Biomarkers mapping of neuropathic pain in a nerve chronic constriction injury mice model

Biochimie 158 (2019) 172e179

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Research paper

Biomarkers mapping of neuropathic pain in a nerve chronic constriction injury mice model S. Vincenzetti a, *, S. Pucciarelli a, Y. Huang a, M. Ricciutelli c, C. Lambertucci b, R. Volpini b, G. Scuppa b, L. Soverchia b, M. Ubaldi b, V. Polzonetti a a b c

School of Biosciences and Veterinary Medicine, Italy School of Pharmacy, University of Camerino, Italy HPLC-MS Laboratory, University of Camerino, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 September 2018 Accepted 8 January 2019 Available online 11 January 2019

Neuropathic pain is caused by a lesion or disease of the somatosensory nervous system and has a considerable impact on the quality of life. Neuropathic pain has a dynamic and complex aetiology and gives heterogeneous symptoms across patients; therefore, it represents an important clinical challenge. Current pharmacological treatment includes tricyclic antidepressant serotonin-noradrenaline uptake inhibitors such as duloxetine, pregabalin, and gabapentin. However, these drugs do not show efficacy in all patients suffering from neuropathic pain. In this work we used a nerve chronic constriction injury mice model based on the ligation of sciatic nerve to analyse, by two-dimensional electrophoresis and mass spectrometry, blood proteins significantly altered by neuropathic pain one-week after surgery. A sham-ligated group of mice acting as control and a group of ligated mice treated with gabapentin were also analysed. The results indicated that four haptoglobin isoforms were significantly more expressed, while transthyretin and alpha-2-macroglobulin expression decreased in the serum of the murine neuropathic pain model with respect to the control mice. Interestingly, the treatment with the gabapentin reversed these conditions. The outcomes of this study can provide a further understanding of the pathophysiological meaning of the biomarkers involved in neuropathic pain. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Keywords: Neuropathic pain Biomarkers Bidimensional electrophoresis Mass spectrometry Haptoglobin

1. Introduction Neuropathic pain (NP) is a continuous pain caused by a lesion or disease of the somatosensory nervous system; it is persistent and refractory to analgesics. General population studies found that 7e8% of adults are afflicted by this pathology [1,2]. NP can be divided into peripheral and central neuropathic pain, depending on the location of the anatomical lesions [3]. Common causes of NP include postherpetic neuralgia (caused by reactivation of latent Herpes varicella-zoster virus), diabetic polyneuropathy, nerve compression (e.g. trigeminal neuralgia), or cutting of nerves (e.g. accidents, amputations, post-surgical procedure). Chronic pain in cancer can be directly due to the tumour itself which invades and destroys bodily structures or can be due to the

* Corresponding author. School of Bioscience and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032, Camerino, MC, Italy. E-mail address: [email protected] (S. Vincenzetti).

cancer treatments (post-chirurgical or post-chemotherapy) or to comorbid diseases (i.e. diabetic neuropathy). About 40% of cancer patients are affected by neuropathic pain but the percentage of cancer patients affected by peripheral neuropathy increases if they are receiving neurotoxic chemotherapy. Usually, the neuropathy induced by a chemotherapy treatment is dose-dependent with cumulative side-effects [4,5]. The resulting sensory symptoms of neuropathic pain are very heterogeneous across patients: pain in an area with partial or complete sensory loss, different types of evoked pain, burning pain, increased pain after repetitive stimulation, and pain persisting after a stimulation [6e9]. Spontaneous pain such as paraesthesia and dysesthesia, paroxysmal pain, and ongoing superficial pain also occur, as well as stimulus-evoked sensation such as allodynia or hyperalgesia. NP involves a complex interaction of mechanisms in the peripheral and central nervous system that comprises a peripheral sensitization of the nociceptors that, together with an abnormal excitability of nociceptive afferent fibres, leads to

https://doi.org/10.1016/j.biochi.2019.01.005 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

