BioMed Research International

Volume 2014 (2014), Article ID 498410, 11 pages

http://dx.doi.org/10.1155/2014/498410

Research Article

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

Francis Sahngun Nahm,¹ Zee-Yong Park,² Sang-Soep Nahm,³ Yong Chul Kim,⁴ and Pyung Bok Lee¹

¹Department of Anesthesiology and Pain Medicine, Seoul National University Bundang Hospital, Seongnam 463707, Republic of Korea

²School of Life Science, Gwangju Institute of Science and Technology, Gwangju 500712, Republic of Korea

³Laboratory of Veterinary Anatomy, College of Veterinary Medicine, Konkuk University, Seoul 143701, Republic of Korea

⁴Department of Anesthesiology and Pain Medicine, Seoul National University Hospital, Seoul 110744, Republic of Korea

Received 6 March 2014; Revised 14 August 2014; Accepted 25 August 2014; Published 16 September 2014

Academic Editor: Livio Luongo

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.

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 [12–14], 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.

(a)

(b)

(c)

(d)

Figure 1: Plantar skin color changes in chronic postischemia pain model rats. (a) Before O-ring application, (b) during O-ring application, (c) 1 hour after reperfusion, and (d) 4 h after reperfusion.

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.

Figure 2: Change in mechanical threshold (%). Group A showed a significant decrease in mechanical threshold compared to group C ( in each group). Asterisk () indicates at each time point. Group A: CPIP group and Group C: control group.

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.

Table 1: Increased cerebral proteins in the chronic post-ischemia pain group; proteins which might be related to cell signaling.

Number
Symbol
Description
value

1
Kctd12
Potassium channel tetramerisation domain containing 12
0.004

2
Ecsit
Evolutionarily conserved signaling intermediate in Toll pathway, mitochondrial
0.004

3
Tns1
Tensin 1
0.004

4
Ccbp2
Chemokine-binding protein 2
0.004

5
Apba1
Amyloid beta A4 precursor protein-binding family A member 1
0.008

6
Tnc
Tenascin C
0.008

7
Rabl2b
RAB, member of RAS oncogene family-like 2B
0.008

8
Epha4
Eph receptor A4
0.035

9
Rab6a
Ras-related protein Rab-6A
0.038

10
Gpr158
G protein-coupled receptor 158
0.042

11
Anxa2
Isoform Short of Annexin A2
0.043

12
Hgs
Isoform 1 of hepatocyte growth factor-regulated tyrosine kinase substrate
0.054

13
Prkcd
Isoform 1 of protein kinase C delta type
0.060

14
Gabarapl2
Gamma-aminobutyric acid receptor-associated protein-like 2
0.065

15
Map2k4
Dual specificity mitogen-activated protein kinase 4
0.066

16
Cacng2
Voltage-dependent calcium channel gamma-2 subunit
0.083

17
Phb2
Prohibitin-2
0.086

18
Camk2b
Calmodulin-dependent protein kinase II beta M isoform
0.086

19
Anxa5
Annexin A5
0.093

20
Scn2a1
Sodium channel Nav1.2
0.095

21
Rab10
Ras-related protein Rab-10
0.097

Table 2: Increased cerebral proteins in the chronic postischemia pain group; proteins which might be related to synaptic plasticity.

Number
Symbol
Description
value

1
Itpr2
Inositol 1,4,5-trisphosphate receptor type 2
0.001

2
Kctd12
Potassium channel tetramerisation domain containing 12
0.004

3
Grid2
Glutamate receptor delta-2 subunit
0.004

4
Baiap3
BAI1-associated protein 3-like isoform 2
0.008

5
Atad1
ATPase family, AAA domain containing 1
0.008

6
Pick1
PRKCA-binding protein
0.008

7
Nlgn3
Isoform 1 of Neuroligin-3
0.056

8
Nudc
Nuclear migration protein nudC
0.085

9
Camk2b
Calmodulin-dependent protein kinase II beta M isoform
0.086

Table 3: Increased cerebral proteins in the chronic postischemia pain group; proteins which might be related to regulation of cell proliferation.

Number
Symbol
Description
value

1
Pik3r4
Phosphoinositide 3-kinase regulatory subunit 4
0.001

2
Itpr2
Inositol 1,4,5-trisphosphate receptor type 2
0.001

3
Anp32b
Acidic leucine-rich nuclear phosphoprotein 32 family member B
0.002

4
Plk1
Serine/threonine-protein kinase
0.004

5
Drg2
Developmentally regulated GTP binding protein 2-like
0.004

6
Dmwd
Dystrophia myotonica-containing WD repeat motif
0.008

7
Acin1
Apoptotic chromatin condensation inducer 1 protein
0.008

8
Pole2
Polymerase (DNA directed), epsilon 2
0.008

9
Cyld
Ubiquitin carboxyl-terminal hydrolase
0.076

10
Csnk2a2
Casein kinase 2, alpha prime polypeptide
0.084

11
Rab10
Ras-related protein Rab-10
0.097

Table 4: Increased cerebral proteins in the chronic postischemia pain group; proteins which might be related to cytoskeletal formation.

