Introduction
Epilepsy is a chronic neurological disease characterized by the onset of recurrent and spontaneous seizures. Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. In humans, studies show that at least half of the individuals who experience the de novo status epilepticus, later develop epilepsy and thereby spontaneous convulsions (cite paper Tsai). The Status Epilepticus (SE) is a crisis of prolonged seizures and is considered the most severe form of seizures. SE is defined as a seizure that persists for a sufficient period, or recur so frequently that it eventually produces a cerebral condition.
Studies have found a relationship between the activation of Calpain and neurodegeneration, which leads to the emergence of spontaneous seizures (cite paper Robert Siman). Calpain is a protein involved in the calcium signaling system in mammalian cells. There are two isoforms of calpain; Calpain I and Calpain II, which needs lower or higher calcium concentrations for their activation respectively (cite paper Kelvin K. W. Wang). As an alternative, researchers have suggested that inhibition of calpain could prevent neuronal death. On the other hand, it is possible to demonstrate the activation of calpain with the presence of the spectrin Breakdown Products (sBDP) (put figure number) (cite paper Kelvin K.). We can also detect the activation of calpain in a brain injury induced by chemoconvulsants, in this case, pilocarpine (cite paper Marco I).
Given the correlation between the activation of calpain and other proteins derived from this protease, inhibition of calpain is attractive as a therapeutic method to treat neural degeneration (cite paper atman). In this research project, we present some of the effects of inhibition of calpain in different aspects related to Epileptogenesis that include cellular pathologies associated with the occurrence of seizures.
Methods
2.1. Western blot
Protein samples were separated in SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots were blocked with 5% non-fat dry milk in Tris-buffered saline (pH 7.4) plus 0.05% Tween 20. Blots were then incubated overnight at 4 °C with primary antibodies diluted in 1% non-fat dry milk. A polyclonal rabbit antibody, AB38, which recognizes calpain-cleaved spectrin fragments of ~150 kDa was produced and characterized previously (Roberts-Lewis et al., 1994). A rabbit monoclonal antibody that detects full-length and cleaved α-spectrin was obtained from Epitomics (Cat. No. 2507-1, Burlingame, CA). To estimate potential variability in protein content and loading, blots were re-probed with an anti-actin antibody (Sigma, St. Louis, MO). Following incubation with the primary antibody, blots were washed and then incubated at room temperature for 1 h with the appropriate secondary antibodies. Anti-rabbit secondary antibodie conjugated to horseradish peroxidase were from GE Health Care (Piscataway, NJ) or Jackson Immunoresearch laboratories (West Grove, PA). Immunoreactive bands were visualized using Super Signal West Dura chemiluminescent substrate (Pierce, Rockford, IL, USA) and film. After scanning the films, immunoreactive bands of the appropriate size were quantified using ImageJ (NIH, Bethesda, MD, USA). Immunoreactivity for the bands of interest was normalized to actin immunoreactivity and compared to control values.
2.2. Histological analysis
Rats were deeply anesthetized and transcardially perfused, first with ice-cold PBS and then with ice-cold 4% PFA in 0.1 M phosphate buffer pH 7.4. Brains were removed from the skull and post-fixed overnight in 4% PFA solution. Fixed brains were cryoprotected in 30% sucrose solution and embedded in OCT compound (Tissue-Tek, Sakura Finetek, Tor- rance, CA). Whole brains were serially sectioned to obtain 15 μm coronal sections. For staining, three mounted sections were selected from a 1-in-15 series starting at approximately the same level of hippocampus (2.8 mm posterior to Bregma). For consistency and to minimize variability in the staining procedure, control and SE brains were processed and stained in parallel. Following staining, cell counts were conducted blinded to the administered treatment. The number of cells counted in two sections was averaged and the average number of cells is the reported value for each animal. Images were obtained using a Nikon Eclipse TE2000-U fluorescence microscope.
To detect degenerating neurons, sections were stained with a simple, reliable, and sensitive technique using the anionic fluorochrome Fluoro-Jade B (FJB, Cat. No. 1FJB, Histo-Chem Inc., Jefferson, AR). Mounted sections were dried at room temperature and rehydrated with 100% ethanol for 10 min, 70% ethanol for 2 min and finally rinsed in distilled water for 2 min. Sections were immersed in 0.06% potassium permanganate for 10 min, rinsed with distilled water for 2 min and finally immersed in 0.0004% FJB staining solution for 10 min. Following staining, sections were rinsed with distilled water, dried and immersed in CitriSolv (Fisher, Pittsburgh, PA). After staining, tissue sections were mounted on slides using Permount (Fisher Scientific, Pittsburg, PA, USA).
To estimate inflammation, brain slices were stained with an antibody to detect Iba-1 (a marker for microglia). Tissue sections were blocked with PBS containing 10% normal goat serum and 0.3% Triton X-100. Sections were then incubated overnight with a rabbit polyclonal anti-Iba1 antibody (Cat. No. 019-19741, Wako, Richmond, VA) diluted 1:500 in blocking buffer. Next day, slices were washed and incubated with a highly cross-adsorbed Alexa Fluor 568 goat anti-rabbit secondary antibody. After staining, tissue sections were mounted on slides using Vectorshield (Vector Laboratories, Bur- lingame, CA, USA).
2.3. Electrode implantation and electroencephalogram (EEG) acquisition
Rats were implanted with intracranial EEG electrodes approximately one week before SE induction. Two screws were used as subdural electrodes and placed bilaterally at ~ 2.5 mm lateral from midline and 4 mm caudal to Bregma over the temporolimbic cortices. In addition, a polyamide coated stainless-steel wire (Plastics-One, Roanoke, VA) was used as a depth electrode and placed ~3.3 mm caudal to Bregma, 1.69 mm lateral from the midline and 2.6 mm below the skull in the right hippocampus. Reference and ground electrodes were placed on the back of the skull slightly behind lambda. Dental acrylic was used to secure a plastic connector (Plastics-One, Roanoke, VA) attached to the electrodes according to standard methods (Zhang et al., 2004; Grabenstatter et al., 2014). Animals were allowed to recover from surgery for one week before further experimentation. For EEG recording, animals were placed in a recording chamber and connected to flexible cables with a commutator to allow free movement. Recordings were obtained 24 h/day using an automatic Pinnacle digital video-EEG system (Pinnacle Technology Inc., Lawrence, KS). Blinded to treatment I examined electrographic recordings off-line in order to identify electrographic seizures. Seizure activity differed from background noise by the presence of EEG signals with progression of spike frequency, large-amplitude and high-frequency activity lasting at least 10 s. Behavioral characterization of seizures was done accordingly with the Racine scale (Racine, 1972) which is based on the behaviors observed during a seizure episode and classify seizures in five categories: mouth and facial movements, stage 1; head nodding, stage 2; forelimb clonus, stage 3; rearing, stage 4; and, rearing and falling, stage 5. Seizures with an electrographic component associated with subtle or no behavioral manifestations were scored as class 2 or below and labeled as “non-convulsive” while seizures with overt behavioral manifestations were scored as class 3 or above and labeled as “convulsive” (Krook-Magnuson et al., 2013; Grabenstatter et al., 2014).
2.4. Statistical analysis
Data is presented as the mean ± SEM. For statistical evaluation, ei- ther parametric or non-parametric tests were used depending on data distribution. Student's t-test was used when to assess differences be- tween two groups. Analysis of variance (ANOVA) followed by post hoc testing was used to assess differences when more than two groups were compared. Values of p ≤ 0.05 were considered significant. GraphPad InStat software (GraphPad Software, Inc., San Diego, CA, USA) was used to perform statistical analysis.