative neurons were GABAergic as indicated with their immunoreactivity to glutamic acid decarboxylase-67 (GAD-67), implying that inhibitory function of GABAergic neural system might by seriously damaged in brains subject to SE attack in this mouse pilocarpine model.Conclusion: This study has first applied FJC staining to demonstrate regional distribution, time-course, and related mechanisms of neuronal death in the mouse pilocarpine model. Taken together with previous studies, time-course and death mechanisms of degenerative GABAergic neurons in the mouse pilocarpine model revealed by FJC staining benefit further understanding of long-term brain pathological changes and recurrent seizure mechanism, and, from the point of possible pharmacological intervention, may also result in finding the most suitable time-window in therapeutic manipulation of the chronic epilepsy in human beings and appropriate neuroprotective treatment to prevent or lessen seizures during the epileptogenic phase.