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- MATERIALS AND METHODS
Summary: Purpose: Prolonged and continuous epileptic seizure (status epilepticus) results in cellular changes that lead to neuronal damage. We investigated whether these cellular changes entail mitochondrial dysfunction and ultrastructural damage in the hippocampus, by using a kainic acid (KA)-induced experimental status epilepticus model.
Methods: In Sprague–Dawley rats maintained under chloral hydrate anesthesia, KA (0.5 nmol) was microinjected unilaterally into the CA3 subfield of the hippocampus to induce seizure-like hippocampal EEG activity. The activity of key mitochondrial respiratory chain enzymes in the dentate gyrus (DG), or CA1 or CA3 subfield of the hippocampus was measured 30 or 180 min after application of KA. Ultrastructure of mitochondria in those three hippocampal subfields during KA-induced status epilepticus also was examined with electron microscopy.
Results: Microinjection of KA into the CA3 subfield of the hippocampus elicited progressive build-up of seizure-like hippocampal EEG activity. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (marker for Complexes I+III) in the DG, or CA1 or CA3 subfields 180 min after KA-elicited temporal lobe status epilepticus. Conversely, the activities of succinate cytochrome c reductase (marker for Complexes II+III) and cytochrome c oxidase (marker for Complex IV) remained unaltered. Discernible mitochondrial ultrastructural damage, varying from swelling to disruption of membrane integrity, also was observed in the hippocampus 180 min after hippocampal application of KA.
Conclusions: Our results demonstrated that dysfunction of Complex I respiratory chain enzyme and mitochondrial ultrastructural damage in the hippocampus are associated with prolonged seizure during experimental temporal lobe status epilepticus.
Mitochondria are ubiquitous intracellular organelles enclosed by a double membrane–bound structure. The primary function of mitochondria is production of cellular energy in the form of adenosine triphosphate (ATP) by way of oxidative phosphorylation through the mitochondrial respiratory chain. Mitochondrial oxidative phosphorylation consists of five enzyme complexes (Complexes I–V) located in the mitochondrial inner membrane (1). Biochemical evidence suggested that the majority of cerebral ATP consumption is used to operate the electrogenic activity of neurons (2). Adequate energy supply by mitochondria is therefore essential for neuronal excitability and neuronal survival.
Seizure activity results in a large number of changes and cascades of cellular events, including gene expression, receptor composition, synaptic physiology, and activation of late cell death pathways (3–9). Prolonged and continuous epileptic seizure (status epilepticus) is a medical emergency that can cause permanent neurologic and mental disability (10,11). Status epilepticus in humans and animal models results in significant cerebral damage and increases the risk of subsequent seizures, along with a characteristic pattern of neuronal cell loss preferentially in the hippocampus (8,11,12). Conversely, relatively few studies addressed the changes in mitochondrial respiratory chain functions or mitochondrial ultrastructure during status epilepticus. Limited reports suggest that mitochondrial dysfunction occurs as a consequence of prolonged epileptic seizures and may play an important role in seizure-induced brain damage (4,13–16).
Systemic or intracerebral injection of kainic acid (KA), a powerful excitotoxic amino acid that activates the KA subtype of ionotropic glutamate receptors, results in sustained epileptic activity in the hippocampus, followed by a selective pattern of neuropathology that is similar to human temporal lobe epilepsy (17–19). We reported previously (20) that microinjection of KA into the CA3 subfield of the hippocampus in anesthetized rats elicits seizure-like hippocampal electroencephalographic (hEEG) activity. Based on this experimental model of temporal lobe status epilepticus, the present study evaluated whether persistent seizure-like hEEG activity leads to dysfunction of key mitochondrial respiratory chain enzymes and ultrastructural damage in the hippocampus. Our electrophysiologic, biochemical, and electron microscopic investigations revealed that both mitochondrial dysfunction and damage took place in the hippocampus during KA-induced temporal lobe status epilepticus.
- Top of page
- MATERIALS AND METHODS
The present study took advantage of an animal model that closely resembles status epilepticus of temporal lobe origin (20), by using spectral analysis of hEEG to quantify epileptic seizure activity in rat hippocampus. By determining the functions of mitochondrial respiratory chain in the hippocampus during experimental status epilepticus, we demonstrated a significant reduction in NCCR activity 180 min after induction of seizure activity by KA. Electron microscopic examination further revealed significant mitochondrial ultrastructural damage in the hippocampus that correlated temporally with mitochondrial respiratory chain dysfunction.
Status epilepticus is a neurologic emergency associated with high mortality and long-term disability (10,11). The outcome is usually worse in patients with status epilepticus of long duration and in those who have severe physiological and metabolic disturbances. Mitochondrial oxidative phosphorylation provides the major source of ATP in cortical neurons (2). Sustained epileptic seizures change the redox potential and reduce the ATP content, leading to collapse in energy production and supply in the brain (11,24,25). We found in this study that whereas the activity of NCCR (Complexes I + III) in the hippocampus underwent a significant decrease during KA-induced status epilepticus, SCCR (Complexes II + III) and CCO (complex IV) remained essentially unchanged. The primary functions of Complex I include oxidizing NADH in the mitochondrial matrix, reducing ubiquinone to ubiquinol, and pumping protons across the inner membrane to drive ATP synthesis (1). Therefore Complex I plays a major role in mitochondrial oxidative phosphorylation (1,26), and its inhibition results in a disturbance of mitochondrial energy metabolism. It follows that prolonged epileptic seizure probably resulted in a dysfunction of Complex I in the mitochondrial electron-transport chain of the hippocampus, leading to incomplete mitochondrial electron transport and decreased ATP production.
Complex I is markedly more susceptible to oxidative stress and glutathionylation than are other respiratory chain complexes (26). As a major source of superoxide, it is a candidate for increasing mitochondrial reactive oxygen species (ROS) production and redox signaling (26,27). Suggestions (28) have been made that Complex I is involved in nitric oxide physiology, induction of the mitochondrial permeability transition, and regulation of apoptosis. Selective loss of Complex I activity contributes to neurodegenerative diseases such as Parkinson and Huntington disease (29). In addition, we demonstrated recently (22) that fatal Escherichia coli lipopolysaccharide-induced endotoxemia is associated with dysfunction of Complexes I and IV in the mitochondrial respiratory chain at the rostral ventrolateral medulla.
Contrary to the more detailed studies on mitochondrial respiratory chain function in other diseases, relatively few investigations (13–16) associate mitochondrial dysfunction with epilepsy or status epilepticus in animals or human subjects. In a perforant-path stimulation model (15), mitochondrial dysfunction and loss of brain glutathione are observed after status epilepticus. Pilocarpine-treated rats with spontaneous seizures exhibit selective decline in the activity of Complexes I and IV in the hippocampal CA1 and CA3 subfields (16). Based on an animal model of status epilepticus that quantifies hEEG activity, the present study extended these findings to demonstrate that selective dysfunction in Complex I took place after a prolonged seizure. The importance of this pattern of mitochondrial respiratory chain dysfunction is strengthened by the finding (14) that Complex I deficiency exists in the CA3 subfield of epilepsy patients with hippocampal sclerosis.
Our electron microscopic examination of mitochondrial ultrastructure in hippocampal neurons, which showed swelling and disruption of mitochondrial membrane that correlated temporally with impaired mitochondrial respiratory function, offers further mechanistic insights into status epilepticus. Both functional impairment and ultrastructural damage of mitochondria in the hippocampus may be a key to the pathogenesis of status epilepticus in this animal model. Overstimulation by KA of postsynaptic N-methyl-d-aspartate (NMDA) receptors results in accumulation of intracellular calcium (13,30,31). A transient intense influx of calcium may lead to mitochondrial swelling, followed by activation of permeability transition pores in the inner membrane and cytochrome c release from the mitochondria (32–34). This kind of damage is difficult to reverse, often accompanied by a loss of mitochondrial function and depletion of ATP level that can cause cell death by necrosis or apoptosis (28,33,34).
