Kainic Acid-induced F-344 Rat model of Mesial Temporal Lobe Epilepsy: Gene Expression and Canonical Pathways

Abstract

Mesial temporal lobe epilepsy (MTLE) is a severe neurological condition of unknown pathogenesis for which several animal models have been developed. To obtain a better understanding of the underlying molecular mechanisms and identify potential biomarkers of lesion progression, we used a rat kainic acid (KA) treatment model of MTLE coupled with global gene expression analysis to examine temporal (four hours, days 3, 14, or 28) gene regulation relative to hippocampal histopathological changes. The authors recommend reviewing the companion histopathology paper (Sharma et al. 2008) to get a better understanding of the work presented here. Analysis of filtered gene expression data using Ingenuity Pathways Analysis (Ingenuity Systems, http://www.ingenuity.com) revealed that a number of genes pertaining to neuronal plasticity (RhoA, Rac1, Cdc42, BDNF, and Trk), neurodegeneration (Caspase3, Calpain 1, Bax, a Cytochrome c, and Smac/Diablo), and inflammation/immune-response pathways (TNF-α, CCL2, Cox2) were modulated in a temporal fashion after KA treatment. Expression changes for selected genes known to have a role in neuronal plasticity were subsequently validated by quantitative polymerase chain reaction (qPCR). Notably, canonical pathway analysis revealed that a number of genes within the axon guidance signaling canonical pathway were up-regulated from Days 3 to 28, which correlated with aberrant mossy fiber (MF) sprouting observed histologically beginning at Day 6. Importantly, analysis of the gene expression data also identified potential biomarkers for monitoring neurodegeneration (Cox2) and neuronal/synaptic plasticity (Kalrn).

Introduction

Mesial temporal lobe epilepsy (MTLE), one of the most common forms of epilepsy, is characterized by seizure generation from the mesial temporal lobe, especially the hippocampus (Falconer, Serafetinides, and Corsellis 1964; Margerison and Corsellis 1966). This disorder is characterized by chronic neuronal hyperexcitability and hypersynchrony that manifests as recurrent, unprovoked partial seizures with or without secondary generalization (Cavazos, Jones, and Cross 2004). Mesial temporal lobe epilepsy is thought to be initiated by lesions and functional alterations secondary to insults such as febrile convulsions, status epilepticus, encephalitis, or trauma, which, after a five- to ten-year latency period, generate spontaneous motor seizures (Engel 1993). Various animal models have been developed to investigate proposed pathogeneses for MTLE (Buckmaster 2004; Coulter, McIntyre, and Loscher 2002; Sharma et al. 2007; White 2002). Kainic acid (KA) administration in rats has frequently been used as an animal model since the epileptic process in KA-administered rats comprises the same three stages as seen in MTLE patients: initial insult, a latency period, and spontaneous motor seizures. In the rat brain, the hippocampal region has been shown to be particularly susceptible to KA-induced seizure generation and was chosen as the focus for our analysis of the temporal changes associated with the genesis of spontaneous seizures (Pinheiro and Mulle 2006).

Aberrant mossy fiber (MF) sprouting is thought to play a causative role in seizure generation in MTLE patients (Houser and Esclapez 1996; Obenaus, Esclapez, and Houser 1993). Mossy fibers are the axons of dentate granule cells, which normally synapse on the dendrites of CA3 pyramidal neurons. Aberrant MF sprouting, which is seen in the hippocampus of KA-treated rats, is a type of neuronal plasticity that is hypothesized to result from the initial insult in MTLE and gives rise to later spontaneous motor seizures (Menks and Sankar 2002; Ribak 1985; Robert 1994; Sharma et al. 2008; Sloviter 1996). The development of aberrant mossy fibers and subsequent MTLE is expected to involve the regulation of a variety of genes. Based on histopathological findings, the gene regulation associated with MTLE lesion development can be readily divided into three major ontological categories: neuronal/synaptic plasticity, neurodegeneration, and inflammation/immune responses. In a companion study as well as in the present work, the term neurodegeneration is used to characterize neurons having pyknotic nuclei, indiscernible nucleoli, and condensed hypereosinophilic neuroplasm (Sharma et al. 2008). Within the neuronal/synaptic plasticity category, the Rho family GTPases such as RhoA, Rac1, and Cdc42 have been shown to play a critical role in axonal growth by modulating changes in the actin cytoskeleton in the neuronal growth cone (Dickson 2001; Luo 2000; Ozdinler and Erzurumlu 2001). The neurotrophin-signaling members, brain-derived neurotrophic factor (BDNF) and its receptor TrkB (Ntrk2), also regulate the actin cytoskeleton via Rac1, which can lead to increased neurite outgrowth (Miyamoto et al. 2006). In addition to aberrant MF sprouting, neurodegeneration may also play a role in MTLE pathogenesis by enhancing neural remodeling. Increased expression of Caspase3, Bax, and the release of cytochrome C (Liu et al. 2001) have been observed during KA-elicited neuronal cell death in hippocampal slice cultures. T. Li, Lou, and Lu (2004) reported an association of Smac/Diablo with neuronal cell death caused by KA-induced seizures in rats, pointing to a possible apoptotic mechanism of neuronal cell death. Cyclooxygenase 2 (Cox2) activity, in neurons as well as in glial cells, may also play a critical role in neurodegeneration (Kelley et al. 1999), as the selective Cox2 inhibitor NS398 was shown to prevent hippocampal neurodegeneration following KA injection (Takemiya et al. 2006). In one model of MTLE, genes pertaining to prostaglandin synthesis (Cox2 induction) were found to be significantly up-regulated (Gorter et al. 2006). Inflammatory and immune responses, as represented by the various interleukins or their receptors, including IL-4, IL-6, IL-1β, and TNF-α, possibly play a role in KA-induced rat models of MTLE (Liu et al. 2000; Oprica et al. 2006; Ravizza et al. 2005). These findings suggest a role for a variety of cell death (apoptotic and non-apoptotic) and inflammation-related genes in KA-induced neurodegeneration.