Number
Symbol
Description
value

1
Krt4
Keratin, type II cytoskeletal 4
0.008

2
Sntb2
Syntrophin, beta 2
0.008

3
Ckap5
Cytoskeleton associated protein 5
0.038

4
Fermt2
Fermitin family homolog 2
0.063

5
Cotl1
Coactosin-like protein
0.065

6
Rps5
40S ribosomal protein S5
0.072

7
Col1a2
Collagen alpha-2(I) chain
0.079

8
Actr10
Actin-related protein 10 homolog
0.080

9
Etl4
Enhancer trap locus 4-like
0.083

10
Farp1
FERM, RhoGEF (Arhgef), and pleckstrin domain protein 1
0.094

Table 5: Decreased cerebral proteins in the chronic postischemia pain group; proteins which might be related to cell signaling.

Number
Symbol
Description
value

1
Vwa1
Von Willebrand factor A domain-containing protein 1
0.002

2
Ppp1r10
Serine/threonine-protein phosphatase 1 regulatory subunit 10
0.003

3
Slc4a8
Isoform 2 of electroneutral sodium bicarbonate exchanger 1
0.003

4
Daam2
Dishevelled associated activator of morphogenesis 2
0.003

5
Trim32
Tripartite motif protein 32
0.007

6
Slc1a1
Excitatory amino acid transporter 3
0.007

7
Spn
Sialophorin
0.007

8
Crbn
Cereblon
0.007

9
Thoc1
Da2-19 THO complex subunit 1
0.007

10
Lmo7
Lim domain only protein 7
0.007

11
Rps27a
Ribosomal protein S27a
0.007

12
Sema4d
Sema domain
0.043

13
Sec3l1
SEC3-like 1
0.047

14
Spna2
Alpha II spectrin
0.065

15
Pde10a
Isoform 3 of cAMP- and cAMP-inhibited cGMP 3′,5′-cyclic phosphodiesterase 10A
0.065

16
Snx2
Sorting nexin 2
0.067

17
Slc25a3
Phosphate carrier protein, mitochondrial
0.068

18
Cox6a1
Cytochrome c oxidase subunit 6A1, mitochondrial
0.090

Table 6: Cerebral proteins with increased expression in the chronic postischemia pain (CPIP) group.

