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Keywords:

  • Kainic acid;
  • Status epilepticus;
  • Hippocampus;
  • Complex I of mitochondrial respiratory chain;
  • Mitochondrial ultrastructure

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

All experimental procedures were carried out in compliance with the guidelines for the care and use of experimental animals endorsed by our institutional animal care committee. All efforts were made to reduce the number of animals used and to minimize animal suffering during the experiment.

General preparation of animals

Experiments were carried out in 36 specific pathogen–free adult male Sprague–Dawley rats (280–350 g). Twenty-eight were used in studies on functions of mitochondrial respiratory enzymes, and eight, for examination of mitochondrial ultrastructure. Rats were obtained from the Experimental Animal Center of the National Science Council (Taiwan, Republic of China) and were housed in an animal room under temperature control (24–25°C) and 12-h light/dark cycle. Standard laboratory rat chow and tap water were available ad libitum.

Animals were anesthetized initially with chloral hydrate (400 mg/kg, i.p.). The trachea was intubated for patency of the airway, and the right femoral artery and vein were cannulated for measurement of systemic arterial pressure and administration of anesthetic agent. At completion of preparatory surgery, the animal was fixed to a stereotaxic headholder (model 1430; Kopf, Tujunga, CA, U.S.A.), and the rest of the body was placed on a heating pad to maintain body temperature at 37°C. Anesthesia was maintained by i.v. infusion of chloral hydrate (40 mg/kg/h) throughout the experiment (21).

Experimental temporal lobe status epilepticus

As in our previous study (20), KA (Tocris, Ellisville, MO, U.S.A.) dissolved in 0.1 M phosphate-buffered saline (PBS, pH 7.4), at a concentration of 0.5 nmol, was microinjected stereotaxically (3.0–3.5 mm posterior to bregma, 1.4–2.2 mm from the midline, and 3.5–4.0 mm below the cortical surface) into the CA3 subfield of the hippocampus on the left side (20, 21). The volume of microinjection was restricted to 50 nl and was delivered via a 27-gauge needle connected to a 0.5-μl Hamilton microsyringe (Reno, NV, U.S.A.). The possible volume effect of microinjection was controlled by injecting the same amount of PBS in a separate group of animals. The hEEG signals were recorded from the right CA3 subfield by a stainless-steel bipolar concentric electrode (SNE-100; Rhodes Medical Instruments, Woodland Hills, CA, U.S.A.; tip diameter: 100 μm), by using the same stereotaxic coordinates as used for microinjection. Bioelectric signals were amplified and filtered by a universal amplifier (20-4615-58; Gould, Valley View, OH, U.S.A.), digitized (model AHA-1520A; Adaptec, Milpitas, CA, U.S.A.), and stored on magnetooptical disk. The response range (1–300 Hz) of the recording system amply covered the range (0.015–32 Hz) in which we were interested. The hEEG signals were simultaneously subject to continuous on-line and real-time power spectral analysis. We quantified (20,21) the magnitude of hEEG activity by calculating the root mean square (RMS) value. The frequency domain of hEEG signals was evaluated by calculating the mean power frequency (MPF) values.

Isolation of mitochondria from the hippocampus

We reported previously (20) that microinjection unilaterally of KA into the hippocampal CA3 subfield results in a progressive increase in both MPF and RMS values of hEEG activity. At 30 or 180 min after microinjection of KA or PBS into the hippocampus, rats were perfused intracardially with 50 ml of warm (37°C) saline that contains heparin (100 U/ml). The brain was rapidly removed under visual inspection and placed on a piece of gauze moistened with ice-cold 0.9% saline for the removal of bilateral dentate gyrus (DG) or CA1 or CA3 subfield of hippocampus. Samples were immediately placed in an ice-cold buffer containing 0.25 M sucrose, 0.5 mM EGTA, and 3 mM Hepes, pH 7.2 (SEH buffer). Isolation of rat mitochondria from the hippocampus was carried out according to our previous report (22), by using a double two-cycle centrifugation procedure. The entire procedure was carried out at 4°C and completed within 2 h. Tissue samples were homogenized in SEH buffer by using a loose-fit 15-mL; glass/Teflon homogenizer (Wheaton, Millville, NJ, U.S.A.). The homogenate was centrifuged at 800 g for 10 min at 4°C, and the supernatant thus obtained was further centrifuged at 8,000 g for 10 min. The precipitate was collected, and the centrifugation was repeated. The final mitochondrial pellet was suspended in minimal amount of SEH buffer and stored at −80°C for measurement of mitochondrial respiratory enzyme activity, which was undertaken within 3 to 4 days.

Determination of protein concentration

Total protein in the mitochondrial suspension was determined by the BCA assay (Pierce Biotechnology, Rockford, IL, U.S.A.) according to the manufacturer's protocol, by using bovine serum albumin as a standard.