Global gene expression analysis approaches, such as serial analysis of gene expression (SAGE) and cDNA and oligonucleotide microarrays, have previously been applied to the rat KA-induced MTLE model. Hevroni et al. (1998) employed a highly sensitive subtractive cDNA protocol to study gene regulation in the rat dentate gyrus following KA administration. Tang et al. (2002) performed oligonucleotide microarrays on parietal cortex of rats at four hours following KA-induced status epilepticus (SE). However, the only long-term gene profiling study on an MTLE model was that of cDNA profiling of electrically induced epilepsy (Lukasiuk, Kontula, and Pitkanen 2003).

In the work described herein, we used global gene expression analysis to evaluate and characterize long-term gene regulation relative to hippocampal histopathological changes as observed in a companion study (Sharma et al. 2008), which the authors recommend reviewing for a better understanding of the work presented here. Since limited information was available on the molecular pathways involved during the different temporal stages in the rat KA-induced MTLE model, we leveraged global gene expression data and subsequent pathway analysis to provide greater mechanistic insight into the role that these molecular pathways and their gene members may play in the pathogenesis of MTLE. The tool we used for this, Ingenuity Pathways Analysis (IPA), is a curated database and analysis system software application. Ingenuity Pathways Analysis was used to determine if the significantly expressed genes in our microarray experiment overlay onto any of the canonical pathways present in the IPA database. Our analysis led us to focus on those pathways and genes encompassing the previously described broad categories of neuronal/synaptic plasticity, neurodegeneration, and inflammation/immune response. In this article, we describe our use of gene expression and pathway analysis to provide mechanistic linkage to histopathological changes observed in the hippocampus. Further, we describe how, by using gene expression analysis coupled with histopathological observations, we have derived insight into the underlying molecular pathogenesis of the different phases of lesion progression in the rat KA-induced MTLE model.

Materials and Methods

Animals

Seven- to eight-week-old male Fischer 344 rats weighing 200 to 300 g were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and housed individually in stainless steel cages. The rats were grouped into two control and two treatment groups for both the gene expression study and a companion histopathology study (Sharma et al. 2008). The rats were allowed free access to a normal laboratory diet (Certified Rodent Diet 5002, Pellet, supplied by PMI Nutrition International, Inc.) and chlorinated potable water. All rats were acclimated for at least one week to housing facilities and diet before being used in the study. Animal room conditions were set to maintain a temperature of 69°F to 75°F and 30% to 70% relative humidity. Rats were maintained on a twelve-hour light/dark cycle. The Animal Care and Use Committee of Lilly Research Laboratories approved all study protocols.

Kainic Acid Treatment

The rats were administered KA (Sigma, USA) subcutaneously at 9 mg/kg. Only those rats with status epilepticus (SE), characterized by stage 4 to 5 limbic seizures, were included in the study. The SE typically developed thirty to forty-five minutes post-KA administration. To minimize the mortality rate, the SE was blocked by diazepam (Hospira, Inc., USA) at 10 mg/kg via the i.p. route approximately one hour and forty-five minutes following initiation of SE. Control rats received similar volumes of normal saline and diazepam. Subcutaneous fluids were administered to dehydrated rats unable to drink water on their own.

Assignment of Rats to Treatment Group

Rats were randomly assigned to treatment and control groups and sequentially numbered. For each time point, three control and six treated rats with the lowest animal identification numbers were selected for hippocampal collection. Selection criteria among the treated rats included previous SE for those terminated on days 3 and 6 and previous spontaneous motor seizures for those terminated on days 14 and 28.

