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Keywords:

  • Benzodiazepine;
  • Development;
  • Pilocarpine;
  • GABA

Abstract

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

Summary: Purpose: Ample evidence exists from both clinical and animal studies that the success of benzodiazepine intervention during status epilepticus (SE) in the mature nervous system is inversely related to seizure duration. This relationship has not been well studied in the developing nervous system.

Methods: The objective of this study was to investigate the relation of age and success of diazepam (DZP) treatment in the lithium-pilocarpine model of secondarily generalized seizure in the rat by using naïve rats of three age groups, roughly corresponding to the human ages of infancy (P15), adolescence (P20), and adult (P60).

Results: In all age groups, the dosage of DZP that stopped the seizures at 5 min was not effective in terminating seizures at 60 min. This decline in efficacy was present as early as 15 min after seizure onset.

Conclusions: These findings demonstrate that the inverse relation between the success of benzodiazepine intervention and seizure duration is observed in young as well as in adult rats and provide further evidence that intervention for SE should commence early.

Status epilepticus (SE) is a neurologic emergency characterized by prolonged, self-sustained seizures. Approximately 126,000 to 195,000 cases of SE occur each year in the United States, with the highest incidence during the first year of life (1). Although the precise relation of SE and outcome is affected by factors such as etiology and age, SE has been associated with mortality (2) as well as neurologic morbidity. In children, the long-term neurologic sequelae can include new-onset motor and intellectual dysfunction (3) and possibly the development of mesial temporal sclerosis and temporal lobe epilepsy (4,5). Therefore prompt recognition and treatment of SE are warranted.

Benzodiazepines (BZDs), which enhance γ-aminobutyric acid subtype A (GABAA) receptor-mediated inhibition, are the first-line therapy for the treatment of SE. However, increasing evidence is found from clinical and animal studies that successful treatment of SE with BZDs is negatively affected by seizure duration (6–10). These studies have been performed predominantly in adults.

Given the developmental changes in the inhibitory GABAA system (11–13), we were interested in determining whether a similar decline in the efficacy of BZDs in treating SE occurs in immature animals.

METHODS

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

These experiments were approved by the Institutional Animal Care and Use Committee of Children's Hospital, Boston, Massachusetts, and performed in accordance with the guidelines set forth by the National Institutes of Health Guiding Principles for the Care and Use of Research Animals.

Epidural electrode implantation

On P13, P18, P58, or later, epidural left frontal and left parietal recording electrodes and a ground electrode (stainless steel jeweler's screws) were implanted into the skull during a sterile surgical procedure in male Sprague–Dawley rats who had received pentobarbital (PTB; 25–50 mg/kg) anesthesia. A dental acrylic cap was used to assure that the electrodes were fixed to the skull. Each rat received postoperative pain relief for a 24-h period.

Status epilepticus induction

SE was induced through pretreatment with intraperitoneal lithium (3 mEq/kg) followed 4–20 h later by pilocarpine on P15 (60 mg/kg), P20 (30 mg/kg), and P60 or later (30 mg/kg). In untreated animals, these dosages produce SE that lasts for >3 h. Four electrographic seizure patterns are observed after the injection of pilocarpine (9). As the latency between the first phase (discrete seizures) and the onset of the next phase (waxing and waning), which heralds the onset of continuous electrographic seizure activity, can be variable and may be >5 min, we choose to define the onset of SE as occurring after 30 s of continuous rhythmic epileptiform activity (i.e., during the waxing and waning phase) and not at the time of the first discrete seizure. The doses of pilocarpine used were chosen in an attempt to shorten the duration between the first and second phase (indeed, in an attempt to bypass the first phase altogether). However, occasional discrete seizures continued to be observed, predominantly in the adult rats and in rats that did and did not respond to diazepam (DZP).

Treatment

Before pilocarpine injection, animals were randomized to time of DZP injection. Treatment times were 5, 15, 30, 60, and 120 min after seizure onset. At the appropriate time, DZP was administered intraperitoneally. The dosage of DZP in each age group was determined as the minimal dose required to terminate clinical and electrographic seizures of 5 min in duration in three consecutive animals (P15, 0.2 mg/kg; P20, 5 mg/kg; P60 and older, 30 mg/kg). Seizure termination was defined as the electrographic absence of continuous or periodic seizure activity.

RESULTS

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

Behavioral changes before the onset of SE commenced within 3 min of pilocarpine injection in all age groups. These behaviors consisted of chewing, grooming, staring, or periods of increased exploration and agitation. None of these behaviors was associated with any changes in the cortical EEG tracing compared with baseline. The cortical electrographic onset of SE did not occur until 15–30 min after the injection of pilocarpine and, in the majority of animals, corresponded to or was observed just before the onset of forelimb clonus.

Based on experimental design, the DZP was effective in terminating all clinical and electrographic seizures of 5 min in duration in all age groups (Table 1). In Fig. 1A, the EEG tracings obtained from a P15 animal during a seizure of 5 min in duration before the administration of DZP are displayed. The top tracing represents a 15-s epoch of EEG obtained before the subcutaneous injection of pilocarpine. In the second tracing, the onset of rhythmic discharges (seizure onset) is observed. At the time of DZP administration (third trace), the seizure was ongoing; 5, 15, and 30 min later (traces 4, 5, and 6, respectively), the EEG tracing is characterized by low-amplitude fast activity, and no epileptiform discharges or rhythmic discharges are evident. This animal, as did all the animals treated at 5 min, recovered and returned to baseline activity.

