BioMed Research International

BioMed Research International / 2014 / Article
Special Issue

Chronic Pain: New Insights in Molecular and Cellular Mechanisms

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Research Article | Open Access

Volume 2014 |Article ID 498410 | https://doi.org/10.1155/2014/498410

Francis Sahngun Nahm, Zee-Yong Park, Sang-Soep Nahm, Yong Chul Kim, Pyung Bok Lee, "Proteomic Identification of Altered Cerebral Proteins in the Complex Regional Pain Syndrome Animal Model", BioMed Research International, vol. 2014, Article ID 498410, 11 pages, 2014. https://doi.org/10.1155/2014/498410

Proteomic Identification of Altered Cerebral Proteins in the Complex Regional Pain Syndrome Animal Model

Academic Editor: Livio Luongo
Received06 Mar 2014
Revised14 Aug 2014
Accepted25 Aug 2014
Published16 Sep 2014

Abstract

Background. Complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP) model, a novel experimental model of CRPS. Materials and Methods. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Conclusion. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

1. Introduction

Complex regional pain syndrome (CRPS) is a rare but serious and painful disorder. Although CRPS can occur following a minor injury, such as a sprain or even soft-tissue blunt trauma, severe intractable pain from CRPS can impair the quality of life. Symptoms and signs of CRPS include sensory changes (allodynia/hyperalgesia), vasomotor changes (temperature asymmetry/skin color change or asymmetry), sudomotor changes (edema/sweating change or asymmetry), and motor or trophic changes [1]. Although the exact pathophysiology of CRPS is not fully understood, several pathological mechanisms, including oxidative stress [2], neurogenic inflammation [3], and alteration in the autonomic nervous system [4, 5], are known to play some roles in its development. Also, psychophysical studies show that CRPS patients have distorted body image and have difficulty in recognizing the size or the position of the affected extremity [6]. The patients get worse when they think about moving the body part, even if they do not move it [7]. Mechanical stimulation of the “virtual (unaffected)” limb reflected in the mirror results in allodynia, which suggests that allodynia and paresthesia can be mediated by the brain [8]. Thus, the distorted body representation of CRPS patients can be treated with mirror therapy [9, 10]. Also, the spreading of symptoms and signs of CRPS from the initial site of presentation to another limb is a well-known phenomenon, which may be due to aberrant central regulation of neurogenic inflammation [11]. These findings highlight the contribution of a cortical pain mechanism in patients with CRPS. Moreover, functional imaging studies provide supporting evidence for the important role of the central nervous system in the pathogenesis of CRPS [1214], and recent research suggests that changes in cortical structures can contribute to the pathophysiology of CRPS [15].

Thus, the brain seems to play an important role in the development and maintenance of symptoms and signs in patients with CRPS. Some researchers insist that the peripheral changes in CRPS must be understood as a manifestation of changes in the brain [16]. Therefore, we postulated that protein expression would be altered in the CRPS-affected brain. However, there have been no studies on the changes of cerebral protein expression in CRPS. Therefore, to verify our hypothesis, we conducted a proteomic analysis using multidimensional protein identification technology (MudPIT) in a chronic postischemia perfusion (CPIP) rat model, a novel and widely used experimental model of CRPS type 1 [17].

2. Materials and Methods

2.1. Animals

This study was approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (IACUC number 52-2009-033). Male Sprague-Dawley rats weighing 200–250 g had free access to food and water and were housed individually in cages with soft bedding under a 12 h night/day light cycle at a constant temperature of 20–22°C and a humidity level of 55–60%. The animals were acclimatized for at least 1 week prior to the CPIP procedure.

2.2. CPIP Model Generation

The CPIP animal model was generated according to previous methods [17]. Briefly, after induction of anesthesia with isoflurane, a tight fitting O-ring (O-ring West, Seattle, WA, USA) with a 5.5 mm internal diameter was applied to the left hind limb of each anesthetized rat just proximal to the left ankle joint for 3 h. The O-ring was then removed from the anesthetized rat, allowing reperfusion of the hind limb (Figure 1). The animals in the control group underwent anesthesia similar to the CPIP animals, but the O-ring was not placed around the hind limb.