S. Vincenzetti et al. / Biochimie 158 (2019) 172e179

enhanced afferent input at the spinal dorsal horn. The synaptic transmission at spinal level is increased by pronociceptive activation and by decreased inhibitory influences. Finally, an alteration of the central processing of somatosensory input occurs by cortical reorganization phenomena [10]. It is known that a central role in neuropathic pain is played by inflammatory and immune mechanisms in both the periphery and the central nervous system. In fact, it has been shown that in response to nerve damage, there is an infiltration of inflammatory cells as well as an activation of resident immune cells which results in the production and in the secretion of different inflammatory mediators which promote neuroimmune activation, sensitizes primary afferent neurons and contribute to pain hypersensitivity [11]. The inflammatory cells implicated in this process are neutrophils, mast cells, T lymphocytes, and macrophages as well as immune-like glial cells such as microglia and astrocytes [12]. Because of the dynamic aetiology, the complex type of neuropathy, as well as the heterogeneous symptoms across patients, NP has been considered as a complex chronic pain syndrome representing a clinical challenge. The first line treatment of neuropathic pain is represented by tricyclic antidepressants, serotoninenoradrenaline uptake inhibitors such as duloxetine, pregabalin, and gabapentin. Carbamazepine, lidocaine patch and capsaicin patch also showed some efficacy. Other drugs in current clinical use but with variable efficacy include tramadol, cannabinoids, lamotrigine, lacosamide, tapentadol [13]. There is great demand for effective neuropathic pain therapy from patients, and obstacles to the development of new efficacious treatments will be lowered only after relationships between the aetiology, mechanisms, and symptoms of neuropathic pain will be completely understood [14]. In this work, a surgical mice model with a sciatic nerve chronic constriction injury (CCI) was used as an NP model. CCI model is one of the most commonly used animal models of NP, it was developed by Bennett and Xie [15] and consists of the three (or four) loose ligation of the sciatic nerve at mid-thigh level with chromic gut (or silk) sutures. CCI model simulates the symptoms of chronic nerve compression in clinical conditions of trauma or tumour developments. This model also mimics a lesion of nerve fibres located mainly at the surface of the peripheral nerve [16]. This constriction of the sciatic nerve is associated with intraneural oedema, focal ischemia, and Wallerian degeneration [17e19]. The proteomic approach could be a very powerful tool for NP research since would help to screen and understand the global changes in protein expression level, that might lead to the identification of pain associated proteins in the serum [20]. In fact, NP is caused often by inflammation and/or nerve injury which can produce changes in the expression of cytokines, neurotransmitters, and structural proteins. Some studies performed using animal models of pain revealed also the involvement of several members of the chemokine family in neurodegeneration, neuroinflammation and neuropathic pain. In particular, it has been shown that during chronic pain state the serum levels of pro-inflammatory cytokines such as IL-1b, IL-6, IL-2, IL-33, and TNF-a, were significantly up-regulated whereas anti-inflammatory cytokines such as IL-10 and IL-4 were found to be down-regulated [21]. Another study has shown that (CCI)induced rats (neuropathic pain induction) have increased serum levels of CCL3 and its receptor CCR5. CCL3 is a chemotactic cytokine crucial for inflammatory cell recruitment in homeostatic and pathological conditions [22]. Binding of chemokine CCL3 to its receptor CCR5 in response to nerve injury activate phosphorylation of p38MAPK and the subsequent production of proinflammatory cytokines, which mediate neurodegeneration (neuronal and glial cell

