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An Analysis of the Effects of a Calpain Inhibitor on Pilocarpine-Induced Status Epilepticus

Janeen Williams, Phillip Lam, Marco I. Gonzlez


Department of Pediatrics, University of Colorado Denver Anschutz Medical Campus

Abstract
Chronic epilepsy can often develop after an acute injury to the brain, namely stroke, ischemia or status epilepticus (SE). Chemoconvulsant animal models of Temporal Lobe Epilepsy can be used to trigger SE and analyze acute and long-term effects of a triggering injury. The current study employed the pilocarpine model epilepsy to evaluate the therapeutic effects of calpain inhibition on the development of chronic epilepsy. Electroencephalography (EEG) was used to analyze the total and integrated power of treated and untreated groups to determine if the drug treatment altered the course of SE. EEG recordings were analyzed using energy comparisons of total and integrated power. Treated and untreated groups showed the same power intensity, similar degradation of power, and comparable integrated power during the first hour of SE. At this time point, animals were treated with diazepam to pharmacologically terminate SE and the calpain inhibitor was administered. Immunohistochemistry was performed to confer the drugs effect on epileptogenic markers and as a supplementary parameter to evaluate SE intensity. The reduction of hippocampal cell death, astrocyte reactivity, and seizure occurrence as well as identical SE intensity between groups show that the use of calpain inhibition for antiepileptogenic treatment is a promising agent for reducing facets associated with epilepsy.

Results
EEG Analysis EEG recordings were used to conduct a power analysis between control and treated groups. Control and variable groups differed by the experimental setup in Figure 1. This analysis required Fourier Transform to compare intensity. A. Total Power in control (pink) and treated animals (blue). B. Power normalized to values detected in control animals.

Results
Quantitative Comparison EEG Analysis. Power intensity was similar in control and treated groups.

Quantitative Comparison Immunocytochemistry. Fluoro-Jade and GFAP stainings showed a decrease in cell death and astrocyte reactivity between control and variable groups.

Fluoro-Jade B Introduction
Epilepsy is a neurological condition resulting in spontaneous seizures. The molecular mechanisms that cause epilepsy are unknown. However, previous research suggests that activation of calpain following a brain injury may catalyze the development of epilepsy. This finding prompted us to investigate if calpain inhibitors might prevent or slow down epileptogenesis. The described research uses EEG analysis and immunohistochemistry stainings to evaluate the validity of using calpain inhibitors to prevent epileptogenesis. Histological Analysis Fluoro-Jade B. Brain tissue was collected and Fluoro-jade B staining performed in fixed tissue. Representative photographs of the signal detected in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus. There is a significant reduction in cell death observed in animals treated with the calpain inhibitor (SE+MDL). GFAP Immunoreactivity. Tissue sections were stained with an antibody for an astrocyte marker (GFAP). There is a reduction in astrocyte reactivity in the SE and SE + treated groups.

GFAP Fluoro-Jade B GFAP

Methods
An intraperitoneal injection of pilocapine was used to induce acute seizures. SE was pharmacologically terminated with diazepam. SE was recorded using an automated system to collect EEG/Video signals simultaneously.

Conclusion
The equivalent changes in power between the EEG signals of control and variable groups show that there is no difference between the different groups as a result of administration of a calpain inhibitor. The reduction in cell death as shown by the Fluoro-jade B staining suggest that calpain inhibition reduced cell death. Neuronal cell death has been correlated with the development of epilepsy. The reduction in astrocyte reactivity as shown by the GFAP staining suggest that the use of a calpain inhibitor limits astrocyte reactivity. Astrocyte reactivity has also been correlated with epileptogenesis. Further study of the effects of calpain inhibition holds promise for the use of calpain inhibitors as agents to prevent epilepsy.

This research was supported by NIH 1R25HL103286-04