Neuroscience Letters 600 (2015) 171–175
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Expression and identification of olfactory receptors in sciatic nerve and dorsal root ganglia of rats Leilei Gong a,b,1 , Qianqian Chen b,1 , Xiaosong Gu a,b,∗ , Shiying Li b,∗ a b
School of Biology and Basic Medical Sciences, Soochow University, Suzhou, JS 215123, China Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS, 226001, China
h i g h l i g h t s • Olfactory receptors (ORs) are present in peripheral nerves. • ORs are differentially expressed in sciatic nerve and DRGs after sciatic nerve injury. • ORs are up-regulated in primary culture of Schwann cells under H2 O2 stimulation.
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 5 June 2015 Accepted 8 June 2015 Available online 10 June 2015 Keywords: Olfactory receptors Sciatic nerve Schwann cells Dorsal root ganglion
a b s t r a c t The olfactory receptor (OR) genes are expressed mainly in the cell membrane of olfactory sensory neurons of the nasal epithelium, and the binding of specific odorant ligands to OR proteins leads to odor detection. ORs are also expressed in non-olfactory tissues and cells, but their functions are often elusive. In this study, microarray analysis was used to detect the presence of ORs in peripheral nerves. We found that a number of ORs were differentially expressed in sciatic nerve and dorsal root ganglia (DRGs) following sciatic nerve injury. The expression and expression profile of several ORs in sciatic nerve were verified by in situ hybridization and real time quantitative RT-PCR. We also observed that the expression of some ORs in primary culture of Schwann cells was up-regulated under H2 O2 stimulation. Overall, all the results suggest that there may be a possible relationship between the differential expression of ORs in injured peripheral nerves and peripheral nerve regeneration. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Olfactory receptor (OR) genes were first identified from rats by Buck and Axel in 1991 [2]. ORs belong to the g-protein–coupled receptor superfamily, which is characterized by seven hydrophobic transmembrane domains [17]. It is through the binding of odorous ligands to OR proteins that the mammalian olfactory system can recognize and discriminate a large number of different odorant molecules [2]. To date, OR gene sequences have been cloned from different species including mammals [2,11,12,23], birds [18], amphibians [7], and fish [4]. ORs are expressed mainly in the cell membrane of olfactory sensory neurons of the nasal epithelium, and they are responsible for odor detection by binding specific odorant ligands. However, there have been reports that ORs are also expressed in non-olfactory tissues and cells, such as taste tis-
sue [8], prostate [26], heart [5], spine [19], red blood cells [6] and male germ cells [24]. Although the functions of some of these ORs are known, for example, a human testicular OR mediates human sperm chemotaxis and may be a critical component of the fertilization process [24], not all of them are functional [12], or the functions of most of them are yet unclear. In this study, we aimed to investigate the expression of ORs in peripheral nerves. The results showed that a number of ORs were differentially expressed in proximal sciatic nerve and dorsal root ganglia (DRGs) following rat sciatic nerve injury. It was further found that the expression of several ORs was up-regulated in primary culture of Schwann cells under H2 O2 stimulation.
2. Materials and methods 2.1. Tissue collection and microarray analysis
∗ Corresponding authors. Fax: +86 513 85051800. E-mail addresses: [email protected] (X. Gu), [email protected] (S. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2015.06.019 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
The experiments in this subsection were performed exactly as described previously [13]. In brief, adult male Sprague–Dawley (SD)
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Fig. 1. The expression of ORs in proximal sciatic nerve (SN) and DRGs following sciatic nerve transection. (A) The number of differentially expressed ORs in proximal sciatic nerve and DRGs following sciatic nerve transection. (B) The number percentage of differentially expressed ORs that demonstrated the maximum change (up- and downregulation) in their respective expression at different times following sciatic nerve transection. (C) Heatmap and cluster dendrogram of differentially expressed ORs that had more than 8 fold changes in their expression at least one time point following sciatic nerve transection as compared to their expression at 0 h following sciatic nerve transection.
