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Kainic Acid Marginally Increases Nav1.6 Expression in a Mouse Model of Epilepsy
Yoan Ganev, Rebecca Shoup, Catherine Harding, and Grant Connor
Department of Neuroscience and Biology
Lake Forest College
Lake Forest, Illinois 60045
Epilepsy is a disease characterized by spontaneous and recurrent seizures that could be either generalized or focal. In temporal lobe epilepsy, seizures begin from the hippocampus and lead to such pathological features as astrogliosis and hippocampal atrophy. Recently, the Nav1.6 sodium channel has been implicated in the propagation of temporal lobe epileptic seizures in the CA3 region of the hippocampus. However, its expression levels in KA-induced epileptic mice remain un-elucidated. Here, we hypothesized that Nav-1.6 staining in the CA3 would be higher following KA-induced epileptogenesis. To test this hypothesis, we performed immunohistochemistry on brain sections from mice injected with KA (epileptic) or saline (non- epileptic). We found that, even though the staining in the KA mice appeared darker, hinting at
an increase in Nav-1.6 in the CA3 of the hippocampus, the statistical comparison did not support that conclusion. The results approached significance when negative optical density differences were removed from the data set. Finally, when a comparison across hemispheres and conditions was performed, no significant differences in any of the possible combinations were found. Overall, our results suggest that with a larger sample size, there may be a significant increase in Nav1.6 expression in epileptic mice. These results support the conclusion that in temporal lobe epilepsy, overexpression of Nav1.6 in response to a seizure could lead to a propagation of future seizures. This could suggest that the Nav1.6 channel could be used as a therapeutic target against temporal lobe epilepsy.
Epilepsy, diagnosed after the occurrence of two or more spontaneous seizures, is the most common brain disorder among all age groups (Banerjee et al., 2009). Some seizures (absence or tonic-clonic) are generalized, beginning with synchronous firing throughout the entire brain, while others are focal, with a defined starting point (Chang et al., 2017). Temporal lobe epilepsy (TLE) is one common type of focal epilepsy. Some physical symptoms of the disease include auras before a seizure, head-jerking, loss of consciousness, linguistic disability, emotional outbursts, and loss of long-term memory (Epilepsy Foundation, 2013). Seizures begin in the temporal lobes (usually in the hippocampal area), and they are often caused by hippocampal sclerosis. They affect such diverse areas as the amygdala, parahippocampal gyrus, and entorhinal cortex (Ladino et al., 2014). However, most research has focused on the hippocampus, showing atrophy and astrogliosis in that part of the brain (Ladino et al., 2014).
Many mechanisms of epileptogenesis in TLE have been proposed. For example, some
studies have focused on mutations in the mTOR signaling pathway, ultimately leading to translation breakdowns, while others have reported an upregulation of PER1, leading to abnormal circadian rhythms (Cho, 2012). Both of these could lead to spontaneous synchronization of neuronal firing, giving rise to a seizure. Another promising line of research investigates the dysfunction of ion channels. One such channel, Nav1.6, encoded by the SCN8A gene, has recently gained attention (O’Brien et al., 2013). The Nav1.6 sodium channel sets action potential thresholds and is localized in axon initial segments (O’Brien et al., 2013). The literature suggests that expression of Nav1.6 (which is encoded by SCN8A) increases in response to a seizure, in a positive feedback loop that generates more seizures (Zhu et al., 2016). After a seizure, Nav1.6 expression is upregulated in the CA3 region of the hippocampus (Wang, 2015). This transient increase could facilitate further seizures, through excess sodium- induced depolarization. Interestingly, the CA3 region of the hippocampus is extremely sensitive to the effects of epileptic seizures, lending itself to pyramidal neuron loss (O’Brien et al., 2013). Finally, a recent study reported that knocking down SCN8A using RNA interference decreased seizures in a kainic acid mouse model of temporal lobe epilepsy (Wong et al., 2018). Thus, the literature suggests that Nav1.6 is a likely mediator of seizures in TLE.
Despite the progress from these studies, the long-term expression levels of the Nav1.6 channel in a kainic acid model have remained un-elucidated. We hypothesize that in TLE, Nav1.6 channels are overexpressed specifically in the CA3 region of the hippocampus. This expression may lead an overall depolarization and an increased likelihood of future epileptic seizures. Here, we evaluated the effects of kainic-acid-induced epilepsy on the expression of the Nav1.6 sodium channel.
Materials and Methods
Mice of the C57BL/6J line were used for these experiments. Half of the mice were injected with kainic acid, and the rest with a saline control solution (sham – 0.9% NaCl dissolved in water). The KA-injected mice developed epileptic seizures that eventually became spontaneous, while the sham-injected mice did not. A week after status epilepticus, the animals were sacrificed. Their brains were salvaged and sectioned into 100 m thick sections using a microtome.