2.3. Behavioral Tests

All behavioral tests were performed during the daylight portion of the regulated circadian cycle between 9 a.m. and 3 p.m. To assess the mechanical threshold, the rats were placed in individual plastic cages with wire mesh bottoms. After 20 min acclimatization, calibrated von Frey filaments (Stoelting Co., Wood Dale, IL, USA) with logarithmically increasing stiffness of 0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 g were applied to the midplantar surface of the hind paw. The mechanical threshold was assessed using an up-down statistical method [18]. Then, the change in the mechanical threshold (CMT, %) was calculated. The mechanical threshold was examined during the postreperfusion period: 1 h, 4 h, 24 h, 48 h, day 7, and day 21. The CMT was calculated by following equation: We used the findings from the neurobehavioral test on day 21 to classify the animals into groups: rats whose CMT was decreased 50% or more after the CPIP procedure were classified as the successful CPIP (A) group. The mechanical threshold of the animals in the control (C) group was also examined and compared using repeated-measures analysis of variance. All animals were sacrificed 3 weeks after the CPIP procedure for proteomic analysis.

2.4. Proteomic Analysis

The difference in cerebral protein expression between Groups A and C was explored using a MudPIT as follows.

2.4.1. Protein Extraction

A total of six animals (three from Groups A and C) were used for the mass spectrometry analysis. On the day 21, right half of each rat cerebrum was grinded using a mortar in liquid nitrogen. The tissue powder was kept at −80°C. The tissue powder was resolubilized in a small volume of 8 M urea, 100 mM Tris-HCl, pH 8.5, and 1 mM dithiothreitol (DTT) for two hours. The homogenates were sonicated and centrifuged at 100,000 ×g for 1 h. Next, 5 mM DTT was added to the homogenate for 30 min at 37°C and alkylated with 25 mM iodoacetamide for 30 min at 37°C in the dark. The samples were then diluted with 2 M urea and with 50 mM Tris-HCl, pH 8.0, and digested at 37°C overnight with sequence grade trypsin (Promega Co., Fitchburg, MA, USA) diluted 1 : 50 in 5 mM CaCl2.

2.4.2. MudPIT

Peptides were separated with an Agilent 1100 series high-performance liquid chromatography (HPLC) pump (Agilent technologies, Santa Clara, CA, USA) connected to a linear quadruple ion-trap mass spectrometer (MS, LTQ, Thermo-Finnigan, San Jose, CA, USA) using an in-house-built nanoelectrospray ionization interface. To identify peptides, the ion-trap mass spectrometer was operated in a data-dependent MS/MS mode ( 400–2000), in which a full MS scan was followed by 10 MS/MS scans and the temperature of the heated capillary was 200°C. MS/MS spectra were generated in the positive ion mode at an electrospray voltage of 2.5 kV and normalized collision energy of 35%. An analytical column-fused (100 μm internal diameter) silica capillary microcolumn (Polymicro technologies, Phoenix, AZ, USA) was pulled to a fine tip using a laser puller and packed with 7 cm of 5 μm C18 reverse-phase resin, which was connected to an internal diameter of 250 μm fused-silica trapping column packed with 2 cm of SCX followed by 2 cm of C18 resin. Each 30 μg peptide mixture was manually loaded onto separate columns using a pressure vessel. A seven-step chromatography run was carried out on each sample and three buffers were used (buffer A: 5% ACN/0.1% formic acid, buffer B: 80% ACN/0.1% formic acid, and buffer C: 5% ACN/0.1% formic acid/500 mM ammonium acetate).