Behavior

Rats were evaluated continuously for clinical signs progressing to SE for four hours post-KA administration. Subsequently, rats were examined cage-side for spontaneous motor seizures by two well-trained technicians and a trained pathologist in a nonblinded fashion for four approximately equally spaced thirty-minute periods between 7:00 a.m. and 5:00 p.m. on weekdays until study termination. Status epilepticus and post-latency spontaneous motor seizures were recorded on a scale of stages 1 through 5 (Racine 1972).

Tissue Collection and RNA Isolation

At four hours post-KA administration and on days 3, 14, and 28, the entire left portion of the hippocampus (approximately 35 mg) from three control and six treated rats was excised and stored in RNAlater (Ambion, Inc., Austin, TX), according to manufacturer recommendations. Total RNA was isolated using the RNA-STAT method (Chomczynski and Sacchi 1987). RNA was further purified using RNeasy columns (Qiagen, Valencia, CA), according to package instructions. The quality and quantity of total and purified RNA were evaluated using agarose gel electrophoresis and UV spectrophotometry (Gene Quant II spectrophotometer, Amersham Biosciences Corp, Piscataway, NJ), respectively.

cDNA and Biotinylated cRNA Synthesis and Hybridization

The following steps were carried out according to procedures found in the GeneChip Expression Analysis Technical Manual. cDNA and biotin-labeled complementary RNA (cRNA) were synthesized from 5 μg of purified total hippocampal RNA. The biotinylated cRNA targets were then cleaned up, fragmented, and hybridized to GeneChip Rat Genome 230 2.0 Array (Affymetrix Inc., Santa Clara, CA). Information on the Rat 230 2.0 array, which comprises more than 31,000 probe sets, analyzes over 30,000 transcripts and variants from over 28,000 well-annotated rat genes, is available on the following Web site: http://www.affymetrix.com/support/technical/datasheets/rat230_2_datasheet.pdf.

Data Analysis

The arrays were scanned with an Affymetrix Gene Chip Scanner, and data analysis and mining were performed using MAS 5.1 software (MicroArray Suite, Affymetrix) and data mining tool (DMT 2.0) software. The measurement of the ratio between perfect match (PM) to mismatch (MM) (PM/MM ratio) was used to define transcripts as present (P), marginal (M), or absent (A). Default settings provided by Affymetrix were used for this purpose. A comparison analysis was performed for each sample collection day for the treated rats (average of six animals/chips), using its corresponding control value (average of three animals/chips) as the baseline.

The effect of KA on each probe set was evaluated using a two-factor analysis of variance model (Winer 1995). Factors in the model included treatment, time, and their interaction. Effects associated with the overall treatment effect and treatment-by-time interaction were tested using F tests. P values were used to control the expected proportion of falsely rejected hypotheses in multiplicity testing. All effects were initially evaluated at the p ≤ .05 significance level. If the treatment-by-time interaction was significant (p ≤ .05), treatment effect at each post-dose time point was tested using a t test.

Pathway Analysis

To more stringently focus the temporal transcript changes prior to pathway analysis, a change significance p value filter of p ≤ .005 was applied to the array data for each time point. The number of transcripts meeting the p value ≤ .005 significance level averaged approximately 700 to 1100 for each time point. The filtered data sets for each time point were up-loaded into IPA for canonical pathway analysis.

This analysis identified pathways from the IPA library of canonical pathways that were most significantly influenced by the input data set. The significance of the association between the data set and the canonical pathway was measured in two ways: (1) a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed and (2) a Fischer exact test was used to calculate a p value, determining the probability that the association between the genes in the data set and the canonical pathway was explained by chance alone. Genes from the input data set that were associated with a canonical pathway in the Ingenuity Pathways Knowledge Base were considered for further analysis.

Quantitative Real-time PCR

Oligonucleotide primers used for qPCR validation studies were identified using the PrimerBank web site (http://pga.mgh.harvard.edu/primerbank/index.html). Total RNA was isolated as described above and treated with DNase I for thirty minutes at 37 °C. DNase I was inactivated (DNAfree, Ambion Inc., Austin), and the RNA was used to generate first-strand cDNA (High Capacity cDNA Archive Kit, Applied Biosystems, Foster City, CA) for use in quantitative (SybrGreen) polymerase chain reaction (PCR) analysis. Quantitative PCR (qPCR) was performed using the ABS 7700 sequence detector. Each sample was assayed in triplicate, and relative quantification was performed using the C_t method (ABS user bulletin number 2) using GAPDH as the normalization gene. The primer/probe sets were determined to have the same amplification efficiencies as the GAPDH control sets (data not shown). Primer concentrations and cycle number were optimized to ensure that reactions were analyzed in the linear phase of amplification. For qPCR, RNA samples from all rats in a group at each time point were pooled together. Genes that were selected for validation by qPCR had regulation at least at one time point and were associated with neuronal plasticity.