rats (180–220 g) were subjected to surgery for sciatic nerve transection, and then proximal sciatic nerve and L4-6 DRGs were collected at different times (0.5, 1, 3, 6, and 9 h, and 1, 4, 7, and 14 days) after surgery. Total RNA was extracted using Trizol (Life technologies, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA was amplified and labeled using a Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA). The cDNA was hybridized using a Gene Expression Hybridization Kit (Agilent Technologies) at 60 ◦ C for 17 h in Hybridization oven. Microarrays were scanned by Agilent Microarray Scanner (Agilent Technologies) and the data were compiled with Agilent feature extraction software. All steps from RNA amplification to the final scanner output were conducted by National Engineering Center for Biochip at Shanghai (China). The raw data were deposited in a MIAME compliant database (NCBI Accession number: Series GSE33175, GSE30165, GSE65053). The differentially expressed ORs were identified via screening when the fold change was greater than 2. Hierarchical clustering was performed on the expression profile of ORs, and the fold change was set at greater than 8.
culture at the final concentration of 0.2 mM to allow incubation for 16 h. 2.3. Quantitative real time polymerase chain reaction (qPCR) and semi-quantitative RT-PCR Total RNA was isolated from the proximal sciatic nerve using Trizol, and the RNA was reversely transcribed to cDNA by using a Prime-Script reagent Kit (TaKaRa Dalian, China) according to manufacturer’s instructions. qPCR was performed using SYBR Green Premix Ex Taq (TaKaRa) on an Applied Biosystems Stepone realtime PCR System. All reactions were run in triplicate. The PCR program was as follows: 95 ◦ C for 10 min; 40 cycles of 95 ◦ C for 15 s, 64 ◦ C for 30 s, and 72 ◦ C for 1 min; and a dissociation cycle consisting of 95 ◦ C for 10 s, 60 ◦ C for 1 min, and 95 ◦ C for 15 s (ramp rate, 1%). The relative expression was calculated using the comparative 2−Ct method. For semi-quantitative RT-PCR, the generated cDNA was used as a template for PCR reaction. The thermocycler program was as follows: 5 min at 94 ◦ C; 30 cycles of 30 s at 94 ◦ C; 45 s at 58 ◦ C; 30 s at 72 ◦ C; and 5 min at 72 ◦ C. The sequences of all primers is listed in Supplementary table 1.1.
2.2. Primary culture of Schwann cells and treatment 2.4. RNA probes and in situ hybridization (ISH) Schwann cells were isolated from the sciatic nerve of 1-day-old SD rats, and purified by removing fibroblasts with anti-Thy1.1 antibody and rabbit complement (Sigma, St Louis, MO) as described previously [14]. The cell preparation contained 98% of Schwann cells, as assessed by immunocytochemistry with anti-S100 (DAKO, Carpinteria, CA). Primary Schwann cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, both from Gibco Life Technologies, Grand Island, NY) at 37 ◦ C in humidified 5% CO2 in air. H2 O2 was added to cell
The fragment of ORs was amplified by PCR from total cDNA derived from Schwann cells, and PCR product was subcloned into pGEM-T vector. The orientation and sequence of the fragment were confirmed by sequencing. RNA labeling with digoxigeninUTP by in vitro transcription with SP6 and T7 RNA polymerase from linearized plasmid containing OR fragment using the DIG RNA Labeling Kit (SP6/T7) (Roche, Mannheim, Germany) according to the manufacturer’s instructions.