Table 1. Animals were treated with intraperitoneal DZP 5, 15, 30, 60, and 120 minutes after seizure onset (seizure duration). For each age group and seizure duration, the number of animals in which the DZP was effective in stopping the electroclinical seizure (numerator) and the total number of animals in each group (denominator) are displayed
 Seizure Duration (minutes)
Age5153060120
P153/30/31/30/31/3
P203/30/30/30/30/3
Adult3/31/31/30/31/3
image

Figure 1. Epidural EEG recording obtained from P15 rats during seizures of 5 (A), 15 (B), 30 (C), and 60 (D) min in duration. Top trace, 15-s baseline epoch before the administration of pilocarpine. Second trace, 15-s epoch at the time of electrographic seizure onset. Third trace, 15-s epoch at the time of diazepam (DZP) administration. Fourth trace, 15-s epoch 5 min after the administration of DZP. Fifth trace, 15-s epoch 15 min after the administration of DZP. Sixth trace, 15-s epoch 30 min after the administration of DZP. Note that the seizure of 5 min in duration was terminated by DZP, whereas the longer seizures were not.

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Seizures of longer duration were treated with the same dose of DZP used to terminate the electroclinical seizures of 5 min in duration. As in the animals treated at 5 min, a loss of postural reflexes was observed and, in the majority of animals, the termination of the clinical seizure. If the clinical seizure continued, the animal was unresponsive, and intermittent clonic movements of the limbs were noted. Although changes in EEG were noted with respect to amplitude, morphology, and frequency after the administration of DZP, in only two of the P15 animals, none of the P20 animals, and three of the adult animals did the changes in the EEG record meet our predefined definition of seizure termination. In panels B, C, and D of Fig. 1, typical EEG tracings recorded during seizures of longer duration in a P15 animal are displayed. In all three panels, it should be noted that 5, 15, and 30 min after DZP administration, electrographic evidence of persistent seizure continues despite behavioral determination that the seizure had ended. In those animals in which DZP was effective in terminating electroclinical seizures of longer duration, some function was recovered, but these animals died several hours or days after the event. Those animals in which the seizure continued at 30 min after DZP treatment were killed with an overdose of pentobarbital.

DISCUSSION

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

In summary, in naïve rats at all ages, DZP was efficacious in treating seizures of 5 min in duration but not of ≥15 min in duration.

One of the predominant factors in determining the sensitivity of the GABAA receptor to BZDs is its subunit composition. The expression of these subunits is known to change during development (13). Kapur and Macdonald (12) and Dunning et al. (11) previously demonstrated that the BZD sensitivity of granule cells and cortical neurons, respectively, increases during development. These findings appear to be contradictory to our finding of a 10-fold increase in the dose of DZP required to terminate the 5-min seizures in the adult rats compared with the P15 animals. Our study was done in vivo where differences in dosage may reflect changes in rates of intraperitoneal absorption, volume of distribution, and maturational changes in the blood–brain barrier, all of which will affect the actual CNS concentration of DZP. Future experiments to measure CNS concentrations of DZP will be required to test this hypothesis. Our findings demonstrate that the dosage of DZP must be determined for each age group. It is important to note that once an age-related dose was established, it was used throughout the study. It should also be noted that DZP was administered only after continuous electrographic seizures were observed. Whether administration of DZP during the period of discrete seizures or before the onset of waxing and waning activity would alter DZP dose requirements is not known.

Our findings are in agreement with and extend the findings of Jones et al. (10) in which it was demonstrated that DZP pharmacoresistance develops soon after the onset of forelimb clonus in P27–40 rats. Therefore the mechanism(s) that result in the loss of DZP efficacy appear to be similar at all ages so far tested. This mechanism(s) does not appear to depend on the initial subunit configuration and function of the GABAA receptor. It was previously shown that whole-cell GABAA-receptor currents in hippocampal dentate granule cells immediately isolated from adult rats after 45 min of status epilepticus are less sensitive to DZP (8); this finding implies that an apparent alteration in the functional properties of the GABAA receptor occurs during SE. However, other mechanisms also may play a role in the decline of GABAA receptor-mediated inhibition during SE, such as a failure in the presynaptic release of GABA or an increased rate of endocytosis of the GABAA receptor in the setting of a prolonged seizure.

There has been a recent trend toward early intervention during prolonged seizures. In their discussion of the definition of SE, Lowenstein et al. (14) outlined several reasons that the time duration of SE should be shortened to 5 min. Their observations are supported by the recent demonstration that the likelihood of a seizure stopping spontaneously is reduced after 5 min (15) and that BZD administration is safe and efficacious in the out-of-hospital setting (16). The findings of this study complement the previous studies in the adult on the efficacy of BZDs and suggest another reason: Given that the mode of action of our current first-line therapy depends on enhancement of the GABAA receptor-mediated inhibition, and the efficacy of these agents quickly declines, intervention should commence early.

Acknowledgments

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

Acknowledgment:  H.P.G. received support from the National Epifellows and T32 NS07473 from NINDS. NS27984 to G.L.H. We thank T. Bleck, J. Kapur, and J. Riviello for their thoughtful comments.

REFERENCES

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