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Gene Expression Profile of the Hippocampus of Rats Subjected to Diffuse Axonal Injury

Abstract:

Background: The patient usually appears some serious complications and the nerve function obstacle after a traffic accident left him with a traumatic brain injury. Because of neuronal death and rupture of nerve fiber conduction, the patients with brain injury have significant limbs powerless, activity obstacle, and memory impairments of logical thought. Numerous studies indicate that the differentially expressed genes (DEGs) of neural signaling pathways are strongly correlated with brain injury. To further analyze the roles of the DGEs in nervous system, we systematically investigated the diffuse axonal injury (DAI) on the hippocampus and its injury mechanism at the whole genome level.

Methodology/Principal Findings:  With the rat whole genome expression chips (Affymetrix), we sequenced gene expression profile in the hippocampus of rats suffered DAI (for 1 day, for 7 days, for 14 or 30 days). Compare with the control group, 315, 326, 327 and 313 DEGs were identified in the hippocampus of rats subjected to DAI for 1 day, 7 days, 14 days and 30 days, respectively. Basis on the gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses, the DEGs were involved in many signaling pathways related to nervous system, especially neuronal survival-related pathways. Finally we verified the microarray results and detected the gene expression of neuronal survival-related genes in the hippocampus by using Real-time quantitative polymerase chain reaction.

Conclusions/Significance:These results suggested that the DEGs and many signaling pathways might play a major role in DAI. It also provided several targeted genes related to DAI for future investigation.

Keywords: hippocampus; diffuse axonal injury; differentially expressed genes; neuronal survival-related pathways

Introduction

Traumatic brain injury (TBI) happens when outside forces traumatically injures the brain, which is also called for intracranial injury [1-3]. Worldwide, more than ten million people die of TBI every year [2]. According to the World Health Organization, TBI is expected to become the world’s major cause of death and disability by 2020 [4-6]. Axonal damage or dysfunction, also known as diffuse axonal injury (DAI), is considered the most important pathologic feature of TBI [4-6]. Because of unclear pathogenesis of DAI, there is no effective treatment for axonal disconnection. DAI can cause several pathological changes, including demyelination, ionic imbalances, lipid peroxidation, mitochondrial alterations and oxidative stresses [7, 8]. Moreover, DAI can lead to a series of biochemical events including electrochemistry, metabolism, neurological disorders and inflammation [9, 10]. Numerous studies indicate that DAI may be in close contact with neuroinflammation and microglial activation and also contribute to cellular damage [11].

The signaling pathways for nerve damage and repair have been well-studied [12-14], such as mitogen activated protein kinase (MAPK) signaling pathways, notch signaling pathway, NF-κB signaling pathway. Toll-like receptors (TLRs) have been found to be expressed in neurons and play an important role in regulating inflammatory response. Membrane-bound TLRs are one kind of the pattern-recognition receptors, which primarily express in microglia and astrocytes in the central nervous system (CNS) [15, 16]. TLRs can make out endogenous danger-associated molecular patterns which are released by dying or damaged cells when under cellular stress [15, 16]. Several CNS disorders, such as Alzheimer disease, DAI, TBI and intracerebral haemorrhage, are associated with TLR-mediated inflammatory reactions [17-21]. Activation of BDNF/ TrkB pathway and notch signaling pathway is beneficial to neurovascular repair after TBI [3, 22].

The hippocampus is located in the medial temporal lobe of brain, which is the major brain component of humans and other vertebrates [23-25]. The hippocampus is one of the most important brain regions related to mediating stress reaction and is also the key encephalic organ that participates in cognition, emotion, learning and memory [23-25]. The high dose of glucocorticoid leads to decreasing hippocampal apoptosis and nerve cell plasticity, thereby causing the atrophy and loss of nerve cell, and eventually leading to local structural and functional damage [24, 25]. At present a large number of researches indicate that DAI can affect the functions of hippocampus at the single gene level [26-28]. As far as we know, there are few investigations published about DAI affects the function of hippocampus at the whole genome level.