2.4.3. Data Searching and Analysis

Acquired MS/MS spectra were searched against an international protein index “rat v. 3.78 FASTA-format decoy database” downloaded from European Bioinformatics Institute (EBI, http://www.ebi.ac.uk/). The SEQUEST algorithm [19] was used to find the best matching sequences from the database with BioWorks 3.3 (Thermo Fisher Scientific Inc., Rockford, IL, USA) for fully tryptic peptides. The mass of the amino acid cysteine was statically modified by +57 Da and the differential modification search was performed for oxidation (+16 Da on Met). Xcorr values were based on tryptic peptides and charge states following 1.8 for singly charged peptides, 2.5 for doubly charged peptides, 3.5 for triply charged peptides, and 0.08 for ΔCn (DTASelect v. 2.0.39). The analysis of protein fold-change was quantified by an overall spectral counting method comparison of label-free methods for quantifying human proteins [20].

3. Results

3.1. Behavioral Tests

A total of 14 animals ( per group) were included in the behavioral test. Before the CPIP procedure, there were no differences in the mechanical threshold between the groups. However, Group A exhibited a significant decrease in the mechanical threshold compared to Group C after the CPIP procedure (, Figure 2). The mean differences of CMT (%) in Group A compared to Group C were −41.5, −73.2, −92.3, −98.2, −92.2, and −95.3 after CPIP procedure 1 h, 4 h, day 1, day 2, day 7, and day 21, respectively.

3.2. Differential Protein Expression in the Rat Cerebrum

A total of 454 proteins were differentially expressed between Groups A and C under the criterion of value <0.1. Among the 454 proteins, we selected those found in the cerebrum of all study animals in either group and excluded “uncharacterized proteins” and “hypothetical proteins.” Finally, we found 155 differentially expressed proteins between Group A and Group C: 125 increased (Table 6) and 30 decreased (Table 7). Specifically, expression of proteins related to cell signaling (Table 1), synaptic plasticity (Table 2), regulation of cell proliferation (Table 3), and cytoskeletal formation (Table 4) was increased in Group A. Also, expression of a group of protein kinases (calmodulin dependent protein kinase II beta M isoform, casein kinase 2, phosphoenolpyruvate carboxykinase 2, mitogen-activated protein kinase 4, protein kinase C delta, N-terminal kinase like protein, uridine kinase-like 1, serine/threonine protein kinase PLK 1, and phosphoinositide 3 kinase regulatory subunit 4) and calcium-related proteins (inositol 1,4,5-triphosphate receptor type 2, annexin A1, annexin A2, annexin A5, voltage-dependent Ca2+ channel gamma-2 subunit, and voltage-dependent Ca2+ channel beta-3 subunit, and coiled-coil domain-containing protein 47) was also elevated in Group A. However, several proteins were decreased in group A. Specifically, expression of proteins related to cell signaling (Table 5) and metabolism of fatty acid (peroxisomal 3,2-trans-enoyl Co A isomerase, acetyl-CoA acyltransferase 1b, and acetyl-CoA acetyltransferase 2) were decreased. Also, proenkephalin A, protein cereblon, and neuroserpin were decreased in Group A.


NumberSymbolDescription value

1Kctd12Potassium channel tetramerisation domain containing 12 0.004
2EcsitEvolutionarily conserved signaling intermediate in Toll pathway, mitochondrial 0.004
3Tns1Tensin 1 0.004
4Ccbp2Chemokine-binding protein 2 0.004
5Apba1Amyloid beta A4 precursor protein-binding family A member 1 0.008
6TncTenascin C 0.008
7Rabl2bRAB, member of RAS oncogene family-like 2B 0.008
8Epha4Eph receptor A4 0.035
9Rab6aRas-related protein Rab-6A 0.038
10Gpr158G protein-coupled receptor 158 0.042
11Anxa2Isoform Short of Annexin A20.043
12HgsIsoform 1 of hepatocyte growth factor-regulated tyrosine kinase substrate 0.054
13PrkcdIsoform 1 of protein kinase C delta type 0.060
14Gabarapl2Gamma-aminobutyric acid receptor-associated protein-like 2 0.065
15Map2k4Dual specificity mitogen-activated protein kinase 4 0.066
16Cacng2Voltage-dependent calcium channel gamma-2 subunit 0.083
17Phb2Prohibitin-2 0.086
18Camk2bCalmodulin-dependent protein kinase II beta M isoform 0.086
19Anxa5Annexin A50.093
20Scn2a1Sodium channel Nav1.2 0.095
21Rab10Ras-related protein Rab-10 0.097