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The proximal sciatic nerve of SD rats was cut into sections (about 12 m thickness), which were treated with 1 g/ml proteinase K for 14 min at 37 ◦ C. After treatment with 0.2% glycine-PBS for 10 min, nerve sections were washed and acetylated with 0.05% acetic anhydride in 0.1 M triethanolamine hydrochloride for 10 min. Hybridization with DIG-labeled probes was carried out for 2 h at 42 ◦ C in hybridization buffer. Afterwards, sections were washed in 2× sodium chloride/sodium citrate (SSC) 3 times for 10 min each at 55 ◦ C, 1 × SSC 2 times for 15 min each at 55 ◦ C and 0.1 × SSC 2 times for 5 min each at 55 ◦ C. Blocking was performed for 12 h at 4 ◦ C with alkaline phosphatase-conjugated Fab anti-DIG antibody (Roche). The slides were stained using 5-bromo4-chloro-3-indolyl-phosphate in alkaline phosphatase (Solarbio, Beijing, China). 2.5. Statistical analysis All data are expressed as means ± S.D. The Student’s t-test was used for statistical analysis between groups by the aid of SPSS 15.0 software. In addition, Pearson’s correlation analysis was used to correlate both qPCR and microarray data. 3. Results 3.1. Global overview of differentially expressed ORs in proximal sciatic nerve and DRGs following sciatic nerve transection By using DNA microarray, a total of 1111 probes of OR genes were screened in proximal sciatic nerve and DRGs of rats. These probes and their expressions at different times are listed in Supplementary table S1.2–1.5. A huge number of ORs were present in proximal sciatic nerve and DRGs, and they were differentially expressed at different times post nerve injury (PNI). The number of up-regulated ORs was significantly larger than that of down-regulated ORs, suggesting that most ORs were activated in response to peripheral nerve injury. The number of up-regulated and down-regulated ORs changed with time during the period of 0.5 h to 14 days PNI (Fig. 1A). In proximal sciatic nerve, the number of up-regulated ORs increased starting from 0.5 h PNI, slightly decreased from 1 h to 6 h PNI, dramatically increased from 9 h until 1 day PNI with a maximum number of up-regulated ORs occurring at 1 day PNI, and then underwent a fluctuant change from 1 day to 14 days PNI. By contrast, in DRGs, the number of up-regulated ORs showed a fluctuant change from 5 h to 14 days PNI with a maximum number of up-regulated OR occurring at 9 h PNI (Fig. 1A). We further observed the relative number of differentially expressed ORs that demonstrated the maximum change (up- and down-regulation) in their respective expression at different times PNI. In proximal sciatic nerve, 51% of total differentially expressed ORs had the maximum change in their respective expression at 1 day PNI, then 15% at 7 days PNI, 7% at 1 h PNI, 6% at 6 h PNI and 9 h PNI, 5% at 0.5 h PNI, 4% at 4 days PNI, and 2% at 14 days PNI. By contrast, in DRGs, 44% of total differentially expressed ORs had the maximum change in their respective expression at 9 h PNI, then 22% at 14 days PNI, 12% at 0.5 h PNI, 5% at 1 h PNI, 3 h and 1 day, 4% at 6 h PNI, 3% at 4 days PNI, and nearly 0 at 7 days PNI (Fig. 1B). Moreover, we selected a number of differentially expressed ORs, whose expression change was greater than 8 fold, for cluster analysis (Fig. 1C). 3.2. Validation of OR expression in sciatic nerve To validate the expression of ORs in peripheral nerves, ISH, as a powerful technique for identifying specific mRNA expression in individual cells or tissues, was used to determine the expression of
Fig. 2. Validation of OR expression in sciatic nerve. In situ hybridization with OR and control scrambled probes showed the expression of 3 ORs in sciatic nerve following sciatic nerve transection.
ORs in sciatic nerve. The results showed that 3 randomly selected ORs (OR132, 477, and 629) were really expressed in the proximal sciatic nerve (Fig. 2). 3.3. Confirmation of the differential expression of several ORs In order to verify the microarray data, 9 differentially expressed ORs (OR132, 349, 463, 477, 526, 629, 1071, 1107 and 1589) were selected for validation by real-time quantitative RT-PCR analysis. The results indicated the expression of these 9 ORs in proximal sciatic nerve showed the change trends similar to those revealed by microarray data, the correlation analysis suggested that qRT-PCR data were strongly correlated to microarray data (Fig. 3). 3.4. Up-regulation of OR expression in Schwann cells following H2 O2 stimulation Following sciatic nerve injury, a large number of ORs were differentially expressed at the different times, which led us to suppose that ORs might play some roles in the regeneration process of peripheral nerves. Reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ), superoxide anion, and hydroxyl radicals, are important mediators of cell apoptosis [15], and so oxidative stress can be considered as one of components of peripheral nerve injury-induced cell damage. In this study, H2 O2 stimulation for 16 h was adopted to mimic the oxidative stress following peripheral nerve injury. The result from semi-quantitative RT-PCR showed that the expressions of 14 ORs (including OR132, 349, 463, 477, 484, 526, 623, 629, 1071,
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Fig. 3. Confirmation of the expression of several ORs by real-time RT-PCR. Relative mRNA levels of 9 ORs were analyzed by real-time RT-PCR at different times post nerve transection (control: 0 h). Data are presented as means ± SD of three dependent assays (each in triplicate). R stands for the correlation coefficient.