Number
Symbol
Description
value

1
Itpr2
Inositol 1,4,5-trisphosphate receptor type 2
0.001

2
Pik3r4
Phosphoinositide 3-kinase regulatory subunit 4
0.001

3
Exoc7
Exocyst complex component 7
0.001

4
Rcor2
REST corepressor 2
0.001

5
Anp32b
Acidic leucine-rich nuclear phosphoprotein 32 family member B
0.002

6
Qrich2
Glutamine rich 2-like
0.002

7
Dnah11
Dynein, axonemal, heavy chain 11
0.002

8
Plk1
Serine/threonine-protein kinase PLK1
0.004

9
Ephx1
Epoxide hydrolase 1
0.004

10
Cacnb3
Voltage-dependent L-type calcium channel subunit beta-3
0.004

11
Anxa1
Annexin A1
0.004

12
Tns1
Tensin 1
0.004

13
Hdac4
Histone deacetylase 4
0.004

14
Osbpl7
Oxysterol binding protein like 7
0.004

15
Ecsit
Evolutionarily conserved signaling intermediate in Toll pathway, mitochondrial
0.004

16
Sorbs3
Sorbin and SH3 domain containing 3, isoform CRA_b
0.004

17
Kctd12
Potassium channel tetramerisation domain containing 12
0.004

18
Ccbp2
Chemokine-binding protein 2
0.004

19
Drg2
Developmentally regulated GTP binding protein 2-like
0.004

20
Grid2
Glutamate receptor delta-2 subunit
0.004

21
Safb
Scaffold attachment factor B1
0.008

22
Dnm3
Isoform 1 of Dynamin-3
0.008

23
Dnajc16
DnaJ homolog subfamily C member 16
0.008

24
Sntb2
Syntrophin, beta 2
0.008

25
Pnpt1
Polyribonucleotide nucleotidyltransferase 1
0.008

26
Eif3g
Eukaryotic translation initiation factor 3 subunit G
0.008

27
Pole2
Polymerase (DNA directed), epsilon 2
0.008

28
Scyl1
N-terminal kinase-like protein
0.008

29
Atad1
ATPase family, AAA domain containing
0.008

30
Krt4
Keratin, type II cytoskeletal 4
0.008

31
Ctsa
Protective protein for beta-galactosidase
0.008

32
Abca1
5 ATP-binding cassette, subfamily A (ABC1), member 15
0.008

33
Dmwd
Dystrophia myotonica-containing WD repeat motif
0.008

34
Baiap3
BAI1-associated protein 3-like isoform 2
0.008

35
Znf512b
Uridine kinase-like 1
0.008

36
Gale
Gale protein
0.008

37
Pick1
PRKCA-binding protein
0.008

38
Acin1
Acin1 protein
0.008

39
Chid1
Chitinase domain containing 1
0.008

40
Pcyox1l
Pcyox1l protein
0.008

41
Rabl2b
RAB, member of RAS oncogene family-like 2B
0.008

42
Serpina3k
Serine protease inhibitor A3K
0.008

43
Glg1
Golgi apparatus protein 1
0.008

44
Tnc
Tenascin C
0.008

45
Lysmd1
LysM and putative peptidoglycan-binding domain-containing protein 1
0.008

46
Apba1
Amyloid beta A4 precursor protein-binding family A member 1
0.008

47
Ckap5
Cytoskeleton associated protein 5
0.038

48
Ndufab1
Acyl carrier protein
0.035

49
Epha4
Eph receptor A4
0.035

50
Kalrn
Isoform 2 of Kalirin
0.035

51
Myh14
Myosin, heavy chain 14
0.035

52
Anxa2
Isoform Short of Annexin A2
0.043

53
Ccdc47
Coiled-coil domain-containing protein 47
0.043

54
Gpr158
G protein-coupled receptor 158
0.042

55
Cugbp1
CUGBP Elav-like family member 1
0.041

56
Hba2
Hemoglobin alpha 2 chain
0.040

57
Acsl3
Isoform long of long-chain-fatty-acid-CoA ligase 3
0.040

58
Rab6a
Ras-related protein Rab-6A
0.038

59
Hbb
Hemoglobin subunit beta-1
0.048

60
Hbb-b1
Zero beta-1 globin
0.044

61
Khsrp
Far upstream element-binding protein 2
0.043

62
Scamp5
Secretory carrier-associated membrane protein 5
0.048

63
Aldh3a2
Fatty aldehyde dehydrogenase
0.049

64
Mesdc2
LDLR chaperone MESD
0.049

65
Rab3d
GTP-binding protein Rab-3D
0.051

66
Vps29
Isoform 2 of vacuolar protein sorting-associated protein 29
0.051

67
Psma3l
Psma3 Proteasome subunit alpha type-3
0.053

68
Hgs
Isoform 1 of hepatocyte growth factor-regulated tyrosine kinase
0.054

69
Nlgn3
Isoform 1 of neuroligin-3
0.056

70
Cygb
Cytoglobin
0.060

71
Pcsk2
Neuroendocrine convertase 2
0.060

72
Prkcd
Isoform 1 of Protein kinase C delta
0.060

73
Fnbp4
Formin binding protein 4
0.062

74
Eif2s3x
Eukaryotic translation initiation factor 2 subunit 3
0.063

75
Fermt2
Fermitin family homolog 2
0.063

76
Vps33a
Vacuolar protein sorting-associated protein 33A
0.063

77
SNX3
Sorting nexin-3
0.063

78
Exoc8
Exocyst complex component 8
0.063

79
Thrap3
Thyroid hormone receptor-associated protein 3
0.063

80
Ndufa1
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1
0.063

81
Gabarapl2
Gamma-aminobutyric acid receptor-associated protein-like 2
0.065

82
Cotl1
Coactosin-like protein