NumberSymbolDescription value

1Itpr2Inositol 1,4,5-trisphosphate receptor type 2 0.001
2Kctd12Potassium channel tetramerisation domain containing 12 0.004
3Grid2Glutamate receptor delta-2 subunit 0.004
4Baiap3BAI1-associated protein 3-like isoform 2 0.008
5Atad1ATPase family, AAA domain containing 10.008
6Pick1PRKCA-binding protein 0.008
7Nlgn3Isoform 1 of Neuroligin-30.056
8NudcNuclear migration protein nudC 0.085
9Camk2bCalmodulin-dependent protein kinase II beta M isoform 0.086


NumberSymbolDescription value

1Pik3r4Phosphoinositide 3-kinase regulatory subunit 4 0.001
2Itpr2Inositol 1,4,5-trisphosphate receptor type 20.001
3Anp32bAcidic leucine-rich nuclear phosphoprotein 32 family member B 0.002
4Plk1Serine/threonine-protein kinase 0.004
5Drg2Developmentally regulated GTP binding protein 2-like0.004
6DmwdDystrophia myotonica-containing WD repeat motif 0.008
7Acin1Apoptotic chromatin condensation inducer 1 protein 0.008
8Pole2Polymerase (DNA directed), epsilon 20.008
9CyldUbiquitin carboxyl-terminal hydrolase 0.076
10Csnk2a2Casein kinase 2, alpha prime polypeptide0.084
11Rab10Ras-related protein Rab-100.097


NumberSymbolDescription value

1Krt4Keratin, type II cytoskeletal 4 0.008
2Sntb2Syntrophin, beta 2 0.008
3Ckap5Cytoskeleton associated protein 5 0.038
4Fermt2Fermitin family homolog 2 0.063
5Cotl1Coactosin-like protein 0.065
6Rps540S ribosomal protein S5 0.072
7Col1a2Collagen alpha-2(I) chain 0.079
8Actr10Actin-related protein 10 homolog0.080
9Etl4Enhancer trap locus 4-like0.083
10Farp1FERM, RhoGEF (Arhgef), and pleckstrin domain protein 1 0.094


NumberSymbolDescription value

1Vwa1Von Willebrand factor A domain-containing protein 1 0.002
2Ppp1r10Serine/threonine-protein phosphatase 1 regulatory subunit 10 0.003
3Slc4a8Isoform 2 of electroneutral sodium bicarbonate exchanger 1 0.003
4Daam2Dishevelled associated activator of morphogenesis 2 0.003
5Trim32Tripartite motif protein 32 0.007
6Slc1a1Excitatory amino acid transporter 3 0.007
7SpnSialophorin 0.007
8CrbnCereblon 0.007
9Thoc1Da2-19 THO complex subunit 1 0.007
10Lmo7Lim domain only protein 7 0.007
11Rps27aRibosomal protein S27a0.007
12Sema4dSema domain 0.043
13Sec3l1SEC3-like 1 0.047
14Spna2Alpha II spectrin 0.065
15Pde10aIsoform 3 of cAMP- and cAMP-inhibited cGMP 3′,5′-cyclic phosphodiesterase 10A 0.065
16Snx2Sorting nexin 2 0.067
17Slc25a3Phosphate carrier protein, mitochondrial 0.068
18Cox6a1Cytochrome c oxidase subunit 6A1, mitochondrial 0.090