Results

Behavioral and Histopathological Observations

Following KA administration, all rats evaluated in this study had SE on day 0. Spontaneous motor seizures were first observed on Day 7. Approximately 80% of the rats dosed developed stage 3 to 5 spontaneous motor seizures after a variable latency period of 7 to 14 days following KA administration. On days 14 and 28, hippocampi were collected for gene array from only those rats that developed spontaneous motor seizures. In the companion study (Sharma et al. 2008), histopathological changes relevant to the categories of neuronal degeneration, aberrant mossy fiber sprouting, microgliosis, and astrogliosis were recorded at days 3, 6, 14, and 28. Severity scores for each of the categories at each time point were generated and are shown in Figure 1. Neuronal degeneration and microgliosis were evident throughout the time course, whereas aberrant mossy fiber sprouting and astrogliosis appeared at day 6 and continued through day 28.

Globally Differentially Regulated Genes

An oligonucleotide array performed on rat brains following KA treatment showed gene regulation at all four observation points in our study. The most differentially expressed genes were observed on day 14, whereas day 3 exhibited regulation of the least numbers of genes. There was an increase in number of regulated genes from day 3 to day 14, which corresponded with the first observed aberrant MF sprouting on day 6 (Figure 1) and the first seizures seen on day 7, as described in the companion study (Sharma et al. 2008). To determine similarity in gene expression changes across the experimental time course, a hierarchical cluster correlation analysis was performed on the 500 most variable transcripts (standard deviations). The clustering analysis output clearly shows that the four hours time point is separated from the later time points and that the day 14 and day 28 time points are most similar in their gene expression patterns (Figure 2).

Validation of Selected Genes by Quantitative PCR

Based on a literature search and data mining, seven differentially regulated genes (DRGs) with putative neuronal plasticity roles and significant regulation in at least one time point were selected for qPCR analysis to validate gene array results. A correspondence in similarity of direction, rather than absolute fold change identity, was used as the criterion for validating the consistency of the two data types. In general, when the significance of the array data was high (p value ≤ .05), the concordance with the qPCR data was also high, indicating that when there is discordance it is most likely a result of low-quality array data. However, when evaluating the overall gene expression response across the entire time course, there is general agreement between the array data and the qPCR data for each of the selected transcripts.

Immediate Early Gene Expression Response

The microarray data revealed that, following KA administration, rats that had developed SE showed a remarkable up-regulation of a number of immediate early genes, as witnessed by their dramatic induction at four hours. At a significance of p ≤ .05, 596 genes were induced twofold or greater. Selected genes, described below and in the discussion section, are shown in the heat map in Figure 2. Well-known immediate early genes involved in transcriptional regulation, such as Fos12, Junb, and Egr1, were up-regulated 29, 9.3, and 6.6-fold, respectively. In addition, three members of Nur77/NGFI-B transcription factor family (Nr4a1, Nr4a2, and Nr4a3) were up-regulated seven-, thirteen-, and seventy-one-fold, respectively. The nucleotide excision repair gene Rad23a was up-regulated 4.7-fold. Additional genes that were significantly up-regulated at four hours were indicative of inflammatory responses, such as Ptgs2 (Cox2) 27.5-fold, Cc12 (monocyte chemoattractant protein-1, MCP-1) 68.2-fold, or neuronal degeneration, Homer1 144-fold, and Bdnf, 2-fold. In contrast, a transcriptional repressor Atf7ip was down-regulated 3.7-fold.

Analyses of Relevant Genes and Canonical Pathways

In an attempt to ascertain biologic meaning from the gene expression data, the transcripts at each time point, exhibiting significant changes relative to time-matched control, were analyzed for their impact on canonical pathways, using IPA. Highly significant pathway impacts seen at four hours, fourteen days, and twenty-eight days were indicative of gene modulation in the categories of neuronal/synaptic plasticity (axonal guidance signaling, ephrin receptor signaling, synaptic long-term potentiation/depression), neurodegeneration (neurotrophin/TRK signaling), and inflammation/immune response (GM-CSF signaling, chemokine signaling) (Table 1 and Figure 3). Based on the output from the pathway analysis as well as additional annotation analysis, sets of relevant genes from the above mentioned categories were selected for further analysis, to determine their possible mechanistic linkage to the temporal histopathological changes observed in the KA induced MTLE model (Tables 2, 3, and 4).