In order to provide clearer information about the differentially expressed genes (DEGs) of neural signaling pathways are strongly correlated with brain injury, we sequenced gene expression profile in the hippocampus of rats suffered DAI (for 1 day, for 7 days, for 14 or 30 days) by the whole genome expression chip. The whole genome expression chip is an efficient method for analyzing the alterations of gene expression, and has been used successfully in many tissues, including adipose tissue [29], corpus cavernosum [30], eye [31], heart [32-34], hippocampus [23], kidney [35-37], limb skeletal muscle [29,38-40], liver [29], pancreas [41] and spleen [42]. In the present study, the rat whole genome expression chip (Affymetrix) was used to detect the DEGs profile of the hippocampus of rats subjected to DAI. This chip contains 31,000 probe sets, and 28,000 of them are obtained from the NCBI Reference and UniGene database. The alterations in gene expression of rats and the mechanism that DAI affects the function of the hippocampus were studied at the whole genome level.

Materials and methods

Animals and grouping

All experiments were performed on male Sprague-Dawley rats (SPF grade, weighing 225g ±10g, 8-10 weeks old) were purchased from the Shanghai Vitalriver Laboratory Animal Research Center (Animal license No. SCXK (Shanghai) 2006-0009). All animal experiments were carried out in accordance to the guidelines of China legislations on the ethical use and care of laboratory animals. After adaptive feeding for one week, the rats were randomly divided into five groups: control group, 1-day DAI group, 7-day DAI group, 14-day DAI group and 30-day DAI group. The rats were housed in groups of five per cage and were raised in a common animal room with a temperature of 22°C ± 2°C and a relative humidity of 35% ± 5%. All of them were given conventional same feed and free access to food and water. Every effort was made to minimize animal suffering during surgery or recovery from surgery.

Animal model of DAI

By use of a lateral head rotation device, the DAI model of rats was built according to several previous studies [20]. The detailed processes were: with an anterior teeth hole and a head clip anchoring two lateral ear bars, rat head was horizontally fixed in the device after anesthetizing with 1% (w/v) pentobarbital sodium (35 mg/kg) through intraperitoneal injecting. The rat head was rapidly gyrated with a sudden acceleration and deceleration when the trigger was pushed. Control rats group was only anesthetized and secured to the device without subjection to injury. DAI rats were under sedation because of injury and regained consciousness after half an hour. In this period, all rats were placed in a clean open area with adequate ventilation, and the vital signs changes of rats were observed to ensure that rats would not get suffocated. For approximately 12 hours after coma, rats in the control group returned to normal behavior, normal activity and rapid responses after anesthesia. Conversely, rats in the DAI group showed some abnormal behaviors, including an unstable gait, a weakened response to stimulation, reduced activity and food intake.

All rats were maintained on free access to food and water at a temperature of 22°C ± 2°C until the time of euthanasia. All rats were decapitated and their hippocampus was dissected on ice in super clean bench, placed into liquid nitrogen, and then transferred into a -80°C low-temperature refrigerator for storage and use.

RNA extraction and purification

Total hippocampus RNA was extracted by use of Trizol Reagent (Cat#15596-018, Life technologies, Carlsbad, CA, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). With RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany), qualified hippocampus RNA was purified.

Analysis of the differential gene expression in thehippocampus by the rat whole genome expression chip

One RNA sample was obtained in each DAI group, and two RNA samples were obtained in control group, giving a total of six total RNA specimens. These RNA specimens were then sent to BioTechnology (Shanghai, China) for the genome expression chip analysis. The rat whole genome expression chip (Affymetrix) was performed as follows: firstly, with using Gene Chip 3’ IVT PLUS Reagent Kit (Cat#902416, Affymetrix, Santa Clara, CA, US), total RNA were amplified, labeled and purified to obtain biotin labeled cRNA. Secondly, array hybridization and wash was performed by using Gene Chip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US), Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US). Thirdly, slides were scanned by Gene Chip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US). Raw data was normalized by MAS 5.0 algorithm, Gene Spring Software12.6.1 (Agilent technologies, Santa Clara, CA, US).  Differences between the DAI and control groups were considered significant at P-value < 0.05.

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotation

The reference genome of Rattus norvegicus, together with gene information, was downloaded from the NCBI database (ftp://ftp.ncbi.nih.gov/genomes/Rattus _ norvegicus/). The information about Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms was downloaded from the UniProtKB database. Both GO and KEGG with P-value <0.05 were considered as significant enriched.

Real-time quantitativePCR

Total RNA was extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). 200 ng of RNA was reverse-transcribed to cDNA with Prime Script^TM RT Master Mix kit (TaKaRa). Real-time quantitative PCR (RT-PCR) was performed on Step One RT-PCR system using the RT-SYBR master mix kit (TaKaRa). Gene-specific primers were listed in Table 1.