NumberSymbolDescription value

1Itpr2Inositol 1,4,5-trisphosphate receptor type 2 0.001
2Pik3r4Phosphoinositide 3-kinase regulatory subunit 4 0.001
3Exoc7Exocyst complex component 7 0.001
4Rcor2REST corepressor 2 0.001
5Anp32bAcidic leucine-rich nuclear phosphoprotein 32 family member B 0.002
6Qrich2Glutamine rich 2-like 0.002
7Dnah11Dynein, axonemal, heavy chain 11 0.002
8Plk1Serine/threonine-protein kinase PLK1 0.004
9Ephx1Epoxide hydrolase 1 0.004
10Cacnb3Voltage-dependent L-type calcium channel subunit beta-3 0.004
11Anxa1Annexin A1 0.004
12Tns1Tensin 1 0.004
13Hdac4Histone deacetylase 4 0.004
14Osbpl7Oxysterol binding protein like 7 0.004
15EcsitEvolutionarily conserved signaling intermediate in Toll pathway, mitochondrial 0.004
16Sorbs3Sorbin and SH3 domain containing 3, isoform CRA_b 0.004
17Kctd12Potassium channel tetramerisation domain containing 12 0.004
18Ccbp2Chemokine-binding protein 2 0.004
19Drg2Developmentally regulated GTP binding protein 2-like 0.004
20Grid2Glutamate receptor delta-2 subunit 0.004
21SafbScaffold attachment factor B1 0.008
22Dnm3Isoform 1 of Dynamin-3 0.008
23Dnajc16DnaJ homolog subfamily C member 16 0.008
24Sntb2Syntrophin, beta 2 0.008
25Pnpt1Polyribonucleotide nucleotidyltransferase 1 0.008
26Eif3gEukaryotic translation initiation factor 3 subunit G 0.008
27Pole2Polymerase (DNA directed), epsilon 2 0.008
28Scyl1N-terminal kinase-like protein 0.008
29Atad1ATPase family, AAA domain containing 0.008
30Krt4Keratin, type II cytoskeletal 4 0.008
31CtsaProtective protein for beta-galactosidase 0.008
32Abca15 ATP-binding cassette, subfamily A (ABC1), member 15 0.008
33DmwdDystrophia myotonica-containing WD repeat motif 0.008
34Baiap3BAI1-associated protein 3-like isoform 2 0.008
35Znf512bUridine kinase-like 1 0.008
36GaleGale protein 0.008
37Pick1PRKCA-binding protein 0.008
38Acin1Acin1 protein 0.008
39Chid1Chitinase domain containing 1 0.008
40Pcyox1lPcyox1l protein 0.008
41Rabl2bRAB, member of RAS oncogene family-like 2B 0.008
42Serpina3kSerine protease inhibitor A3K 0.008
43Glg1Golgi apparatus protein 1 0.008
44TncTenascin C 0.008
45Lysmd1LysM and putative peptidoglycan-binding domain-containing protein 1 0.008
46Apba1Amyloid beta A4 precursor protein-binding family A member 1 0.008
47Ckap5Cytoskeleton associated protein 5 0.038
48Ndufab1Acyl carrier protein 0.035
49Epha4Eph receptor A4 0.035
50KalrnIsoform 2 of Kalirin 0.035
51Myh14Myosin, heavy chain 14 0.035
52Anxa2Isoform Short of Annexin A2 0.043
53Ccdc47Coiled-coil domain-containing protein 47 0.043
54Gpr158G protein-coupled receptor 158 0.042
55Cugbp1CUGBP Elav-like family member 1 0.041
56Hba2Hemoglobin alpha 2 chain 0.040
57Acsl3Isoform long of long-chain-fatty-acid-CoA ligase 3 0.040
58Rab6aRas-related protein Rab-6A 0.038
59HbbHemoglobin subunit beta-1 0.048
60Hbb-b1Zero beta-1 globin 0.044
61KhsrpFar upstream element-binding protein 2 0.043
62Scamp5Secretory carrier-associated membrane protein 5 0.048
63Aldh3a2Fatty aldehyde dehydrogenase 0.049
64Mesdc2LDLR chaperone MESD 0.049
65Rab3dGTP-binding protein Rab-3D 0.051