1107, 1316, 1493, 1501, 1589) were all up-regulated after exposure to H2 O2 for 16 h (Fig. 4).
4. Discussion Since ORs were first discovered in the early 1990’s, the expression of ORs has been extensively observed in not only olfactory tissues but also non-olfactory tissues [2,12]. ORs in the olfactory epithelium have explicit functions: they recognize volatile odorants and send a chemical signal as an action potential to the brain, generating the perception of smells [12,17]. In contrast, the function of ORs in non-olfactory tissues, on the whole, remains largely elusive, although it has been reported that ORs in non-olfactory tissues may be involved in hormone secretion [22] or in cell movement, such as sperm chemotaxis [21,24], muscle cell adhesion [9] and cytokinesis [27], and they may play other functional roles in the brain [1], skin [3], and prostate cancer cells [20]. Our previous study have demonstrated the presence of a large number of ORs in peripheral nerves [13]. To continue this work, here we further examined and validated the expression of ORs in sciatic nerve and DRGs following sciatic nerve injury. As is well known, sciatic nerve is the longest and largest peripheral nerve in the body, and it consists of sensory and motor nerves. The sensory nerve conveys sensory stimuli from tissues and organs
Fig. 4. The expression of 14 ORs was up-regulated in primary culture of Schwann cells after exposure to H2 O2 .
L. Gong et al. / Neuroscience Letters 600 (2015) 171–175
into the central nervous system, and the motor nerve transmits signals from the central nervous system to the periphery of the body [10]. The sensory neurons extending into the sciatic nerve are located in the L4-6 DRGs. In this study, we validated the differential expression of many ORs in sciatic nerve and L4-6 DRGs after sciatic nerve injury. To have a preliminary insight into the functional significance of ORs in peripheral nerves, we analyzed the expression changes of ORs in primary cultured Schwann cells after exposure to H2 O2 stimulation. Here H2 O2 , as a species of ROS, was used to mimic oxidative stress caused by nerve injury [14,15]. Schwann cells are the principal glial cells of the peripheral nervous system, and they play an important role in axonal growth and myelination. Damage to Schwann cells could affect axonal conductivity and lead to pathologic changes in neurons [25]. After primary Schwann cells were exposed to H2 O2 , the expression of a number of ORs in the cells was up-regulated. This finding may help to understand the implications of ORs in peripheral nerve injury and regeneration. We observed that after sciatic nerve injury, ORs in sciatic nerve and DRGs were differentially expressed at the mRNA level. Since the protein is an ultimate executor of biological functions, it is worthwhile to examine the expression of ORs at the protein level. Unfortunately, highly purified, specific antibodies to the relevant ORs are commercially unavailable or difficult to produce due to the structurally similarity of these different ORs [12]. Although the change trend of mRNAs is generally positively correlated to that of proteins [16], it is required in the future to develop the corresponding antibodies to various ORs and then determine the protein expression of these ORs in sciatic nerve and DRGs after sciatic nerve injury. On the other hand, the functional identification of ORs in nonolfactory tissues is a very important issue, but the resolution of this issue is hindered by several limitations, such as the small number of antibodies available for similar OR genes, and the relatively lower expression level of OR transcript in non-olfactory tissues than in olfactory tissues [12]. Therefore, it is necessary to further investigate the functional significance of ORs in the peripheral nerve system, which is far from complete clarification. Our current observations suggest a possible relationship between the differential expression of ORs in injured peripheral nerve and peripheral nerve regeneration. In this sense, the results in this study may provide a foundation for future intense work. Competing interests The authors declare no competing interests. Acknowledgments This study was supported by National Key Basic Research Program of China (973 and 863 programs, Grant Nos. 2014CB542202, 2012AA020502), National Natural Science Foundation of China (Grant No. 81130080, 31300879), Jiangsu Provincial Natural Science Foundation (BK2012230), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We thank Professor Jie Liu for assistance in the preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2015.06. 019
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Expression and identification of olfactory receptors in sciatic nerve and dorsal root ganglia of rats Leilei Gong a,b,1 , Qianqian Chen b,1 , Xiaosong Gu a,b,∗ , Shiying Li b,∗ a b
School of Biology and Basic Medical Sciences, Soochow University, Suzhou, JS 215123, China Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS, 226001, China
h i g h l i g h t s • Olfactory receptors (ORs) are present in peripheral nerves. • ORs are differentially expressed in sciatic nerve and DRGs after sciatic nerve injury. • ORs are up-regulated in primary culture of Schwann cells under H2 O2 stimulation.