Neuronal Plasticity-related Genes

Forty transcripts with known roles in neuronal/synaptic plasticity were chosen from the filtered set of significantly changed transcripts (Table 2). Nine of these genes, which were significantly regulated on at least one time point, were mapped to the axonal guidance signaling pathway (Table 1 and Figure 3a). The transcription of Kras2 and Bdnf showed significant up-regulation only at four hours, whereas Ntrk2 the cognate receptor of Bdnf was significantly up-regulated on days 3 through 28. Sema4D was significantly up-regulated at four hours and day 14. Transcripts downstream of Bdnf signaling, including Kras, Sema4D (which binds to Plexin B), and the Rho GTPases Rac1 and RhoA, were up-regulated, respectively, from days 3 and 14 through day 28, whereas the RhoGTPase Cdc 42 was significantly up-regulated on day 14. In a similar fashion to the RhoGTPases, the Rac/Rho regulator Kalirin (Kalrn) was significantly up-regulated on days 3 and 14. Ncam1 and Pak3, which may play a role in neuritogenesis, were significantly down-regulated on day 14 (Ncam1) and day 28 (Pak3), respectively. Egr1 and Syb11, two other transcripts with roles in neuritogenesis, also showed significant down-regulation on day 28. In the companion histopathology study, aberrant MF sprouting was first observed on day 6, was maximal on day 14, and was slightly decreased thereafter (Figure 1). The down-regulation of these neuritogenesis-related transcripts at day 28 is therefore reflective of the diminishment of the aberrant MFs generation that was observed at that time (Figure 1).

Neurodegeneration-related Genes

A total of thirty-six significantly regulated genes were compiled based on their roles in neurodegeneration (Table 3). Several of these genes, including Bdnf, Ntrk2, and Kras 2, map to the neurotrophin/TRK signaling pathway (Table 1 and Figure 3c). It should be noted that these genes are also members of the axonal signaling canonical pathway, demonstrating that these transcripts have other roles in neuronal function in addition to their role in neurodegeneration. Some of the other roles attributed to these three genes include axonal guidance, synaptic plasticity, apoptosis, cell survival, and cell proliferation (Alcantara et al. 1997; Ishikawa, Ikeuchi, and Hatanaka 2000; Minichiello and Klein 1996; Rossler, Giehl, and Thiel 2004).

Seven transcripts associated with apoptotic signaling showed significant regulation (Table 3). Caspase 3 was up-regulated at four hours and day 28, whereas Bax and Smac/Diablo were up-regulated on day 14 and Cytochrome c at four hours and days 14, and 28. Calpain small subunit (Capns1) was up-regulated on days 3 and 14, whereas TNF-α and Kras were up-regulated only at four hours. The up-regulation of these apoptosis-related genes correlates well with the neurodegeneration observed from day 3 onward (Figure 1).

Cited2, a pro-survival gene, was up-regulated 7.8- and 2-fold at four hours and day 28 respectively, whereas the anti-apoptosis gene Bag 3 was up-regulated 4.8- and 2.3-fold at four hours and day 3, respectively. The induction of these anti-apoptotic genes is most likely a compensatory response to injury by impacted neurons.

Inflammation/Immune-response–related Genes

A total of 40 significantly regulated genes were compiled based on their role in inflammation/immune-response (Table 4). Tumor necrosis factor-α, an important pro-inflammatory mediator, was up-regulated 4.7 fold at four hours but showed no further induction during the 28-day time course. Tumor necrosis factor-α may act as an early trigger for apoptotic and inflammatory responses (Barker et al. 2001; Knoblach, Fan, and Faden 1999). The mRNA levels for two genes that have critical roles in prostaglandin synthesis, Ptgs2 (Cox2) and Alox5ap, were increased at four hours and at all post-KA treatment collection times. This gene expression evidence for prostaglandin synthesis elevation correlated well with an inflammatory response (microgliosis and astrogliosis) that was observed from days 3 through 28 (Figure 1) This early inflammatory response probably plays a role in mediating the neurodegeneration observed from day 3 onward.

We saw additional transcript changes indicative of glial cell activation, which correlate with the observed glial cell infiltration seen in the companion study. Glial fibrillary acidic protein (GFAP), a marker of cells of glial lineage (Hansen et al. 1991) showed a significant transcriptional up-regulation from Days 3 to 28 (Table 4). Immunohistochemical evidence of GFAP-labeled astrocytes was observed from day 6 to day 28 in the companion study (Figure 1). CD68, a gene associated with microglial cell activation, was significantly up-regulated on day 3 (Table 4), when an activated microgliosis was first observed and which continued through day 28 (Figure 1).