66Vps29Isoform 2 of vacuolar protein sorting-associated protein 29 0.051
67Psma3lPsma3 Proteasome subunit alpha type-3 0.053
68HgsIsoform 1 of hepatocyte growth factor-regulated tyrosine kinase 0.054
69Nlgn3Isoform 1 of neuroligin-3 0.056
70CygbCytoglobin 0.060
71Pcsk2Neuroendocrine convertase 2 0.060
72PrkcdIsoform 1 of Protein kinase C delta 0.060
73Fnbp4Formin binding protein 4 0.062
74Eif2s3xEukaryotic translation initiation factor 2 subunit 30.063
75Fermt2Fermitin family homolog 20.063
76Vps33aVacuolar protein sorting-associated protein 33A 0.063
77SNX3Sorting nexin-3 0.063
78Exoc8Exocyst complex component 8 0.063
79Thrap3Thyroid hormone receptor-associated protein 3 0.063
80Ndufa1NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1 0.063
81Gabarapl2Gamma-aminobutyric acid receptor-associated protein-like 2 0.065
82Cotl1Coactosin-like protein 0.065
83Gad1Glutamate decarboxylase 1 0.065
84Ehd1EH domain-containing protein 1 0.066
85Map2k4Mitogen-activated protein kinase 4 0.066
86Mug1Murinoglobulin (alpha-1-inhibitor 3) 0.070
87Pck2Phosphoenolpyruvate carboxykinase 2 0.072
88Rps540S ribosomal protein S5 0.072
89Ap2s1Adaptor protein complex 2 subunit sigma 0.075
90Tpp1Tripeptidyl-peptidase 1 0.076
91CyldUbiquitin carboxyl-terminal hydrolase 0.076
92NucNucleolin-like protein 0.079
93Col1a2Collagen alpha-2(I) chain 0.079
94Slc6a17Orphan sodium- and chloride-dependent neurotransmitter transporter NTT40.079
95Actr10Actin-related protein 10 homolog 0.080
96Cacng2Voltage-dependent calcium channel gamma-2 subunit 0.083
97Ampd3AMP deaminase 3 0.083
98Eif5b-ps1Eif5b Eukaryotic translation initiation factor 5B 0.083
99Timm9Mitochondrial import inner membrane translocase subunit Tim9 0.083
100Etl4Enhancer trap locus 4-like 0.083
101Csnk2a2Casein kinase 2, alpha prime polypeptide 0.084
102Cct6aChaperonin containing TCP1 subunit 6a 0.084
103NudcNuclear migration protein nud 0.085
104Ndufa13NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 130.085
105Camk2bCalmodulin-dependent protein kinase II beta M isoform 0.086
106CltaIsoform brain of clathrin light chain A 0.086
107Asah1Acid ceramidase 0.086
108Phb2Prohibitin-2 0.086
109Sod1Superoxide dismutase [Cu-Zn] 0.088
110Ndufs8NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 80.090
111Slc17a7Isoform 1 of vesicular glutamate transporter 1 0.091
112Ugp2UDP-glucose pyrophosphorylase 2, isoform CRA-b 0.091
113RalaRas-related protein Ral-A 0.091
114Anxa5Annexin A5 0.093
115Hnrph1Isoform 1 of heterogeneous nuclear ribonucleoprotein H 0.093
116Stxbp5lSyntaxin binding protein 5-like 0.093
117Abcd3ATP-binding cassette subfamily D member 3 0.094
118Farp1FERM, RhoGEF (Arhgef), and pleckstrin domain protein 1 0.094
119Leng4Leng4 protein 0.094
120Scn2a1Sodium channel Nav1.2 0.095
121Rab10Ras-related protein Rab-10 0.097
122Aldh7a1Alpha-aminoadipic semialdehyde dehydrogenase 0.097
123CltbIsoform Brain of Clathrin light chain B 0.097
124PhyhiplIsoform 1 of phytanoyl-CoA hydroxylase-interacting protein-like 0.098
125SynpoIsoform 1 of synaptopodin 0.099


