Transcriptome analysis of the hippocampal CA1 pyramidal cell region after kainic acid-induced status epilepticus in juvenile rats

Abstract

Molecular mechanisms involved in epileptogenesis in the developing brain remain poorly understood. The gene array approach could reveal some of the factors involved by allowing the identification of a broad scale of genes altered by seizures. In this study we used microarray analysis to reveal the gene expression profile of the laser microdissected hippocampal CA1 subregion one week after kainic acid (KA)-induced status epilepticus (SE) in 21-day-old rats, which are developmentally roughly comparable to juvenile children. The gene expression analysis with the Chipster software generated a total of 1592 differently expressed genes in the CA1 subregion of KA-treated rats compared to control rats. The KEGG database revealed that the identified genes were involved in pathways such as oxidative phosporylation (26 genes changed), and long-term potentiation (LTP; 18 genes changed). Also genes involved in Ca(2+) homeostasis, gliosis, inflammation, and GABAergic transmission were altered. To validate the microarray results we further examined the protein expression for a subset of selected genes, glial fibrillary protein (GFAP), apolipoprotein E (apo E), cannabinoid type 1 receptor (CB1), Purkinje cell protein 4 (PEP-19), and interleukin 8 receptor (CXCR1), with immunohistochemistry, which confirmed the transcriptome results. Our results showed that SE resulted in no obvious CA1 neuronal loss, and alterations in the expression pattern of several genes during the early epileptogenic phase were comparable to previous gene expression studies of the adult hippocampus of both experimental epileptic animals and patients with temporal lobe epilepsy (TLE). However, some changes seem to occur after SE specifically in the juvenile rat hippocampus. Insight of the SE-induced alterations in gene expression and their related pathways could give us hints for the development of new target-specific antiepileptic drugs that interfere with the progression of the disease in the juvenile age group.

Figure 1. Neuronal CA1 pyramidal cell damage and the region-specific laser microdisecction one week after SE.

Fluoro-Jade B staining of a representative control (A) and KA-treated rat (B) one week after SE. Fig.1B shows positively stained CA1 pyramidal neurons (see also the inset in B). Figs.1C and D show hematoxylin-eosin staining of the CA1 pyramidal layer of a KA-treated rat before and after laser microdissection, respectively. Scale bars: A and B: 200 µm; C and D: 75 µm. Abbreviations: CA1 pyr, stratum pyramidale of CA1; DG, dentate gyrus; KA, kainic acid.

Figure 2. Heatmap of the samples assayed by microarrays.

The figure illustrates the highest contrasts between the samples that were clustered by similarity in the gene expression levels. The control samples were clearly distinguished as a coherent group separate from the samples of KA-treated rats. Controls showed up-regulation of a specific set of genes (as indicated by yellow) in contrast to the samples of KA-treated rats presenting down-regulated levels of the same genes (as indicated by blue), while a distinct set of genes were down-regulated in the control samples, and up-regulated in the samples of KA-treated rats. The clustering of the genes (gene names omitted) is shown in the left vertical panel, and the clustering of the samples is indicated in the top horizontal panel of the figure. Abbreviations: CTRL, control rat; KA, kainic acid-treated rat.

Figure 3. K-means clustering of the genes by their expression pattern.

The K-means clustering method is visualized with the 10 distinct clusters as plotted by the log of the gene expression level of all the genes analyzed by the microrarrays. Our interest was to reveal the major differences between the KA-treated and control rats. In the figure the samples of the control and KA-treated rats are indicted by the numbers 1–4 and 5–8, respectively, in the x-axis. Abbreviation: KA, kainic acid.

Figure 4. The KEGG-derived long-term potentiation pathway.

The concerted action of glutamate on NMDA and AMPA receptors in CA1 pyramidal neurons is illustrated in this pathway, as well as the activation of different subpathways. The genes activated on the microarray are encircled. The table below shows the entire list of up- or down-regulated genes related to LTP on the microarray. Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA, N-methyl-D-aspartate.

Figure 5. Validation of the microarray results by immunohistochemical staining in control and KA-treated rats.

Immunoreactivity of GFAP in a representative control (A) and KA-treated rat (B). Note the increase in GFAP immunoreactivity and the morphological change in astrocytes of the KA-treated rat (insets in A and B). Figs 5 C and D show the immunoreactivity of apo E in a control and KA-treated rat, respectively. Note the increase in apo E immunostaining in some cells within the CA1 pyramidal cell layer of the KA-treated rat compared to the control rat (insets in C and D, respectively). The CB1 immunoreactivity was enhanced more pronouncedly in a KA-treated rat (F) than in a control rat (E) in the borders of the CA1 pyramidal layer with both the stratum oriens and radiatum (white arrows in F). Scale bars: 75 µm in A–D, and 100 µm in E-F. Abbreviations: apo E, apolipoprotein E; CB1, cannabinoid type 1 receptor; CA1 pyr, stratum pyramidale of CA1; GFAP, glial fibrillary protein; KA, kainic acid; str., stratum.

Figure 6. Validation of the microarray results by immunohistochemical staining of PEP-19 and CXCR1 in control and KA-treated rats.

Immunoreactivity of PEP-19 in the whole hippocampus in a representative control (A) and KA-treated rat (B). Figs. 6C and Dshow the CA1 region of A and B, respectively, at the higher magnification. Note the lack of PEP-19 immunoreactivity in the CA1 pyramidal layer in the control rat (C), while the staining was weak in the CA1 layer (D, arrowheads), and greatly enhanced in single neurons (D, arrows) in the KA-treated rat. Figs. 6E and Fshow the CXCR1 immunoreactivity in the CA1 region of a control and KA-treated rat. In control rats, immunoreactivity occurred in discrete neurons in the CA1 pyramidal layer (E, arrows and insert 1), and in vascular endothelial cells (E, arrowheads and insert 2). In KA-treated rats, no immunoreactive neurons could be detected (F). Scale bars: 200 µm in A–B, 40 µm in C–D, and 75 µm in E-F. Abbreviations: CXCR1, interleukin 8 receptor; PEP-19, Purkinje cell protein 4; pyr, stratum pyramidale.