Discussion

In this study, we provided an overview of temporal gene regulation in the KA-induced rat model of MTLE by evaluating global gene expression (microarray) data relative to histopathology changes observed in the companion study, and to previous work described in the literature. As such, we administered KA at 9 mg/kg subcutaneously to F-344 rats to generate an MTLE effect and to allow us to document the clinical signs, and to investigate the morphology, distribution, and temporal progression of the resulting lesions. Spontaneous motor seizures were first observed on day 7 after KA administration. Six treated rats, which were necropsied on day 14, exhibited a total of twenty-seven seizures (excludes those seizing immediately post-KA administration) prior to day 14. Four of six treated rats had five seizures within the two weeks prior to necropsy on day 28. The hippocampal lesions were characterized by neuronal degeneration and microgliosis in the CA1, CA3, and the dentate hilus regions and by aberrant MF sprouting and were similar to those observed in MTLE patients (Babb and Brown 1986). Neurodegeneration was first observed on day 3, but the severity continuously decreased thereafter until study termination at day 168. The gliosis that started parallel to the neurodegeneration was most intense on day 28 and persisted until the termination of the study. Aberrant MF sprouting was first observed on day 6, immediately preceding the spontaneous motor seizure generation, was of increased severity on days 14 and 28, and decreased until study termination.

In this model, expression of numerous genes was modulated as judged by microarray, therefore, to verify the validity of the gene expression changes seen in the microarray data, we evaluated seven neuronal plasticity-related differentially regulated genes by qPCR. When comparing qPCR and array fold change data, we noted that microarray fold changes with a p value ≤ .05 showed regulation in the same direction as qPCR-derived fold changes. Based on this association, we concluded that microarray data based on p value ≤ .05 (the criterion used for all our gene list generation) could be considered reliable. Using time course array data with an even more stringent p value filter (p ≤ .005), we undertook a canonical pathway analysis using IPA software. With the analysis, we were able to identify significantly impacted pathways that were both mechanistically relevant and correlated with temporal histopathological observations (Table 1 and Figure 3). From the pathway analysis and additional annotation mining, we compiled lists of genes that pertained to the broad categories of neuronal/synaptic plasticity, neurodegeneration, and inflammation/immune-response. In the ensuing sections, we discuss in more detail the identified pathways and associated genes and their relevance to the pathophysiology of MTLE.

Immediate Early and Early Gene Responses

Initial analysis of the gene expression data revealed the striking induction of a large number of (immediate early) genes following KA-induced SE (four hours) (Figure 4). A significant number of induced genes were found to be broadly involved in transcriptional regulation, cell differentiation and proliferation, apoptosis, and inflammation. Fos12 and Junb, which were up-regulated 29 and 9.3-fold respectively, are immediate early genes that heterodimerize to form the AP-1 transcription factor. Through its role in regulating gene transcription (Kaminska et al. 2000), AP-1 has been shown to affect many cellular processes including cell differentiation, apoptosis, and regulating the function of extracellular matrix proteases (Jaworski et al. 1999). The effect of AP-1 on extracellular protease function may ultimately have a role in neuronal plasticity, as controlled proteolysis is essential for this process (Monard 1988). The gene Egr1, an early growth response transcription factor, which was up-regulated 6.6-fold, is known to modulate synaptic plasticity by regulating Arc ( L. Li et al. 2005). The immediate early genes belonging to the NR4A group were also remarkably up-regulated (NR4A2, thirteen-fold; NR4A3, seventy-one-fold). These genes may be induced by a variety of signals, such as stress, growth factors, cytokines, peptide hormones, and neurotransmitters (Maxwell and Muscat 2006).

In addition to the classic early response genes described above, at four hours we also saw an early neuronal response to an excitatory stimulus, as evidenced by the induction of the Homer1 and Bdnf genes (Figure 4). Both genes are known to have roles in modulating synaptic responses. The dramatic induction (144-fold) of Homer1 could be a response to increased synaptic activity during SE, as previous studies have shown that Homer1 up-regulation occurs in response to physiological synaptic activity in the hippocampus, particularly during cortical development (Brakeman et al. 1997; Kato et al. 1997). Homer1, which binds metabotropic glutamate receptors as well as downstream signaling elements, has been shown to have a dampening effect on postsynaptic neuronal signaling. BDNF, which showed a 22.4 fold increase at four hours, is a nerve growth factor that promotes survival of specific neurons and regulates synaptic transmission (Middlemas et al. 1999).

Evidence for cell injury and an immune response are also seen at the earliest time point. Rad23a and Xpa, which were up-regulated at four hours, both play a role in nucleotide excision repair (NER) (de Boer et al. 2002; Sugasawa et al. 1997). Their up-regulation may be an attempt to repair damaged DNA following KA-generated excitotoxicity. At the same time, the Cc12 (monocyte chemoattractant protein-1, MCP-1) gene was remarkably up-regulated (72.6-fold). Cc12 plays a critical role in the innate immune response by directing the migration of macrophages to sites of inflammation (Huang et al. 2001). This increase in Cc12 expression may have been a major initiating factor for the microgliosis observed from Day 3 through Day 14 in the companion study.