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 5 June 2015 Accepted 8 June 2015 Available online 10 June 2015 Keywords: Olfactory receptors Sciatic nerve Schwann cells Dorsal root ganglion
a b s t r a c t The olfactory receptor (OR) genes are expressed mainly in the cell membrane of olfactory sensory neurons of the nasal epithelium, and the binding of specific odorant ligands to OR proteins leads to odor detection. ORs are also expressed in non-olfactory tissues and cells, but their functions are often elusive. In this study, microarray analysis was used to detect the presence of ORs in peripheral nerves. We found that a number of ORs were differentially expressed in sciatic nerve and dorsal root ganglia (DRGs) following sciatic nerve injury. The expression and expression profile of several ORs in sciatic nerve were verified by in situ hybridization and real time quantitative RT-PCR. We also observed that the expression of some ORs in primary culture of Schwann cells was up-regulated under H2 O2 stimulation. Overall, all the results suggest that there may be a possible relationship between the differential expression of ORs in injured peripheral nerves and peripheral nerve regeneration. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Olfactory receptor (OR) genes were first identified from rats by Buck and Axel in 1991 [2]. ORs belong to the g-protein–coupled receptor superfamily, which is characterized by seven hydrophobic transmembrane domains [17]. It is through the binding of odorous ligands to OR proteins that the mammalian olfactory system can recognize and discriminate a large number of different odorant molecules [2]. To date, OR gene sequences have been cloned from different species including mammals [2,11,12,23], birds [18], amphibians [7], and fish [4]. ORs are expressed mainly in the cell membrane of olfactory sensory neurons of the nasal epithelium, and they are responsible for odor detection by binding specific odorant ligands. However, there have been reports that ORs are also expressed in non-olfactory tissues and cells, such as taste tis-
sue [8], prostate [26], heart [5], spine [19], red blood cells [6] and male germ cells [24]. Although the functions of some of these ORs are known, for example, a human testicular OR mediates human sperm chemotaxis and may be a critical component of the fertilization process [24], not all of them are functional [12], or the functions of most of them are yet unclear. In this study, we aimed to investigate the expression of ORs in peripheral nerves. The results showed that a number of ORs were differentially expressed in proximal sciatic nerve and dorsal root ganglia (DRGs) following rat sciatic nerve injury. It was further found that the expression of several ORs was up-regulated in primary culture of Schwann cells under H2 O2 stimulation.
2. Materials and methods 2.1. Tissue collection and microarray analysis
∗ Corresponding authors. Fax: +86 513 85051800. E-mail addresses: [email protected] (X. Gu), [email protected] (S. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2015.06.019 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
The experiments in this subsection were performed exactly as described previously [13]. In brief, adult male Sprague–Dawley (SD)
172
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Fig. 1. The expression of ORs in proximal sciatic nerve (SN) and DRGs following sciatic nerve transection. (A) The number of differentially expressed ORs in proximal sciatic nerve and DRGs following sciatic nerve transection. (B) The number percentage of differentially expressed ORs that demonstrated the maximum change (up- and downregulation) in their respective expression at different times following sciatic nerve transection. (C) Heatmap and cluster dendrogram of differentially expressed ORs that had more than 8 fold changes in their expression at least one time point following sciatic nerve transection as compared to their expression at 0 h following sciatic nerve transection.