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injury and death) and induce pain hypersensitivity [22]. The aim of the present work was the identification of significantly altered proteins in the serum of mice subjected to sciatic nerve ligation one-week after surgery with respect to a control serum by a proteomic approach based on two-dimensional electrophoresis, in order to find possible NP biomarkers. In addition, gabapentin, a drug that proved to be effective against NP, has been administered to the nerve-ligated mice in order to assess if an eventual variation of the serum proteins expression pattern could give an indication on the drug efficacy. 2. Materials and methods 2.1. Animals C57BL/6 male mice weighting around 25e30 g at the beginning of the experiment were used. Animals were housed under a normal day/night cycle, in rooms with constant temperature (20e22
C) and humidity (45e55%) and given ad libitum access to food and water throughout the experiments. In these experiments, a total number of 18 mice were used, separated into three different groups, each consisting of 6 mice. The first group of mice was subjected to the Chronic Constriction Injury procedure (CCI-operated) (n ¼ 6); a second group of mice was sham-operated (control), (n ¼ 6); the third group consisted of CCIoperated mice treated with 50 mg/kg/10 mL of gabapentin (Teva Pharma, Assago, Italy) (CCI-GABA) (n ¼ 6). The dose of gabapentin was chosen based on data from the literature showing an efficacy of gabapentin in the range 10e100 mg/kg in different animal models of inflammation [23]. Gabapentin was dissolved in water and administered orally, twice a day (at 9 a.m. and 6 p.m.) for 7 consecutive days. 2.2. Chronic constriction injury (CCI) of the sciatic nerve The Chronic Constriction Injury of the sciatic nerve was performed as described in Bennett and Xie [15]. Briefly, mice were anesthetized by inhalation of a mixture of isoflurane and oxygen. The anaesthetic was delivered by a facemask connected to a gas anaesthesia machine in turn connected to the oxygen source and equipped with a precision vaporizer. Isoflurane was used in a concentration of 4e5% for anaesthesia induction and of 1e2% for maintenance. The mice were constantly monitored to avoid excess cardiac or respiratory depression and insufficient anaesthesia. To perform the CCI, the right sciatic nerve of the mice was exposed at the level of the mid-thigh. In the CCI-operated mice (ligated mice), three ligatures were loosely tied around the nerve, using a nonabsorbable surgical suture thread, whereas in sham-operated controls, the sciatic nerve was exposed but not ligated. As reported in literature three ligatures are sufficient to induce NP in smaller rodents such as mice [24]. The pharmacological treatment started the day after the surgery. 2.3. Assessment of mechanical hypersensitivity after CCI Changes in nociceptive thresholds after the CCI surgery were evaluated using an Electronic von Frey device (Ugo Basile, Gemonio (VA), Italy) at 7 days post-injury. Immediately after the morning drug or vehicle administration, mice were placed in individual plastic boxes with a metal mesh floor and allowed to acclimatize for 30 min. After this period the animals were quiet and did not show exploratory movements thus the test began. Gradually increased strength was applied to filaments through the mesh floor perpendicularly to the plantar surface of the hind

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paw. The intensity of mechanical stimuli was increased (range from 0.1 to 5.0 g) until the hind paw was withdrawn. Threshold pressure values were recorded. Both ipsilateral (paw of the same side of the operated leg) and contralateral (paw opposite to the operated leg) threshold were recorded. The nociceptive threshold for each paw was obtained in triplicate and the mean of the three measurements was calculated. 2.4. Blood samples collection One week after surgery and after the behavioural Von Frey test, animals were subjected to an intracardiac puncture for blood collection. Briefly, mice were deeply anesthetized with isoflurane and 1 mL of blood for each mouse was collected directly from the heart, using a 1 mL syringe with a 25-gauge needle. After collection, blood samples were stored at room temperature for 45 min in order to allow blood to clot. Samples were centrifuged at 3000g for 10 min and the supernatant containing the serum was stored at 80
C until use. Protein determination on the serum samples was performed with the Bradford method [25]. 2.5. Two-dimensional electrophoresis (2-DE) In this proteome analysis, the experimental design was based on the complete sample pooling strategy where all samples from one treatment group are pooled and any replicates are technical replicates of this pooled sample [26,27]. Sample pooling minimizes individual variation and therefore reduces variability, furthermore it represents an alternative approach to biological replicates in those experiments when the interest is the characteristic of the population (common changes in expression patterns) [26,27]. For total protein extraction, 25 mL of each serum sample, obtained from each six CCI-operated mice, were pooled together and 1 mg of total proteins from the pool was treated for 2-DE. The same procedure was performed for serum samples from control mice (sham-operated) and serum samples from CCI-GABA mice. To ensure statistical significance for quantitative analyses, three technical replicates were performed for each group (CCI-operated, sham-operated and CCI-GABA). Before 2-DE 1 mg of total protein was first cleaned up with a 2-D Clean-Up Kit (GE-Healthcare Life Sciences, Uppsala, Sweden), according to the manufacturer's instructions and then dissolved in a 350 mL rehydration solution containing: 8 M urea; 2% (w/v) 3-[(3Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS); 65 mM dithiothreitol (DTT); 0.001% (w/v) bromophenol blue; 0.5% (v/v) IPG buffer, pH range 3e10. The protein sample, resuspended in the rehydration solution, was then loaded on the Isoelectric focusing (IEF) which represent the first dimension. IEF was performed as previously described [28,29] using a precast immobilized pH gradient gel strip Immobiline DryStrip, (IPGstrip, length 18 cm) with a linear pH gradient range of 3e10. The IPG-strips were placed on the IPGphor isoelectric focusing cell (GEHealthcare) and were rehydrated for 12 h at 20
C without voltage. The focusing was then performed at 20
C in 3 steps: first step, voltage of 500 V for one hour; second step, voltage of 1000 V for one hour; third step, voltage of 8000 V for 4 h. The current limit per IPGstrip was 50 mA. After IEF, IPG strips were equilibrated for 15 min in the equilibration buffer (50 mM Tris-HCl, pH 8.8; 6M urea; 30% glycerol; 2% SDS; 65 mM DTT and a few grains of bromophenol blue) and loaded on a 3% SDS-PAGE, using a Protean II apparatus (Bio-Rad, Hercules, CA, USA), performed as previously described [28,29]. The gels (180  200  1.5 mm) were run at 30 mA per gel for 6e7 h. Proteins were stained for 1 h with 0.1% Coomassie Brilliant Blue R250 in 50% methanol and 10% acetic acid. The gels were de-stained (50% methanol; 10% acetic acid) until the protein spots