Five KA and five sham sections were used for staining. Additionally, one additional sham section served as the no-primary-anti-body control. The brain samples were first incubated in Blocking Serum, consisting of 5% bovine serum albumin (Lampire Biological Laboratories
17E54002), 2% goat serum, 0.5% Triton X-100 (Sigma Aldrich 121C-1630), and Phosphate-
Buffered Saline (PBS) solvent (VWR Life Science 02-0119-0500). After this initial wash, all sections (except for the no-primary antibody control) were incubated overnight in Rabbit-Anti- Nav1.6 Primary Antibody (ASC-009 from the George Lab at Northwestern University). The no- primary antibody control was left in Blocking Serum. The primary antibody was used at a 1:100 dilution (with blocking serum as the solvent). After another PBS wash, all sections (including the no-primary antibody control) were incubated in Goat-Anti-Rabbit secondary antibody (Millipore 2316538), diluted 1:200 in PBS. The sections were then washed with 0.3% H2O2 (diluted in PBS)
To set up for chromogenic detection, a reaction between Reagent A and Reagent B (in PBS) from the ABC kit (VECTASTAIN, Vector laboratories, PK-6100) was set up, and the brain slices were incubated in the solution. Afterwards, the slices were washed in PBS and in Tris- Buffered Saline (TBS – pH = 7.4). To achieve chromogenic detection, a diaminobenzidine reaction was performed (DAB taken from Sigma Aldrich D4293-50SET). A gold and silver tablet were dissolved in deionized water before use. All sections were kept in the DAB solution for 7 minutes and later washed with TBS. Afterwards, the sections were mounted onto WR North American Micro Slides using a paint brush and kept overnight in the dark. A cresyl violet counterstain was then performed to visualize neuronal cell bodies. The slides with the DAB-stained brain sections were put through progressively more dilute ethanol washes (95% and 70%) and then incubated in water. They were dropped in cresyl violet (1:10 dilution) and put through the wash solutions in reverse order (water, 70% ethanol, and 95% ethanol). Finally, coverslips were mounted onto the slides using copious amounts of Mowiol solution.
KA, sham, and no-primary-antibody control brain sections were imaged with a Nikon ECLIPSE LV100N microscope using a 40X objective. NIS software was used to processes the images. The images were then analyzed for optical density of DAB staining using FIJI software (set for H DAB vector). Optical densities from the beginning of the CA3 (test region) and the dentate gyrus (background staining region) were taken. A difference between these optical densities was calculated, and the final data were obtained by finding log(225/difference). All data were subsequently analyzed using Student’s t-tests in SPSS (with = 0.05 as the significance level).
The goal of our immunohistochemical analysis was to look for differences in the expression of Nav1.6 in the CA3 region of the hippocampus in epileptic and control mice. Representative images of hippocampi in the KA, sham, and no-primary antibody conditions are shown in Figure 1. It appears that the KA sections (Figure 1b) are qualitatively darker than the sham (Figure 1a) and no-primary-antibody (Figure 1c) sections. The bottom images show the beginning of the CA3 (yellow arrow) and the dentate gyrus (green arrow), used in the analysis as described in the methods. When the optical density differences in the KA (M = 0.088, SD = 0.300) and sham (M = 0.057, SD = 0.095) conditions were compared using an independent-samples t-test, no significant result was obtained: t(9) = -0.19, p = 0.852. Although it appears that the results are in the direction predicted by the hypothesis (Figure 2), the statistics do not support greater Nav1.6 staining in the CA3 of the hippocampus in the KA condition. It was unusual to obtain negative optical density differences, and we suspected that it was due to sections folding in on themselves, giving darker Nav1.6 staining in the dentate gyrus than in the CA3. To remedy this problem, we removed the negative optical density differences from our data and re-ran the t-test. This time, the KA condition (M = 0.249, SD = 0.250) was almost significantly higher in Nav1.6 staining in the CA3 than the sham condition (M = 0.065, SD = 0.097): t(6) = -1.885, p = 0.082. We believe that with better technique and more brain sections, this value could reach significance (Figure 3). Finally, as an exploratory analysis, we split all of the data into left and right hemispheres (for both KA and sham conditions) and compared to the no-primary antibody control. Negative optical density differences were retained. No statistically significant differences in any of the comparisons were found (showing that there was no difference in Nav1.6 staining in the left vs. right hemispheres for the KA, sham, and no- primary antibody control). Additionally, there was no effect of condition (no difference between KA and sham staining). These data are displayed graphically in Figure 4. This analysis was performed mainly to determine which hemisphere was injected with KA, but since no significant result was obtained, no conclusion could reliably be drawn.