0.065

83
Gad1
Glutamate decarboxylase 1
0.065

84
Ehd1
EH domain-containing protein 1
0.066

85
Map2k4
Mitogen-activated protein kinase 4
0.066

86
Mug1
Murinoglobulin (alpha-1-inhibitor 3)
0.070

87
Pck2
Phosphoenolpyruvate carboxykinase 2
0.072

88
Rps5
40S ribosomal protein S5
0.072

89
Ap2s1
Adaptor protein complex 2 subunit sigma
0.075

90
Tpp1
Tripeptidyl-peptidase 1
0.076

91
Cyld
Ubiquitin carboxyl-terminal hydrolase
0.076

92
Nuc
Nucleolin-like protein
0.079

93
Col1a2
Collagen alpha-2(I) chain
0.079

94
Slc6a17
Orphan sodium- and chloride-dependent neurotransmitter transporter NTT4
0.079

95
Actr10
Actin-related protein 10 homolog
0.080

96
Cacng2
Voltage-dependent calcium channel gamma-2 subunit
0.083

97
Ampd3
AMP deaminase 3
0.083

98
Eif5b-ps1
Eif5b Eukaryotic translation initiation factor 5B
0.083

99
Timm9
Mitochondrial import inner membrane translocase subunit Tim9
0.083

100
Etl4
Enhancer trap locus 4-like
0.083

101
Csnk2a2
Casein kinase 2, alpha prime polypeptide
0.084

102
Cct6a
Chaperonin containing TCP1 subunit 6a
0.084

103
Nudc
Nuclear migration protein nud
0.085

104
Ndufa13
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 13
0.085

105
Camk2b
Calmodulin-dependent protein kinase II beta M isoform
0.086

106
Clta
Isoform brain of clathrin light chain A
0.086

107
Asah1
Acid ceramidase
0.086

108
Phb2
Prohibitin-2
0.086

109
Sod1
Superoxide dismutase [Cu-Zn]
0.088

110
Ndufs8
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 8
0.090

111
Slc17a7
Isoform 1 of vesicular glutamate transporter 1
0.091

112
Ugp2
UDP-glucose pyrophosphorylase 2, isoform CRA-b
0.091

113
Rala
Ras-related protein Ral-A
0.091

114
Anxa5
Annexin A5
0.093

115
Hnrph1
Isoform 1 of heterogeneous nuclear ribonucleoprotein H
0.093

116
Stxbp5l
Syntaxin binding protein 5-like
0.093

117
Abcd3
ATP-binding cassette subfamily D member 3
0.094

118
Farp1
FERM, RhoGEF (Arhgef), and pleckstrin domain protein 1
0.094

119
Leng4
Leng4 protein
0.094

120
Scn2a1
Sodium channel Nav1.2
0.095

121
Rab10
Ras-related protein Rab-10
0.097

122
Aldh7a1
Alpha-aminoadipic semialdehyde dehydrogenase
0.097

123
Cltb
Isoform Brain of Clathrin light chain B
0.097

124
Phyhipl
Isoform 1 of phytanoyl-CoA hydroxylase-interacting protein-like
0.098

125
Synpo
Isoform 1 of synaptopodin
0.099

Table 7: Cerebral proteins with decreased expression in the chronic postischemia pain (CPIP) group.

Number
Symbol
Description
value

1
Vwa1
Von Willebrand factor A domain-containing protein 1
0.002

2
Ppp1r10
Serine/threonine-protein phosphatase 1 regulatory subunit 10
0.003

3
Poldip2
DNA-directed polymerase delta interacting protein 2
0.003

4
Slc4a8
Isoform 2 of electroneutral sodium bicarbonate exchanger 1
0.003

5
Daam2
Dishevelled associated activator of morphogenesis 2
0.003

6
Cep350
Centrosome-associated protein 350
0.003

7
Tra2b
Transformer-2 protein homolog beta
0.007

8
Epb4.1l1
Isoform L of band 4.1-like protein 1
0.007

9
Trim32
Tripartite motif protein 32
0.007

10
Slc1a1
Excitatory amino acid transporter 3
0.007

11
Spn
Sialophorin
0.007

12
Crbn
Cereblon
0.007

13
Thoc1
Da2-19 THO complex subunit 1
0.007

14
Lmo7
Lim domain only protein 7
0.007

15
Rps27a
Ribosomal protein S27a
0.007

16
Sema4d
Sema domain, immunoglobulin domain (Ig), transmembrane domain
0.043

17
Sec3l1
SEC3-like 1
0.047

18
Ikbkap
Elongator complex protein 1
0.058

19
Peci
Peroxisomal 3,2-trans-enoyl-CoA isomerase
0.058

20
Penk
Proenkephalin-A
0.058

21
Bles03
basophilic leukemia expressed protein
0.058

22
Spna2
Alpha II spectrin
0.065

23
Pde10a
Isoform 3 of cAMP and cAMP-inhibited cGMP 3′,5′-cyclic phosphodiesterase 10A
0.065

24
Snx2
Sorting nexin 2
0.067

25
Slc25a3
Phosphate carrier protein, mitochondrial
0.068

26
Serpini1
Neuroserpin
0.070

27
Acaa1b
Acetyl-CoA acyltransferase 1b
0.077

28
H2afz
Histone H2A.Z
0.079

29
Cox6a1
Cytochrome c oxidase subunit 6A1, mitochondrial
0.090

30
Acat2
Acetyl-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 [25–28]. Moreover, CaMKII is thought to be important in central sensitization [29–31] 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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