NumberSymbolDescription value

1Vwa1Von Willebrand factor A domain-containing protein 1 0.002
2Ppp1r10Serine/threonine-protein phosphatase 1 regulatory subunit 10 0.003
3Poldip2DNA-directed polymerase delta interacting protein 2 0.003
4Slc4a8Isoform 2 of electroneutral sodium bicarbonate exchanger 1 0.003
5Daam2Dishevelled associated activator of morphogenesis 2 0.003
6Cep350 Centrosome-associated protein 350 0.003
7Tra2bTransformer-2 protein homolog beta 0.007
8Epb4.1l1Isoform L of band 4.1-like protein 1 0.007
9Trim32Tripartite motif protein 32 0.007
10Slc1a1Excitatory amino acid transporter 3 0.007
11SpnSialophorin 0.007
12CrbnCereblon 0.007
13Thoc1Da2-19 THO complex subunit 1 0.007
14Lmo7Lim domain only protein 7 0.007
15Rps27aRibosomal protein S27a0.007
16Sema4dSema domain, immunoglobulin domain (Ig), transmembrane domain 0.043
17Sec3l1SEC3-like 1 0.047
18IkbkapElongator complex protein 1 0.058
19PeciPeroxisomal 3,2-trans-enoyl-CoA isomerase 0.058
20PenkProenkephalin-A 0.058
21Bles03basophilic leukemia expressed protein 0.058
22Spna2Alpha II spectrin 0.065
23Pde10aIsoform 3 of cAMP and cAMP-inhibited cGMP 3′,5′-cyclic phosphodiesterase 10A 0.065
24Snx2Sorting nexin 2 0.067
25Slc25a3Phosphate carrier protein, mitochondrial 0.068
26Serpini1Neuroserpin 0.070
27Acaa1bAcetyl-CoA acyltransferase 1b 0.077
28H2afzHistone H2A.Z 0.079
29Cox6a1Cytochrome c oxidase subunit 6A1, mitochondrial 0.090
30Acat2Acetyl-CoA acetyltransferase 2 0.094

4. Discussion

In our study, various proteins were differentially expressed in the cerebrum of CPIP animals. Specifically, expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in Group A. These findings suggest that both functional and structural changes may occur in the cerebrum of CPIP animals, and altered protein expression can be related to the development of CRPS. This is the first study of cerebral protein expression changes in the CPIP rat model.

We also found that inositol 1,4,5-triphosphate (IP3) receptor type 2 and phosphoinositide 3 kinase (PI3K) regulatory subunit were also increased in Group A. IP3 receptor is intracellular calcium release channel and is regulated by calcium and calmodulin (CaM) [21]. And it is known that PI3K is an important mediator of central sensitization in painful inflammatory condition [22], and many tumorous conditions are related to this enzyme [23, 24]. Based on these findings, cerebral overexpression of IP3 receptor type 2 and PI3K can be related to the sustained pain after rat CPIP model.

Among the 155 proteins expressed differentially, calcium related proteins, including calcium calmodulin kinase II (CaMKII), were increased in group A. Calcium plays a crucial role in many physiological processes, including signal transduction, cell growth, and proliferation. CaMKII is one of the most prominent protein kinases, present in every tissue, but most concentrated in the brain. CaMKII plays various roles, including synthesis and release of neurotransmitter modulation of ion channel activity, synaptic plasticity, learning, and memory [2528]. Moreover, CaMKII is thought to be important in central sensitization [2931] and is implicated in central neuropathic pain [31] and long term potentiation (LTP) [32]. LTP is initiated when NMDA receptors allow Ca2+ into the postsynaptic neuron, and this Ca2+ influx activates CaMKII. LTP in nociceptive spinal pathways shares several features with hyperalgesia, and LTP at synapses constitutes a contemporary cellular model for pain [33, 34]. And it was reported that the overexpression of CaMKII was observed in the dorsal root ganglia of rat model of type 1 diabetes [35], and the inhibition of CaMKII can reverse the chronic inflammatory pain [36]. These findings are consistent with the result of our study. Therefore, overexpression of cerebral CaMKII implicates cerebral involvement in CRPS, and CaMKII can be a target for the prevention and treatment of CRPS.