became evident and the gel background transparent. 2.6. Protein visualization and image analysis After de-staining the gels were scanned at 600 dpi resolution, and the gel images were analysed using PDQuest software (Version 7.1.1; Bio-Rad, Hercules, CA, USA) according to the protocols provided by the manufacturer in order to define spot-intensity calibration, spot detection, calculation of molecular mass and isoelectric point (pI). The gels were cropped to frame the same cluster of spots and subsequently a set of spot generation conditions such as faintest spot, smallest spot, size of the largest spot, and a selected region of the background, were used. The quantity of each spot was normalized as a percentage of the total quantity of all spots in the gel and evaluated in terms of OD. After the generation of a master gel, based on a representative gel for each type of sample, the spots were detected and matched automatically by the software. The spot detection and matching were also edited manually. Finally, a spot quantity table containing all matched spots was generated. The isoelectric point (pI) for each spot was determined using a linear 3e10 distribution, and molecular mass determinations were based on the markers Bio-Rad low range (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin 45.0 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa). 2.7. In-gel protein digestion In-gel digestion of proteins was carried out according to the protocol of Shevchenko and co-workers [30]. Briefly, each spot was excised from the gel and cut into small pieces (ca. 1  1 mm) and treated with trypsin as described previously [29]. The combined extracts were recovered and dried in a vacuum concentrator at room temperature. A piece of blank gel, without spots, and a piece of lysozyme from the molecular mass markers were submitted to the same procedure, and used as negative and positive controls, respectively. The samples were then subjected to LC-MS/MS analysis. 2.8. LC-MS/MS analysis After the digestion, the tryptic peptides were dissolved in 100 mL of 0.1% (v/v) trifluoroacetic acid and subjected to a reversed phase chromatography (C18 Gemini-NX, ml particle size, 110 Å pore size, 250  4.6 mm, Phenomenex, Torrance, CA.) connected to a HPLC Agilent Technologies 1100 Series (Agilent Technologies, Santa Clara, CA.). The column effluent was analysed by MS using an electrospray ion trap mass spectrometer (Agilent Technologies LC/MSD Trap SL) operating in positive ion mode over the mass range 300e2200 amu (atomic mass units). MS spray voltage was 3.5 kV and the capillary temperature was maintained at 300
C. Obtained spectra were extracted and analysed by the MASCOT software (www. matrixscience.com) with the following search parameters: database, NCBInr taxonomy: Mus musculus; enzyme, trypsin; peptide tolerance, 1.2 Da; MS/MS tolerance, 0.6 Da and allowance of one missed cleavage. 2.9. Statistical analysis All experiments were performed in triplicate and all the electrophoretic patterns resulted similarly. Quantitative data of protein level in serum presented as means ± SE. The significance of differences was evaluated by one-way analysis of variance (ANOVA) and Tukey's test multiple comparison analysis was performed by GraphPad Prism6 software. Behavioural data were analyzed using a