rats (180–220 g) were subjected to surgery for sciatic nerve transection, and then proximal sciatic nerve and L4-6 DRGs were collected at different times (0.5, 1, 3, 6, and 9 h, and 1, 4, 7, and 14 days) after surgery. Total RNA was extracted using Trizol (Life technologies, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA was amplified and labeled using a Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA). The cDNA was hybridized using a Gene Expression Hybridization Kit (Agilent Technologies) at 60 ◦ C for 17 h in Hybridization oven. Microarrays were scanned by Agilent Microarray Scanner (Agilent Technologies) and the data were compiled with Agilent feature extraction software. All steps from RNA amplification to the final scanner output were conducted by National Engineering Center for Biochip at Shanghai (China). The raw data were deposited in a MIAME compliant database (NCBI Accession number: Series GSE33175, GSE30165, GSE65053). The differentially expressed ORs were identified via screening when the fold change was greater than 2. Hierarchical clustering was performed on the expression profile of ORs, and the fold change was set at greater than 8.
culture at the final concentration of 0.2 mM to allow incubation for 16 h. 2.3. Quantitative real time polymerase chain reaction (qPCR) and semi-quantitative RT-PCR Total RNA was isolated from the proximal sciatic nerve using Trizol, and the RNA was reversely transcribed to cDNA by using a Prime-Script reagent Kit (TaKaRa Dalian, China) according to manufacturer’s instructions. qPCR was performed using SYBR Green Premix Ex Taq (TaKaRa) on an Applied Biosystems Stepone realtime PCR System. All reactions were run in triplicate. The PCR program was as follows: 95 ◦ C for 10 min; 40 cycles of 95 ◦ C for 15 s, 64 ◦ C for 30 s, and 72 ◦ C for 1 min; and a dissociation cycle consisting of 95 ◦ C for 10 s, 60 ◦ C for 1 min, and 95 ◦ C for 15 s (ramp rate, 1%). The relative expression was calculated using the comparative 2−Ct method. For semi-quantitative RT-PCR, the generated cDNA was used as a template for PCR reaction. The thermocycler program was as follows: 5 min at 94 ◦ C; 30 cycles of 30 s at 94 ◦ C; 45 s at 58 ◦ C; 30 s at 72 ◦ C; and 5 min at 72 ◦ C. The sequences of all primers is listed in Supplementary table 1.1.
2.2. Primary culture of Schwann cells and treatment 2.4. RNA probes and in situ hybridization (ISH) Schwann cells were isolated from the sciatic nerve of 1-day-old SD rats, and purified by removing fibroblasts with anti-Thy1.1 antibody and rabbit complement (Sigma, St Louis, MO) as described previously [14]. The cell preparation contained 98% of Schwann cells, as assessed by immunocytochemistry with anti-S100 (DAKO, Carpinteria, CA). Primary Schwann cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, both from Gibco Life Technologies, Grand Island, NY) at 37 ◦ C in humidified 5% CO2 in air. H2 O2 was added to cell
The fragment of ORs was amplified by PCR from total cDNA derived from Schwann cells, and PCR product was subcloned into pGEM-T vector. The orientation and sequence of the fragment were confirmed by sequencing. RNA labeling with digoxigeninUTP by in vitro transcription with SP6 and T7 RNA polymerase from linearized plasmid containing OR fragment using the DIG RNA Labeling Kit (SP6/T7) (Roche, Mannheim, Germany) according to the manufacturer’s instructions.