Neuronal Plasticity

Neuronal plasticity is a dynamic process and includes several events including axonal and dendritic growth and synaptic alterations. The two most significantly impacted canonoical pathways in the Ingenuity analysis were Axonal guidance signaling and Ephrin signaling, both at Day 14 (Table 1), when significant aberrant mossy fiber generation was evident (Figure 1). Gene members of both of these pathways play significant roles in the axonal and denditic outgrowth seen at this stage. It is notable that a number of Rho GTPases (RhoA, Rac 1, and Cdc42), which have been implicated in axonal sprouting (Dickson 2001; Luo 2000), are up-regulated at times that correlate well with aberrant mossy fiber generation (Table 2). These Rho GTPases cause changes in the actin cytoskeleton in the neuronal growth cones, leading to axonal growth (Dickson 2001; Luo 2000). Rac and RhoA regulate axonal growth in trigeminal brainstem whole-mount cultures (Ozdinler and Erzurumlu 2001). Sema4D-induced RhoA activation has a role in actin filament organization and axonal growth cone collapse, preventing axonal growth (Swiercz, Kuner, and Offermanns 2004). In addition, plexin-B inhibits Rac-induced PAK activation (Vikis, Li, and Guan 2002), thus inhibiting axonal growth. Sema4D mRNA was significantly up-regulated at four hours and at Day 3, and thus, the action of Sema4D probably prevented MF growth during that early time frame. Conversely, a significant up-regulation of Bdnf at four hours, and its cognate receptor Ntrk2 on Days 3, 14, and 28, support the roles of this neurotrophin and its receptor in neurite growth (Miyamoto et al. 2006). High Bdnf levels at four hours likely activate the Ntrk2 receptor (Middlemas et al. 1999). We observed two- and four-fold increases in Ntrk2 expression on Days 14 and 28, respectively, by qPCR. During the later time points, an increase in Ntrk2 level likely led to increased activation of GTPase Ras protein (Kras) (Reuther et al. 2000), which was up-regulated on Day 0, and possibly activated PI3 kinase. PI3 kinase phosphorylates Rho GTPase family member Cdc42 and Rac (Benard, Bohl, and Bokoch 1999; Jiang et al. 2000). Inactive GDP-bound RhoA is converted to active GTP-bound RhoA by Rac. RhoA-binding kinase Rock induces neurite retraction acting downstream of Rho in neurons (Katoh et al. 1998). Rac and RhoA mRNA levels were significantly high on Days 3, 14 and 28, which overlapped with the time when aberrant MF sprouts developed and persisted in our model. This pathway may have a role in both growth and retraction of axons (Katoh et al. 1998; Ozdinler and Erzurumlu 2001). Ntn1, which is up-regulated on Day 28, also has a role in RhoA-, Rac-, and Cdc42-mediated actin cytoskeleton reorganization, resulting in neurite elongation (Meyerhardt et al. 1999; Xie et al. 2006). Downstream to Ntn binding to its receptor DCC, Nck1 activates Rac and Cdc42 (Govek et al. 2005; X. Li et al. 2002), which ultimately brings about actin dynamics changes and resultant axon attraction toward its target.

In axonal guidance signaling, which was the most significantly impacted pathway according to our input gene data (Table 1), the majority of genes, especially the Rho GTPases (RhoA, Rac, and Cdc42) and Ntrk2 were up-regulated on Days 3 to 28 (Table 2). In the companion study, aberrant MF sprouting was first observed on Day 6 and persisted until Day 28 (Figure 1). The concordance of the up-regulation of Rho GTPases and Ntrk2 with the occurance of aberrant MF sprouting alludes to the potential importance of these gene products, which have demonstrated roles in axonal guidance, in the formation of aberrant mossy fibers.

Kalrn showed approximately two-fold up-regulation by qPCR on Day 14 and was significantly induced in the microarray analysis on Days 3 and 14 (Table 2). Kalrn is a brain-specific multidomain GDP/GTP exchange factor necessary for the maintenance of hippocampal pyramidal neuron dendrites and dendritic spines (Ma et al. 2003) as well as new axon outgrowths (May et al. 2002). The Kalrn transcript, as it is brain specific and induced coincident with the observed aberrant mossy fiber formation, may be a possible marker for dendritic and axonal outgrowth. Rac was significantly up-regulated on Days 3, 14, and 28. Kalrn and Rac likely play a role in aberrant MF sprouting and morphogenesis of dentate granule cell spines following activation of Eph receptor B (EPHB) by Ephrin B (EFNB) (Zhou et al. 2001). Similar neuronal dendritic and axonal outgrowth process generation may also be mediated via the Rho GTPase Cdc42, which was up-regulated on Day 14. Considered together, the up-regulation of the Rho GTPases and their associated regulator (Kalrn) at times associated with aberrant MF generation, supports their role in mediating axonal/dendritic sprouting.