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2-way ANOVA and Newmann-Keuls was used as post-hoc. Significance was set at P < 0.05. 3. Results 3.1. Assessment of mechanical hypersensitivity after CCI To evaluate the presence of mechanical hypersensitivity the hind paw withdrawal threshold was measured 7 days post-injury was tested with a 2-ways ANOVA with the gabapentin treatment as between factor and paw tested (ipsilateral vs contralateral) as within factor. The statistical analysis revealed an effect of treatment [F (2,15) ¼ 15.05, p < 0.001], an effect of paw tested [F (1,15) ¼ 150.61, p < 0.001] and of the paw tested  treatment interaction [F (2,15) ¼ 27.01, p < 0.001]. In particular, post hoc analysis revealed a statistical difference between the CCI-operated group and the CCI-GABA group (p < 0.001) and between the CCIoperated and sham-operated groups (p < 0.001). These results show that nerve-ligated animals were strongly allodynic compared to the sham-operated group and that gabapentin exerts an antinociceptive effect on CCI-operated group at the dose used (50 mg/ kg) (Fig. 1). Importantly, repeated administration of gabapentin at doses of 100 mg/kg, or even higher, over 6 days did not lead to the induction of tolerance to its anti-hyperalgesic action [31]. 3.2. Two-dimensional electrophoresis (2-DE) The protein expression profile samples obtained from the sera of CCI-operated mice and CCI-operated mice treated with gabapentin, a drug that proved to be effective against NP (CCI-GABA) were analysed by 2-DE in pH range 3e10 and compared with those of the sham-operated mice (controls). Fig. 2, shows a representation of the comparison of protein spots between these three groups. Image analysis, performed by the PDQuest software, revealed that the spots A, J, K, L, M, O, and Q were differentially expressed in the CCIoperated mice with respect to the control group and the CCI-GABA mice (Figs. 2 and 3). For this reason, we focused our attention only on these spots differentially expressed between the abovementioned mice group. Therefore, the spots A, J, K, L, M, O, and Q were successfully identified with significant scores using LC-MS/

Fig. 1. Paw withdrawal threshold in response to von Frey filaments after 7 days of surgery in saline-treated sham group, saline-treated ligated group and gabapentintreated ligated group. ***P < 0.001. Contralateral paw threshold is showed as control for each group.

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MS in combination with a database search by MASCOT software. The experimental pI values and the molecular weights of the proteins identified are shown in Table 1 and were compared with the theoretical values found by the MASCOT software. In particular, spot A was identified as haptoglobin, isoform 2; spots J, K, L, and M were also identified as haptoglobin (four isoforms which differ for the isoelectric point, see Fig. 2 and Table 1), spot O was identified as transthyretin (TTR) and spot Q as alpha-2-macroglobulin (a2M), 35 kDa subunit. Fig. 3 shows the normalized quantity (which is related to the protein expression) of the protein spots in the three serum samples (sham-operated, CCI-operated and CCI-GABA mice). 4. Discussion In the present work, significantly altered proteins in the blood of CCI-operated mice (a surgical mice model with a sciatic nerve chronic constriction injury) with respect to the control mice were identified. In addition, gabapentin was tested in the nerve-ligated mice (CCI-GABA) in order to test any variation of protein pattern which could give an indication of the drug efficacy. Spot A (Fig. 3A) is significantly more expressed in the serum of the CCI-operated mice with respect to control serum (sham-operated mice) (CCI-operated vs Sham-operated; P < 0.001). Interestingly, the protein expression decreased considerably in the serum of CCI-GABA mice (CCI-GABA vs CCI-operated p < 0.01). Spot A has been identified as haptoglobin, isoform 2, its experimental molecular weight is 15.4 kDa (Table 1) and could correspond to the a2chain of human haptoglobin which shows a molecular weight of 16.5 kDa [32]. Human haptoglobin molecular structure has been well elucidated, and therefore can be used as a model to understand the structure of this protein from other species. It is known from literature that human haptoglobin consists of three phenotypes namely HP1-1, HP2-1, and HP2-2 and that all of them share a bchain of 40 kDa. When a b-chain crosslink with an a1-chain (9 kDa), they form an a1b unit. The phenotype HP1-1 corresponds to the a1b dimer. The haptoglobin a2-chain contains the same residues of the a1-chain plus a redundant copy of the residues 17e70 resulting in 142 total amino acids (16.5 kDa). The phenotypes HP2-1 and HP2-2 are respectively linear polymers formed by the a1b and a2b units and cyclic complexes formed by a2b units [32]. Even Bellei and co-workers [33] reported the presence of an increased protein band corresponding to haptoglobin in the SDSPAGE protein profile of ligated mice with respect to controls. In the present work other four isoforms of haptoglobin (spots J, K, L, M; Fig. 3B) were found significantly more expressed in the serum of CCI-operated mice if compared with those of the control mice (CCIoperated vs sham-operated; P < 0.01). Notably, in the serum of the CCI-GABA mice, the expression level of these haptoglobin isoforms decreased significantly with respect to the serum of CCI-operated mice (CCI-GABA vs CCI-operated mice, P < 0.01) reaching almost the level of expression detected in the control mice serum (CCI-GABA vs sham-operated, not significant). Furthermore, the four haptoglobin isoforms (J, K, L, M) showed a molecular weight of 38 kDa, similar to the human haptoglobin bchain [32]. The observed isoelectric point heterogeneity of the four isoforms may be due to post-translational modification (PTM) of this protein. In human haptoglobin, several different modification sites for phosphorylation and glycosylation have been demonstrated by 2-DE, giving rise to seven major isoforms with pI ranging from 4.78 to 5.44 [34]. In addition, it seems that there is a link between human haptoglobin polymorphism and some pathological conditions, which may reflect functional differences among the phenotypes [35]. Five isoforms of haptoglobin were found