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The proximal sciatic nerve of SD rats was cut into sections (about 12 m thickness), which were treated with 1 g/ml proteinase K for 14 min at 37 ◦ C. After treatment with 0.2% glycine-PBS for 10 min, nerve sections were washed and acetylated with 0.05% acetic anhydride in 0.1 M triethanolamine hydrochloride for 10 min. Hybridization with DIG-labeled probes was carried out for 2 h at 42 ◦ C in hybridization buffer. Afterwards, sections were washed in 2× sodium chloride/sodium citrate (SSC) 3 times for 10 min each at 55 ◦ C, 1 × SSC 2 times for 15 min each at 55 ◦ C and 0.1 × SSC 2 times for 5 min each at 55 ◦ C. Blocking was performed for 12 h at 4 ◦ C with alkaline phosphatase-conjugated Fab anti-DIG antibody (Roche). The slides were stained using 5-bromo4-chloro-3-indolyl-phosphate in alkaline phosphatase (Solarbio, Beijing, China). 2.5. Statistical analysis All data are expressed as means ± S.D. The Student’s t-test was used for statistical analysis between groups by the aid of SPSS 15.0 software. In addition, Pearson’s correlation analysis was used to correlate both qPCR and microarray data. 3. Results 3.1. Global overview of differentially expressed ORs in proximal sciatic nerve and DRGs following sciatic nerve transection By using DNA microarray, a total of 1111 probes of OR genes were screened in proximal sciatic nerve and DRGs of rats. These probes and their expressions at different times are listed in Supplementary table S1.2–1.5. A huge number of ORs were present in proximal sciatic nerve and DRGs, and they were differentially expressed at different times post nerve injury (PNI). The number of up-regulated ORs was significantly larger than that of down-regulated ORs, suggesting that most ORs were activated in response to peripheral nerve injury. The number of up-regulated and down-regulated ORs changed with time during the period of 0.5 h to 14 days PNI (Fig. 1A). In proximal sciatic nerve, the number of up-regulated ORs increased starting from 0.5 h PNI, slightly decreased from 1 h to 6 h PNI, dramatically increased from 9 h until 1 day PNI with a maximum number of up-regulated ORs occurring at 1 day PNI, and then underwent a fluctuant change from 1 day to 14 days PNI. By contrast, in DRGs, the number of up-regulated ORs showed a fluctuant change from 5 h to 14 days PNI with a maximum number of up-regulated OR occurring at 9 h PNI (Fig. 1A). We further observed the relative number of differentially expressed ORs that demonstrated the maximum change (up- and down-regulation) in their respective expression at different times PNI. In proximal sciatic nerve, 51% of total differentially expressed ORs had the maximum change in their respective expression at 1 day PNI, then 15% at 7 days PNI, 7% at 1 h PNI, 6% at 6 h PNI and 9 h PNI, 5% at 0.5 h PNI, 4% at 4 days PNI, and 2% at 14 days PNI. By contrast, in DRGs, 44% of total differentially expressed ORs had the maximum change in their respective expression at 9 h PNI, then 22% at 14 days PNI, 12% at 0.5 h PNI, 5% at 1 h PNI, 3 h and 1 day, 4% at 6 h PNI, 3% at 4 days PNI, and nearly 0 at 7 days PNI (Fig. 1B). Moreover, we selected a number of differentially expressed ORs, whose expression change was greater than 8 fold, for cluster analysis (Fig. 1C). 3.2. Validation of OR expression in sciatic nerve To validate the expression of ORs in peripheral nerves, ISH, as a powerful technique for identifying specific mRNA expression in individual cells or tissues, was used to determine the expression of
Fig. 2. Validation of OR expression in sciatic nerve. In situ hybridization with OR and control scrambled probes showed the expression of 3 ORs in sciatic nerve following sciatic nerve transection.
ORs in sciatic nerve. The results showed that 3 randomly selected ORs (OR132, 477, and 629) were really expressed in the proximal sciatic nerve (Fig. 2). 3.3. Confirmation of the differential expression of several ORs In order to verify the microarray data, 9 differentially expressed ORs (OR132, 349, 463, 477, 526, 629, 1071, 1107 and 1589) were selected for validation by real-time quantitative RT-PCR analysis. The results indicated the expression of these 9 ORs in proximal sciatic nerve showed the change trends similar to those revealed by microarray data, the correlation analysis suggested that qRT-PCR data were strongly correlated to microarray data (Fig. 3). 3.4. Up-regulation of OR expression in Schwann cells following H2 O2 stimulation Following sciatic nerve injury, a large number of ORs were differentially expressed at the different times, which led us to suppose that ORs might play some roles in the regeneration process of peripheral nerves. Reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ), superoxide anion, and hydroxyl radicals, are important mediators of cell apoptosis [15], and so oxidative stress can be considered as one of components of peripheral nerve injury-induced cell damage. In this study, H2 O2 stimulation for 16 h was adopted to mimic the oxidative stress following peripheral nerve injury. The result from semi-quantitative RT-PCR showed that the expressions of 14 ORs (including OR132, 349, 463, 477, 484, 526, 623, 629, 1071,
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Fig. 3. Confirmation of the expression of several ORs by real-time RT-PCR. Relative mRNA levels of 9 ORs were analyzed by real-time RT-PCR at different times post nerve transection (control: 0 h). Data are presented as means ± SD of three dependent assays (each in triplicate). R stands for the correlation coefficient.