Assays for activity of mitochondrial respiratory enzymes

Assays for mitochondrial respiratory enzyme activity were the same as those in our previous study (22). All assays were performed by using a thermostatically regulated UV/visible spectrophotometer (Utrospec 400; Amersham Pharmacia Biotech, Uppsala, Sweden). Duplicate determination was carried out on each tissue sample. All reagents used in enzyme assays were purchased from Sigma (St. Louis, MO, U.S.A.).

Nicotinamide adenine dinucleotide (NADH) cytochrome c reductase (NCCR, marker for Complex I + III) activity was determined by the reduction of oxidized cytochrome c measured at 550 nm, and calculated as the difference in the presence or absence of rotenone (22, 23). The activity was assayed in 50 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 1.0 mMβ-NADH, and 20 μL (50–100 μg protein) of mitochondrial suspension in the presence or absence of rotenone (20 μM). The reaction was initiated after 2 min of stabilization by adding 0.1 mM cytochrome c, and absorbance at 550 nm was measured at 5-s intervals over the first 3 min at 37°C. The molar extinction coefficient of cytochrome c at 550 nm is 18,500 M/cm.

Determination of succinate cytochrome c reductase (SCCR, marker for Complex II + III) activity was performed in 40 mM K2HPO4 buffer (pH 7.4) containing 20 mM succinate, 1.5 mM KCN, and 30 μL (100–200 μg protein) of mitochondrial suspension (22,23). After 5 min of incubation at 37°C, the reaction was initiated by adding 50 μM cytochrome c, and absorbance at 550 nm was followed at 5-s intervals over the first 2 min at 37°C.

Cytochrome c oxidase (CCO, marker for Complex IV) activity was estimated by recording the oxidation of reduced cytochrome c at 550 nm (22, 23). The activity of CCO is defined as the first-order rate constant and is calculated from the known concentration of ferrocytochrome c and enzyme amount in the assay mixture (22). The activity was assayed in 10 mM K2HPO4 buffer (pH 7.4) containing 30 μL (100–200 μg protein) of mitochondrial suspension. After 5 min of incubation at 30°C, 45 μM ferrocytochrome c was added to start the reaction. The background rate was measured after the addition of K3Fe(CN)6 (1.0 mM). Ferrocytochrome c was prepared from 1 mM oxidized cytochrome c by reduction with excess amount of Na2S2O4 at 25°C for 5 min. The mixture was applied to a Sephadex G-25 column to separate ferrocytochrome c from Na2S2O4.

As a routine, samples from the CA1 or CA3 subfield on both sides of each animal were used to assay for the activity of all three mitochondrial respiratory enzymes. However, because of the size of the sample, the activity of only two enzymes in the bilateral DG of each animal was determined. To avoid day-to-day or batch variability, hippocampal samples were obtained in parallel from KA- or PBS-treated rats.

Electron microscopic evaluation of mitochondrial damage

At 30 or 180 min after microinjection of KA or PBS into the hippocampal CA3 subfield, the bilateral DG or CA1 or CA3 subfield of the hippocampus were removed and processed for electron microscopy. Tissue samples were diced and immediately submerged in 4% gluteraldehyde (0.1 M sodium cacodylate buffer, pH 7.2). Each specimen was trimmed and embedded in Spurr's medium. Tissue blocks were postfixed with osmium, en bloc stained with uranyl acetate, and poststained with uranyl acetate and lead citrate. Tissue sections were cut to a thickness of 90 nm and viewed on 300-mesh coated grids by using a JEOL JEM-2000 EXII (Tokyo, Japan) electron microscope.

Statistical analysis

All values are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used to assess group means, followed by the Dunnett or Scheffé multiple range test for post hoc assessment of individual means. A value of p < 0.05 was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Temporal changes in hEEG after microinjection of KA into the hippocampus

Unilateral application by microinjection of KA into the CA3 subfield of hippocampus resulted in a progressive buildup of hEEG activity (Fig. 1) in all animals studied (n = 28). Simultaneous power spectral analysis revealed a concomitant increase in both magnitude (MPF value) and frequency (RMS value) of these seizure activities. As we reported previously (20), these manifestations of experiment temporal lobe status epilepticus endured more than 180 min. Conversely, local microinjection of PBS into the hippocampus of control animals that received the same preparatory surgery and were similarly maintained under chloral hydrate anesthesia elicited minimal alterations in hEEG activity over the same observation period. Throughout the course of our experiments, mean systemic arterial pressure was stable (>70 mm Hg), and the animals showed no convulsive behavior. At the same time, none of the animals died as a result of KA or PBS treatment.

image

Figure 1. Representative continuous tracings showing changes in hippocampal EEG (hEEG) signals elicited by microinjection of kainic acid (KA, 0.5 nmol; at arrow) into the left hippocampal CA3 subfield and their corresponding root mean square (RMS) and mean power frequency (MPF) values.