In addition, we observed that proenkephalin A, cereblon, and neuroserpin decreased in CPIP animals. Proenkephalin is an endogenous opioid hormone which produces the enkephalin peptide. Enkephalin provides a role as inhibiting neurotransmitters in the pathway for pain perception to reduce pain perception. Therefore, decreased proenkephalin A in the cerebrum of CPIP animals seems to reflect the blunted ability to pain modulation and exaggerated response to the pain. For the cereblon, it is known to be related to memory, learning, and intelligence [37], and anomalous cereblon expression can lead to memory and learning deficit [38]. The defect in cereblon gene is associated with mental retardation [39]. Therefore, decreased expression of cereblon in CPIP animals in our study might be related to the deficit in the learning and memory. Neuroserpin, which was known to be related to neurogenesis [40] was decreased in CPIP animals in our study. Neuroserpin plays a role of neuronal protection in pathologic state, and point mutation can cause encephalopathy [41]. Also it has been known that deficiency in neuroserpin exacerbates ischemic brain injury [42]. Therefore, the decreased expression of neuroserpin in CPIP animals in our study might be related to the altered or defected cerebral function.

In our study, we used the CPIP model because CRPS develops after a minor injury without distinguishable nerve lesions. This model is considered a novel animal model of CRPS type 1, in which nerve injury or bone fracture usually does not exist. The previous proteomic studies in neuropathic pain research usually used the nerve ligation model or nerve crush injury model [43, 44]. Since the CPIP model exhibits similar features of human CRPS type 1, our results may have an implication for cerebral involvement in human CRPS. The mechanical threshold was similar at the beginning (day 1) and after 21 days. This is because we took no actions for treatment on the CPIP animals and therefore the initial pain seemed to persist without change. We did not measure the mechanical threshold in the contralateral paws, because it has been already known that the mechanical threshold decreases in the contralateral paws of the CPIP animals, and ipsilateral plantar allodynia is known to be the most characteristic feature of the CPIP animals [17]. The CMT in the ipsilateral paw was used for the criterion of the successful establishment of the animal model.

This study had some limitations. First, the differentially expressed cerebral proteins may not be specific to CPIP animals. These proteins may also change in response to peripheral noxious stimuli. However, CPIP animals exhibit many features of human CRPS type 1, and thus our findings can be extrapolated to human CRPS. Second, because of the complexity of protein interactions in many physiologic pathways in the brain, it is still unclear which is the key protein in the development of CRPS.

Third, we performed proteomic analysis only 21 days after CPIP model generation. However, protein expression related to the development and maintenance of CRPS can differ according to the time course. A proper time course to evaluate a possible correlation between pain behavior development and protein modulation may be useful to discriminate protein changes associated with the early inflammation from that one responsible for possible structural or functional alterations (neural sensitization) occurring at central level. Further investigation of the cerebral mechanism of CRPS is required.

5. Conclusion

In conclusion, the cerebral proteome is altered after CPIP injury; many functional and structural changes seem to occur in the cerebrum. These findings support the notion of cerebral involvement in CRPS. Therefore, treatment of CRPS should target not only the periphery, but also the brain.

Conflict of Interests

The authors have no conflict of interests to declare.

Acknowledgment

This study was financially supported by Grant no. 02-2009-038 from the Seoul National University Bundang Hospital.

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Copyright © 2014 Francis Sahngun Nahm et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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