Data analyses

Data was analyzed through using SPSS 19.0 (SPSS Inc., Chicago, IL, USA), which using analysis of variance (ANOVA) or Contingency Table Analysis. Multiple comparisons of the means were carried out using Fisher’s Protected Least Significant Difference only after ANOVA showed a significant effect (P < 0.05).

Results

The DEG profile of the hippocampus of rats subjected to DAI

Compared with the control group, 315 DEGs in the 1-day DAI group were identified, of which 179 were up-regulated and 136 were down-regulated. A total of 326 DEGs in the 7-day DAI group were identified, of which 190 were up-regulated and 136 were down-regulated. A total of 327 DEGs in the 14-day DAI group were identified, of which 177 were up-regulated and 150 were down-regulated. A total of 326 DEGs in the 30-day DAI group were identified, of which 171 were up-regulated and 142 were down-regulated (Fig.1 and Table 2). As shown in Fig.2 and Table 2, there were only 6 same up-regulated genes and 4 same down-regulated genes in all DAI groups.

GO function analysis of rats subjected to DAI

The results of GO function analysis were summarized in three main categories: biological process, cellular component and molecular function. As shown in Fig.3A, significant biological processes of the DEGs in the 1-day DAI hippocampus on the aspects of positive regulation of cAMP biosynthetic process, phototransduction, regulation of notch signaling pathway, tumor necrosis factor production, MAPK cascade, innate immune response, regulation of ERK1 and ERK2 cascade, positive regulation of Ras protein signal transduction, and cellular response to cAMP. As shown in Fig.3B, significant biological processes of the DEGs in the 7-day DAI hippocampus on the aspects of synaptic transmission, innate immune response- activating signal transduction, synaptic vesicle coating, neural precursor cell proliferation, synaptic transmission, phototransduction, and hippocampus development. As shown in Fig.3C, significant biological processes of the DEGs in the 14-day DAI hippocampus on the aspects of neurological system process, positive regulation of Ras protein signal transduction, tight junction assembly, regulation of neuron death, negative regulation of RIG-I signaling pathway, and positive regulation of synaptic transmission. As shown in Fig.3D, significant biological processes of the DEGs in the 30-day DAI hippocampus on the aspects of phototransduction, synaptic transmission, G-protein coupled receptor signaling pathway, neuronal action potential, regulation of growth, regulation of cell growth, negative regulation of RIG-I signaling pathway, negative regulation of neurotransmitter secretion, neuromuscular process, neuromuscular process, hippocampus development, and wnt signaling pathway. Based on these data, several biological processes and signaling pathways for nerve damage and repair had changed in the hippocampus of rats subjected to DAI.

KEGG analysis of rats subjected to DAI

As shown in Fig.4A and Table 3, compared with the control group, the DEGs in the 1-day DAI hippocampus were mainly enriched in biosynthesis of unsaturated fatty acids (Acot2, Acox3, Acot7), natural killer cell mediated cytotoxicity (Bid, Zap70, Cd244, Ncr3, Lcp2), complement and coagulation cascades (F13a1, Kng2, F8, Cd46), primary immunodeficiency (Zap70, Icos), PPAR signaling pathway (Acox2, Acox3, Apoa1), cell adhesion molecules (CAMs) (RT1-EC2, Icos, RT1-CE5, Cldn11, Selp), ErbB signaling pathway (Stat5b, Nrg1, Nck2), and neuroactive ligand-receptor interaction (Grin3a, Prlhr, P2rx3, Crhr2, Gabrg3, Adra2b). As shown in Fig.4B and Table 3, the DEGs in the 7-day DAI hippocampus were concentrated in natural killer cell mediated cytotoxicity (Cd247, Klrd1, Ncr3, Cd244), synaptic vesicle cycle (Unc13c, Cltb, Atp6v1g3), olfactory transduction (Pde1c, Olr442), Toll-like receptor signaling pathway (Casp8, Tlr1, Tlr4), neuroactive ligand-receptor interaction (P2rx3, Glra1, F2, Grin3a, Thrb, Grm5), Glutamatergic synapse (Grin3a, Gnb3, Grm5), Calcium signaling pathway (P2rx3, Pde1c, Nos3, Grm5). As shown in Fig.4C and Table 3, the DEGs in the 14-day DAI hippocampus mainly concentrate in neuroactive ligand-receptor interaction (Npy2r, Ghrhr, Tshb, P2rx3,Trhr, Grin2d, Grm8, Sstr5), notch signaling pathway (Notch4, Ep300), natural killer cell mediated cytotoxicity (Sh2d1a, Lcp2, Ncr3), long-term potentiation (Ep300, Grin2d) and many signaling pathways involved in the processing of metabolism. As shown in Fig.4D and Table 3, the DEGs in the 30-day DAI hippocampus were primarily involved with neuroactive ligand-receptor interaction (Gabrg3, Gria4, Trhr, P2rx3, Pth1r, Prlhr, Tshb), primary immunodeficiency (Zap70,Ada), protein digestion and absorption (Col22a1, Col9a1, Mme), mTOR signaling pathway (Irs1, Rictor), cell cycle (Mcm2, Ccnb2, Rbl1), PPAR signaling pathway (Gk, Acox3). We thus identified that “neuroactive ligand- receptor interaction” may play an important role in individual variations in nociception response to DAI from 1 day to 30 days.

Regulation of neurotransmitter receptors-related gene expression in DAI

With respect to the neuroactive ligand-receptor interaction, two genes (P2rx3, Prlhr) were up-regulated, and four genes (Adra2b, Crhr2, Gabrg3, Grin3a) were down-regulated in the 1-day DAI group, four genes (F2, Glra1,Grm5,P2rx3) were up-regulated, and two genes (Grin3a, Thrb) were down-regulated in the 7-day DAI group, five genes (Ghrhr, Npy2r, P2rx3, Sstr5, Trhr) were up-regulated, and three genes (Grin2d, Grm8, Tshb) were down-regulated in the 14-day DAI group, three genes (P2rx3, Prlhr, Trhr) were up-regulated, and four genes (Gabrg3, Gria4, Pth1r, Tshb) were down-regulated in the 30-day DAI group (Table 4). In order to validate the sequencing results, these DEGs in the neuroactive ligand-receptor interaction were selected for RT-PCR analysis (Fig.5). These results showed that the RT-PCR results of all these genes were consistent with the sequencing data.

Discussion

In general, with major degrees of DAI, the patients are rendered comatose at the time of trauma and subsequently only have limited recovery [1-3]. Although rats with DAI have provided an invaluable model for investigating neurobehavioral deficits in brain damage in DAI-patients [4-6], the complete gene expression profile in the hippocampus of this animal model is undetermined. By contrast of several previous studies, our results detected the gene expression in hippocampus of the DAI rat that was a specific brain region important in motor and motivation control, learning and memory. We used the rat whole genome expression chip (Affymetrix) and bioinformatics technology to study the gene expression profile of the hippocampus exposed to DAI for 1 day, 7days, 14 days or 30 days. Our data showed that DAI primarily altered the expression of genes and pathways related to neurotransmission, neuronal survival, immune responses, and other biological processes.

During the 1-day, 7-day, 14-day and 30-day DAI processes, many significant biological processes, cellular components and molecular functions of the DEGs in the hippocampus had changed, including innate immune response, synaptic vesicle coating, synaptic transmission, phototransduction, hippocampus development, tight junction, regulation of neuron death (Fig. 3). Synaptic transmission, synaptic vesicle coating, regulation of neuron death, hippocampus development, notch signaling pathway, wnt signaling pathway, RIG-I signaling pathway, G-protein coupled receptor signaling pathway and cAMP signaling pathway might participate in neural regeneration and recovery. Ras/ ERK1/2/MAPK pathway was particularly associated with cellular signal transduction. Regulation of tumor necrosis factor production, innate immune response-activating signal transduction, and RIG-I signaling pathway mightbe involved in innate immune response.

With the increase of injury time, more and more DEGs were mainly enriched in neuroactive ligand-receptor interaction (Fig.4 and Table 4). In the functional pathway of neuroactive ligand-receptor interaction, 19 genes Adra2b, Crhr2, F2, Gabrg3, Ghrhr, Glra1,Gria4, Grin2d, Grin3a, Grm5, Grm8, Npy2r, P2rx3, Prlhr, Pth1r, Sstr5, Thrb, Trhr and Tshb were involved (Table 4). RT-PCR was used to verify the gene expression of these 19 genes on the hippocampus of various rat groups. These results were consistent with the results from gene chip assay. Down-regulated Crhr2 could enhance the activity of hypothalamic-pituitary-adrenal (HPA) axis, the levels of blood plasma adrenocorticotropic hormone and corticosterone [43]. Crhr2 could maintain and regulate the effect of HPA axis [44] and participate in the recovery regulation of HPA axis response [45]. The expression of Crhr2 in hippocampus of 7-day and 21-day stressed rats was significantly down-regulated [23]. In this study, the expression of Crhr2 in 1-day DAI hippocampus was significantly down-regulated as well. This result suggests that the daily exposure to DAI for 1 day causes functional disorder in the HPA axis of the DAI rats. P2RX3 is one of the purinoceptors for Adenosine Triphosphate (ATP), which functions as a ligand-gated ion channel to activate ATP-evoked nociceptor [46]. The expression of P2RX3 was of great significance to the restoration process of sciatic nerve injury [47]. With the increase of injury time, compared with the control group, the expression of P2rx3in the DAI hippocampus was always significantly up-regulated. This result indicates that the up-regulated P2rx3 may promote the neural regeneration and recovery after DAI.

The notch signaling pathway, PPAR signaling pathway, ErbB signaling pathway play critical role in the neural stem cell differentiation, maintains a healthy balance between cell proliferation and differentiation/apoptosis, and determines the cell fate in the differentiation [48-50]. The activated notch signaling pathway can inhibit apoptosis for neural stem cells and promote the neural regeneration and recovery after cerebral ischemia. PPAR signaling pathway can be combined with NF-B to regulate the neural regeneration. Compared with the control group, three DEGs in the 1-day DAI and 14-day DAI were mainly enriched in notch signaling pathway (Cd46,Notch4, Ep300), three DGEs in the 1-day DAI and 30-day DAI were mainly enriched in PPAR pathway (Acox2,Acox3,Apoa1, Gk), three DGEs in the 1-day DAI were mainly enriched in ErbB pathway (Stat5b, Nrg1, Nck2).

The patient usually appears some serious complications and the nerve function obstacle after a traffic accident left him with a traumatic brain injury. Because of neuronal death and rupture of nerve fiber conduction, the patients with brain injury have significant limbs powerless, activity obstacle, and memory impairments of logical thought. Numerous studies indicate that the DEGs of neural signaling pathways are strongly correlated with brain injury. In this study, we systematically investigated the DEGs in nervous system of DAI on the hippocampus at the whole genome level. Further studies are needed to understand how DEGs are translated into nervous system changes in the DAI rats.

Conclusions

In a word, we provide clearer information about the DEGs and many signaling pathways can play a major role in DAI by the rat whole genome expression chip. In addition, our experimental results show that these DEGs were involved in many signal pathways of nerve damage and repair such as notch, PPAR, ErbB and wnt signaling pathways. It also provided several targeted genes related to DAI for future investigation.

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Figure Legends

Fig. 1. A scatter plot graph for differential expressed genes in the 1-day, 7-day, 14-day and 30-day DAI groups in comparison with the control group. Expression levels are indicated on Y (injury group) or X (control group). With an estimated P-value < 0.05 and |log2Ratio| > 1 (Ratio: injury group / control group), the red shows the parts of up-regulated genes in injury group, the green shows the parts of down-regulated genes in injury group.

Fig. 2.  A venn diagram for differential expressed genes in the 1-day, 7-day, 14-day and 30-day DAI groups in comparison with the control group.

Fig. 3. Gene Ontology classification of down- and up-regulated genes in the 1-day, 7-day, 14-day and 30-day DAI groups in comparison with the control group. The results are summarized in three main categories: biological process, cellular component and molecular function.

Fig. 4. Kyoto Encyclopedia of Genes and Genomes classification of down- and up- regulated genes in the 1-day, 7-day, 14-day and 30-day DAI groups in comparison with the control group.

Fig.5. Verification of 19 differentially expressed genes between DAI groups and control group by RT-PCR. Different letters on top of bars indicate significant difference (P<0.05) with Fisher’s Protected Least Significant Difference. Each bar corresponds to a single group represented as the mean ± S.E. of its biological replicates.

Tables

Table1Gene-specific primers used in real-time quantitative PCR.

Table2Differential expressed genes in the 1-day, 7-day, 14-day and 30-day injury groups in comparison with the control group.

Table3 KEGG analysis of rats subjected to DAI.

Table4 Differential expressed genes in neuroactive ligand-receptor interaction.