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Mitochondrial respiratory enzyme functions in the hippocampus during experimental temporal lobe status epilepticus

We evaluated the changes of mitochondrial respiratory functions in DG or CA1 or CA3 subfield of the hippocampus by examining the activity of marker enzymes in respiratory Complex I + III (NCCR), II + III (SCCR), and IV (CCO) during experimental temporal lobe status epilepticus. The specific enzyme activity of NCCR or SCCR was expressed in nmol of cytochrome c reduced/min/mg protein, and in nmol of cytochrome c oxidized/min/mg protein for CCO.

Compared with the PBS-treated group, the activity of NCCR (Fig. 2) in the DG or CA1 or CA3 subfield of the hippocampus showed no significant change 30 min after microinjection of KA (0.5 nmol) into the CA3 subfield. Conversely, NCCR activity in these three hippocampal regions underwent a significant decrease 180 min after elicitation by KA of experimental temporal lobe status epilepticus. Compared with PBS-treated rats, the activity of NCCR showed an ∼24.1%, 38.3%, or 40.5% reduction in the DG or CA1 or CA3 subfields. Measurement of activities of SCCR or CCO in the three regions of the hippocampus revealed an entirely different picture (Figs. 3 and 4). No significant alteration was found 30 or 180 min after microinjection of KA into the hippocampus.

image

Figure 2. Enzyme assay for activity of nicotinamide adenine dinucleotide (NADH) cytochrome c reductase (marker for Complex I + III) in mitochondria isolated from dentate gyrus (DG) or CA1 or CA3 subfield of hippocampus 30 or 180 min after microinjection of kainic acid (KA) or phosphate-buffered saline (PBS) into the hippocampal CA3 subfield. Values are presented as mean ± SEM of duplicate analysis on CA1 or CA3 samples from five (PBS group) or nine (KA group) animals; or DG samples from three to four (PBS group) or six (KA group) animals, obtained at each time interval. *p < 0.05 vs. PBS-treated controls in the Scheffé analysis.

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image

Figure 3. Enzyme assay for activity of succinate cytochrome c reductase (marker for Complex II + III) in mitochondria isolated from dentate gyrus (DG) or CA1 or CA3 subfields of the hippocampus 30 or 180 min after microinjection of kainic acid (KA) or phosphate-buffered saline (PBS) into the hippocampal CA3 subfield. Values are presented as mean ± SEM of duplicate analysis on CA1 or CA3 samples from five (PBS group) or nine (KA group) animals; or dentate gyrus (DG) samples from three to four (PBS group) or six (KA group) animals, obtained at each time interval. No significant difference was detected (p > 0.05) among all treatment groups.

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image

Figure 4. Enzyme assay for activity of cytochrome c oxidase (marker for Complex IV) in mitochondria isolated from dentate gyrus (DG) or CA1 or CA3 subfields of the hippocampus 30 or 180 min after microinjection of kainic acid (KA) or phosphate-buffered saline (PBS) into the hippocampal CA3 subfield. Values are presented as mean ± SEM of duplicate analysis on CA1 or CA3 samples from five (PBS group) or nine (KA group) animals; or DG samples from three to four (PBS group) or six (KA group) animals, obtained at each time interval. No significant difference was detected (p > 0.05) among all treatment groups.

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Mitochondrial ultrastructural damage in the hippocampus during experimental temporal lobe status epilepticus

We also observed significant mitochondrial ultrastructural damage in the hippocampus 180 min after microinjection of KA into the CA3 subfield. Although the degree varied from mild to profoundly severe, mitochondrial damage was invariably associated with significant swelling of all mitochondrial spaces, including cristae (Fig. 5B). In the more severe cases, mitochondrial swelling was accompanied by a disruption in membrane integrity (Fig. 5C). It should be mentioned that mitochondrial damage was observed in all three hippocampal regions examined, and no discernible regional difference was noted in the degree of severity induced. Conversely, we found no evidence of mitochondrial damage in the DG or CA1 or CA3 subfields of the hippocampus 30 min after microinjection of either KA (Fig. 5A) or PBS into the hippocampus.

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Figure 5. Representative electron photomicrographs of mitochondrial ultrastructure in hippocampal CA3 subfield. A: Intact mitochondrial ultrastructure 30 min after microinjection of kainic acid (KA) into the hippocampus. B: Mild mitochondrial damage 180 min after microinjection of KA into the hippocampus. Note swelling of all mitochondrial spaces, particularly in cristae (*). C: Severe mitochondrial damage 180 min after hippocampal application of KA. Note severe mitochondrial swelling accompanied by a disruption in membrane integrity (arrows). Scale bar, 200 nm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Acknowledgment:  This study was supported in part by research grant NSC-92-2314-B-182A-170 to Y.C.C. from the National Science Council. S.H.H.C. is National Chair Professor of Neuroscience appointed by the Ministry of Education, and Sun Yat-sen Research Chair Professor appointed by the National Sun Yat-sen University, Taiwan, Republic of China.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES