1. Introduction {#sec1}
===============

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 \[[@B1]\]. Although the exact pathophysiology of CRPS is not fully understood, several pathological mechanisms, including oxidative stress \[[@B2]\], neurogenic inflammation \[[@B3]\], and alteration in the autonomic nervous system \[[@B4], [@B5]\], 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 \[[@B6]\]. The patients get worse when they think about moving the body part, even if they do not move it \[[@B7]\]. 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 \[[@B8]\]. Thus, the distorted body representation of CRPS patients can be treated with mirror therapy \[[@B9], [@B10]\]. 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 \[[@B11]\]. 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 \[[@B12]--[@B14]\], and recent research suggests that changes in cortical structures can contribute to the pathophysiology of CRPS \[[@B15]\].

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 \[[@B16]\]. 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 \[[@B17]\].

2. Materials and Methods {#sec2}
========================

2.1. Animals {#sec2.1}
------------

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 {#sec2.2}
--------------------------

The CPIP animal model was generated according to previous methods \[[@B17]\]. 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](#fig1){ref-type="fig"}). 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 {#sec2.3}
---------------------

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 \[[@B18]\]. 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: $$\begin{matrix}
{\text{CMT}\left( \% \right) = \frac{M_{\text{post}} - M_{\text{pre}}}{M_{\text{pre}}} \times 100.} \\
\end{matrix}$$ 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 {#sec2.4}
-----------------------

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

### 2.4.1. Protein Extraction {#sec2.4.1}

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 CaCl~2~.

### 2.4.2. MudPIT {#sec2.4.2}

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 (*m*/*z* 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 *μ*m 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 *μ*m C18 reverse-phase resin, which was connected to an internal diameter of 250 *μ*m fused-silica trapping column packed with 2 cm of SCX followed by 2 cm of C18 resin. Each 30 *μ*g 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 {#sec2.4.3}

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 \[[@B19]\] 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 \[[@B20]\].

3. Results {#sec3}
==========

3.1. Behavioral Tests {#sec3.1}
---------------------

A total of 14 animals (*n* = 7 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 (*P* \< 0.01, [Figure 2](#fig2){ref-type="fig"}). 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 {#sec3.2}
--------------------------------------------------------

A total of 454 proteins were differentially expressed between Groups A and C under the criterion of *P* 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](#tab6){ref-type="table"}) and 30 decreased ([Table 7](#tab7){ref-type="table"}). Specifically, expression of proteins related to cell signaling ([Table 1](#tab1){ref-type="table"}), synaptic plasticity ([Table 2](#tab2){ref-type="table"}), regulation of cell proliferation ([Table 3](#tab3){ref-type="table"}), and cytoskeletal formation ([Table 4](#tab4){ref-type="table"}) 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 Ca^2+^ channel gamma-2 subunit, and voltage-dependent Ca^2+^ 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](#tab5){ref-type="table"}) 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.

4. Discussion {#sec4}
=============

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) \[[@B21]\]. And it is known that PI3K is an important mediator of central sensitization in painful inflammatory condition \[[@B22]\], and many tumorous conditions are related to this enzyme \[[@B23], [@B24]\]. 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 \[[@B25]--[@B28]\]. Moreover, CaMKII is thought to be important in central sensitization \[[@B29]--[@B31]\] and is implicated in central neuropathic pain \[[@B31]\] and long term potentiation (LTP) \[[@B32]\]. LTP is initiated when NMDA receptors allow Ca^2+^ into the postsynaptic neuron, and this Ca^2+^ influx activates CaMKII. LTP in nociceptive spinal pathways shares several features with hyperalgesia, and LTP at synapses constitutes a contemporary cellular model for pain \[[@B33], [@B34]\]. And it was reported that the overexpression of CaMKII was observed in the dorsal root ganglia of rat model of type 1 diabetes \[[@B35]\], and the inhibition of CaMKII can reverse the chronic inflammatory pain \[[@B36]\]. 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 \[[@B37]\], and anomalous cereblon expression can lead to memory and learning deficit \[[@B38]\]. The defect in cereblon gene is associated with mental retardation \[[@B39]\]. 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 \[[@B40]\] was decreased in CPIP animals in our study. Neuroserpin plays a role of neuronal protection in pathologic state, and point mutation can cause encephalopathy \[[@B41]\]. Also it has been known that deficiency in neuroserpin exacerbates ischemic brain injury \[[@B42]\]. 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 \[[@B43], [@B44]\]. 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 \[[@B17]\]. 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 {#sec5}
=============

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.

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

Conflict of Interests
=====================

The authors have no conflict of interests to declare.

![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.](BMRI2014-498410.001){#fig1}

![Change in mechanical threshold (%). Group A showed a significant decrease in mechanical threshold compared to group C (*n* = 7 in each group). Asterisk (\*) indicates *P* \< 0.05 at each time point. Group A: CPIP group and Group C: control group.](BMRI2014-498410.002){#fig2}

###### 

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

  Number   Symbol      Description                                                                      *P* 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

###### 

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

  Number   Symbol   Description                                              *P* 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

###### 

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

  Number   Symbol    Description                                                     *P* 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

###### 

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

  Number   Symbol   Description                                              *P* 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

###### 

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

  Number   Symbol    Description                                                                     *P* 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

###### 

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

  Number   Symbol      Description                                                                      *P* 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

###### 

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

  Number   Symbol     Description                                                                    *P* 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

[^1]: Academic Editor: Livio Luongo