The neurotrophin/TRK signaling pathway, one of the most significantly impacted canonical pathways according to the input gene expression data (Table 1), mediates multiple responses including apoptosis; cell survival, growth, and proliferation; neurite outgrowth; and synaptic plasticity. In our study, a total of six significantly regulated genes were present in the neurotrophin/TRK pathway (Table 1 and 3). BDNF binding with the Ntrk2 receptor dimer leads to apoptosis, cell growth and proliferation, and neurite growth and differentiation (Alcantara et al. 1997; Barnabe-Heider and Miller 2003; Encinas et al. 1999; Ishikawa, Ikeuchi, and Hatanaka 2000; Minichiello and Klein 1996; Rossler, Giehl, and Thiel 2004). In the present study, up-regulation of Ntrk2 mRNA on Days 3, 14, and 28 suggests that Ntrk2 signaling plays a role in neurodegeneration, neuronal survival, and neurite growth, which are critical processes occurring during epileptogenesis and epilepsy.

Neurodegeneration

Seven significantly regulated genes related to cell death and apoptosis are present in the neurodegeneration category (Table 3). Caspases and calpains are two cysteine protease families with roles in neuronal cell death (Chan and Mattson 1999). mRNA levels of Caspase 3 and calpain subunit 1 (Capns1) were significantly increased at four hours and Days 3 and 28, respectively. In contrast, Smac/Diablo and Bax mRNA levels were up-regulated only on Day 14. It is possible that initial (four hours) KA-induced neurodegeneration occurred primarily as a result of caspase 3 activation. These data would suggest that neuronal cell death following KA administration is largely mediated by an apoptotic mechanism and is triggered by calpain, TNF-α, and mitochondrial-mediated Caspase 3 activation at various time points.

The down-regulation of Otx2 at four hours is also consistent with a pro-apoptotic environment. Forebrain Otx2 mutant neurons undergo apoptosis (Rhinn et al. 1999), supporting a possible pro-apototic effect as a result of the down-regulation of this gene at four hours. Pro-survival genes Cited 2 (Shetty et al. 2005) and Bag3 (Lee et al. 1999) were up-regulated at four hours and Day 28 and four hours and Day 3, respectively. The pro-survival gene up-regulation may be an attempt by neurons to offset the neuronal degeneration observed, beginning on Day 3.

Inflammation/Immune Response

Consistent with a rapid and continuing inflammatory response in post-KA treatment, hippocampal tissue is the induction of two genes that have important roles in prostaglandin signaling, Ptgs2 (Cox2) and Alox5ap. mRNA levels for these two genes were increased at four hours and at all post-KA treatment time points (Table 4). Cox2 is an immediate early gene that is constitutively expressed in pyramidal neurons of the hippocampus and cortex, and in the amygdala (Yamagata et al. 1993). Excitatory stimuli such as seizures and KA (Chen et al. 1995; Tocco et al. 1997; Yamagata et al. 1993), and cerebral ischemia (Sanz et al. 1997), markedly and transiently up-regulate Cox2 expression in neurons. Since the selective Cox2 inhibitor NS398 prevents hippocampal neurodegeneration following KA injection (Takemiya et al. 2006), it is thought that Ptgs2 has a pivotal role in increasing neuronal cell death. Alox5ap increases oxygenation of arachidonic acid, leading to increased chemotaxis, cell proliferation, and vascular permeability. Increased Alox5ap mRNA expression along with the increased Cox2 expression across the time course are clear indicators of a continuing inflammatory response in the hippocampus, especially microgliosis, which was evident from Days 3 to 28 (Figure 1). The rapid response of the Ptgs2 gene to KA-induced SE, as well as its postulated role in neurodegeneration, make it an attractive candidate as an injury biomarker for MTLE.

Conclusion and Future Directions

This study, coupled with the associated companion study, to our knowledge, is the first set of experiments to provide a temporal overview of gene regulation relative to histopathologic findings in the KA-induced rat model of MTLE. The array data demonstrate that there is temporal regulation of a number of genes/pathways that correlate with the observed development of lesions and with previously described mechanisms of MTLE. Our data support the hypothesis that regulation of a variety of genes pertaining to neuronal plasticity, neurodegeneration, and inflammation/immune-response processes occur during different stages of MTLE development. Although regulation of genes known or predicted to have roles in the above processes occurred at times that correlated with observed lesions, additional experimental evaluation will be needed to verify the precise role these genes may play in the KA-induced MTLE process. The use of knockout or knockdown approaches with some of the key gene products identified in this study may be an appropriate way to assess the role these genes and their associated pathways play in MTLE. We also believe that results of this study and follow-up work will provide better insight into the pathogenesis of MTLE and will eventually lead to the discovery of new therapeutic targets.