Free radical generation induced by activation of glutamate receptors has been implicated in cell death in both acute and chronic neurologic diseases (29,34,35). Impaired mitochondrial respiratory chain function and calcium-dependent depolarization of the mitochondrial membrane potential may further lead to incomplete O2 consumption, reduced production of ATP, and exacerbated overproduction of ROS (25,27,30,34). Free radicals can damage all cell structures, including lipids, proteins, DNA, and mitochondrial membrane structure (27). As inhibition of mitochondrial respiratory chain results in excess free radical production, and free radicals themselves are direct inhibitors of the mitochondrial respiratory chain, this can result in a vicious cycle that leads to oxidative cell damage (15,27). Overall, data from animal studies suggest that impaired mitochondrial calcium handling, significant free radical overproduction, and increased generation of nitric oxide and peroxynitrite after prolonged seizures precede neuronal death in vulnerable brain regions (13,36–40). Thus our observed selective dysfunction of Complex I may be a crucial mechanistic link to KA-elicited neuronal damage.
A crucial determinant in the present study is the consistency of mitochondrial contents that were isolated from our hippocampal samples. One method that has been used to correct for potential variations in mitochondrial contents in the samples analyzed is to express the mitochondrial respiratory enzyme activity as a ratio to citrate synthase (16). Instead, we chose to enhance the purity of our mitochondrial isolate by subjecting our samples to a double two-cycle centrifugation procedure. Whereas the possibility remains for diseased or abnormal mitochondria to be separated more readily than healthy ones or vice versa, the validity of our experimental approach is demonstrated by the consistent activities that we were able to detect from the three mitochondrial marker enzymes. We also noted that our measured mitochondrial activities are an order of magnitude higher than those reported for rat brain (15).
Intracerebral injection of KA has been used as an experimental model for investigation of the vulnerability of the hippocampus, particularly during status epilepticus (18, 19). We recognize that one inherent concern is that results obtained may be linked to direct excitotoxic effects of KA rather than to hippocampal seizure activity. Whereas our experimental design did not allow us to exclude this confounding factor, our results indicated that its contribution may be minimal for three reasons. First, unilateral microinjection of KA to the CA3 subregion resulted in significant and sustained epileptiform activities recorded from the contralateral CA3 subfield. Second, the reduction in NCCR activity was comparable in CA1 (38.3%) or CA3 (40.5%) subfields. Third and more important, no significant alteration in mitochondrial respiratory chain function or mitochondrial morphology was detected in the DG or CA1 or CA3 subfields of the hippocampus during the first 30 min of KA-induced hEEG activation.
To conform to the guidelines for the care and use of experimental animals endorsed by our institutional animal care committee, all animals were maintained under i.v. infusion of chloral hydrate. We confirmed that this is not a confounding factor, because hippocampal application of PBS in animals that received the same anesthetic maintenance and preparatory and experimental procedures as our KA-treated rats did not result in discernible hEEG activation or changes in mitochondrial respiratory enzyme functions or ultrastructure. These same observations also ascertained that hypoxia, which may result in animals under anesthesia, also is not a confounding factor.
In conclusion, our results demonstrate that dysfunction of mitochondrial Complex I respiratory enzyme and mitochondrial ultrastructural damage in the hippocampus are associated with prolonged seizure during experimental temporal lobe status epilepticus induced by KA in the hippocampus. The establishment of a functional and structural link between mitochondria and status epilepticus may offer a new vista in the development of more effective neuroprotective strategies to reduce brain damage caused by status epilepticus and novel treatment perspectives for therapy-resistant forms of epilepsy.