1107, 1316, 1493, 1501, 1589) were all up-regulated after exposure to H2 O2 for 16 h (Fig. 4).
4. Discussion Since ORs were first discovered in the early 1990’s, the expression of ORs has been extensively observed in not only olfactory tissues but also non-olfactory tissues [2,12]. ORs in the olfactory epithelium have explicit functions: they recognize volatile odorants and send a chemical signal as an action potential to the brain, generating the perception of smells [12,17]. In contrast, the function of ORs in non-olfactory tissues, on the whole, remains largely elusive, although it has been reported that ORs in non-olfactory tissues may be involved in hormone secretion [22] or in cell movement, such as sperm chemotaxis [21,24], muscle cell adhesion [9] and cytokinesis [27], and they may play other functional roles in the brain [1], skin [3], and prostate cancer cells [20]. Our previous study have demonstrated the presence of a large number of ORs in peripheral nerves [13]. To continue this work, here we further examined and validated the expression of ORs in sciatic nerve and DRGs following sciatic nerve injury. As is well known, sciatic nerve is the longest and largest peripheral nerve in the body, and it consists of sensory and motor nerves. The sensory nerve conveys sensory stimuli from tissues and organs
Fig. 4. The expression of 14 ORs was up-regulated in primary culture of Schwann cells after exposure to H2 O2 .
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into the central nervous system, and the motor nerve transmits signals from the central nervous system to the periphery of the body [10]. The sensory neurons extending into the sciatic nerve are located in the L4-6 DRGs. In this study, we validated the differential expression of many ORs in sciatic nerve and L4-6 DRGs after sciatic nerve injury. To have a preliminary insight into the functional significance of ORs in peripheral nerves, we analyzed the expression changes of ORs in primary cultured Schwann cells after exposure to H2 O2 stimulation. Here H2 O2 , as a species of ROS, was used to mimic oxidative stress caused by nerve injury [14,15]. Schwann cells are the principal glial cells of the peripheral nervous system, and they play an important role in axonal growth and myelination. Damage to Schwann cells could affect axonal conductivity and lead to pathologic changes in neurons [25]. After primary Schwann cells were exposed to H2 O2 , the expression of a number of ORs in the cells was up-regulated. This finding may help to understand the implications of ORs in peripheral nerve injury and regeneration. We observed that after sciatic nerve injury, ORs in sciatic nerve and DRGs were differentially expressed at the mRNA level. Since the protein is an ultimate executor of biological functions, it is worthwhile to examine the expression of ORs at the protein level. Unfortunately, highly purified, specific antibodies to the relevant ORs are commercially unavailable or difficult to produce due to the structurally similarity of these different ORs [12]. Although the change trend of mRNAs is generally positively correlated to that of proteins [16], it is required in the future to develop the corresponding antibodies to various ORs and then determine the protein expression of these ORs in sciatic nerve and DRGs after sciatic nerve injury. On the other hand, the functional identification of ORs in nonolfactory tissues is a very important issue, but the resolution of this issue is hindered by several limitations, such as the small number of antibodies available for similar OR genes, and the relatively lower expression level of OR transcript in non-olfactory tissues than in olfactory tissues [12]. Therefore, it is necessary to further investigate the functional significance of ORs in the peripheral nerve system, which is far from complete clarification. Our current observations suggest a possible relationship between the differential expression of ORs in injured peripheral nerve and peripheral nerve regeneration. In this sense, the results in this study may provide a foundation for future intense work. Competing interests The authors declare no competing interests. Acknowledgments This study was supported by National Key Basic Research Program of China (973 and 863 programs, Grant Nos. 2014CB542202, 2012AA020502), National Natural Science Foundation of China (Grant No. 81130080, 31300879), Jiangsu Provincial Natural Science Foundation (BK2012230), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We thank Professor Jie Liu for assistance in the preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2015.06. 019
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