Summary of Results
We found that qualitatively, the KA sections appeared darker than the sham sections and the no-primary antibody control. This indicates that there may be an increase in Nav1.6 expression in response to the induction of epilepsy. The differences were not statistically significant, though. The staining in the CA3 was almost significantly higher in the epileptic condition, indicating that the Nav1.6 sodium channel may be upregulated after seizures. However, when the data were broken down by hemisphere and condition, no significant differences were found.
Discussion of Trends
Overall, the results trended in the direction predicted by the hypothesis, suggesting that epilepsy might induce an upregulation of Nav1.6 channels. It is likely that the results were not statistically significant because of inconsistency in staining technique. The data were plagued by very large standard deviations. The fact that there were only five brain sections per condition could have led to this low statistical power. Another contributor could be poor execution of the protocols and inconsistent DAB staining. It was difficult to make sure that the DAB solution was left for the same amount of time in the wells containing the brain sections. This likely led to unequal staining in the brain sections and might have contributed to the large standard deviations in the data. In addition, it appeared that some sections (in both the KA and the sham conditions) folded in on themselves while they were being mounted onto slides. This would lead to inaccurate readings of the optical density and would dilute the data even further.
Another factor that could have contributed to the lack of a significant difference is that the Nav1.6 channel is ubiquitously expressed throughout the brain (Burbidge et al., 2002). As a result, it is likely that an increase in the expression in the CA3 also correlated with an increase in expression in the dentate gyrus, which was taken as the neutral background region. Therefore, any effect of KA on the overall expression of the channel would have been lost when a difference in staining between the CA3 and dentate was taken. To remedy this problem, perhaps it would be useful to analyze the staining in the CA3 region of the hippocampus alone. Finally, it is possible that the sections were left in the DAB solution for too long. This made the brown precipitate too prominent, overpowering the cresyl violet stain. It would have been interesting to observe whether or not the Nav1.6 staining overlaps with cresyl violet (such an overlap would suggest that neuronal Nav1.6 is affected). Some literature suggests that astrocytic Nav1.6 may be overexpressed in epilepsy, hinting that the expected overlap might not be observed (Zhu et al., 2016). However, this analysis was not possible, as the DAB staining overpowered the cresyl violet, obscuring neuronal cell bodies. To find support for the hypothesis, this study needs to be replicated with several modifications. More brain sections need to be used (for both the KA and sham conditions). The brains would be left in the DAB solution for a shorter time, and mounting would be more careful to prevent sections from folding onto themselves.
Applications and Future Directions
If a significant result had been discovered, several implications for epilepsy would become apparent. For example, the Nav1.6 channel could be used as a potential treatment to curtail future seizures. An allele of Nav1.6 that could be conditionally knocked out using a Cre- lox has been developed (Levin et al., 2003). If safely expressed in the human hippocampus, such a genetic manipulation could be used in place of the most drastic temporal lobe epilepsy treatments (such as medial temporal lobectomy, as performed on patient H. M). By reducing the expression of Nav1.6 in the hippocampus, the vicious cycle of seizure-driven hyperexcitability would be broken. In addition, finding out more about the effects of the Nav1.6 channel in temporal lobe epilepsy would contribute to the body of knowledge on epilepsy in general. It might shed some light on other seizure-based diseases, such as benign infantile epilepsy or paroxysmal dyskinesia. Preliminary research has found a glutamic acid to lysine substitution in Nav1.6 in both of these illnesses (Gardella et al., 2016). The connection of this channel to epilepsy could explain why these two diseases also present with seizures.
The present study could be expanded in multiple ways. For example, recent work has suggested that an increase in Nav1.6 after an epileptic seizure is associated with an upregulation in Ankyrin-G (Chen et al., 2009). This study was only correlational, and it would be interesting to investigate which upregulation (Nav1.6 or Ankyrin-G) causes the other one. At the same time, it may be fruitful to replicate the study using induced pluripotent stem cells from epilepsy and control patients. This would eliminate the major confound created by cannulating mice to administer the kainic acid. This procedure gives mice a traumatic injury that could affect the expression of many proteins (including Nav1.6 in the brain). Using cultured neurons from patients would represent a workaround, and it would make the research more applicable to humans.
In conclusion, the present study demonstrates that there may be an increase in Nav1.6 channels following an epileptic seizure. However, this result is not definitive, and it requires much future work for validation.
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Illustrations and Figures
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