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Fig. 2. Representative map of the protein expression pattern of the plasma from Sham-operated (control), CCI-operated and CCI-GABA mice. Three independent 2-DE experiments were performed for the three samples. The selected regions show the spots differentially expressed. Proteins were first separated on an immobilized pH 3e10 linear gradient strip followed by separation with 13% SDS-PAGE. Gels were stained with Coomassie blue. The standards were Bio-Rad low molecular weight (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin 45.0 kDa; carbonic anhydrase, 31.0 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa).

Fig. 3. Quantitative analysis of haptoglobin (A), haptoglobin isoforms (B), Transthyretin (C), Alpha-2-macroglobulin (D), in the serum of Sham-operated (controls), CCI-operated, and CCI-GABA mice. Data are shown as mean values ± SD. ***P < 0.001; **P < 0.01; *P < 0.05. The analyses were performed by using the PDQuest software, as described under the Materials and Methods section.

Table 1 Identification of differentially expressed protein in CCI-operated and sham-operated groups. Spot IDa

Protein nameb

Scorec

Species

Mr. (kDa)/pId

Mr. (kDa)/pIe (mean ± s.d.)

Sequencesd

A J K L M O Q

Haptoglobin, isoform 2 Haptoglobin Haptoglobin Haptoglobin Haptoglobin Transthyretin Alpha-2-macroglobulin

24 55 183 60 78 73 49

Mus Mus Mus Mus Mus Mus Mus

13.1/5.12 38.7/5.89 38.7/5.90 38.7/5.91 38.7/5.92 15.8/5.77 28.5/7.03

15.4 ± 1.06/4.9 ± 0.15 42.9 ± 0.84/4.7 ± 0.06 42.5 ± 0.90/4.9 ± 0.06 41.9 ± 0.77/5.2 ± 0.11 41.7 ± 0.56/5.5 ± 0.17 15.5 ± 1.12/7.6 ± 0.58 39.2 ± 1.11/7.1 ± 0.42

AEGDGVYTLNDEK IIGGSMDAKGSFPWQAK DITPTLTLYVGKNQLVEIEKV IIGGSMDAKGSFPWQAK DITPTLTLYVGKNQLVEIEK KVFKKTSEGSWEPFASGKTA KEVLVTSRSSGTFSKT

a b c d e

musculus musculus musculus musculus musculus musculus musculus

Assigned spot ID as indicated in Fig. 2. MASCOT results (SwissProt & NCBInr databases). MASCOT score reported. From SwissProt & NCBInr databases. Experimental values were calculated from the 2-DE maps by the PDQuest software.

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significantly upregulated in the cerebrospinal fluid of patients suffering from chronic neuropathic pain due to trauma or surgery with respect to the healthy controls [36,37]. Ding et al. [38], by two-dimensional electrophoresis followed by western blotting analysis, evidenced in mice plasma the presence of four spots corresponding to the alpha subunits of haptoglobin (14 kDa). In addition, these authors found that the isoforms 2 and 3 of haptoglobin increased during mice aging, indicating a state of increased inflammation in older ages. In mice serum only three locations for PTM have been identified experimentally [39], but more post-translational modification sites of mouse haptoglobin may be predicted which could explain the heterogeneity of haptoglobin observed in this proteomic study, in fact, it is possible to predict five ε-amino groups of lysine which could be glycated in mouse haptoglobin, as shown in Fig. 4 (NetGlycate 1.0 [40]). Haptoglobin is involved in various biological processes which include: acute-phase response, defence response, immune system process, negative regulation of hydrogen peroxide catabolic process, negative regulation of oxidoreductase activity, positive regulation of cell death, receptor-mediated endocytosis, response to hydrogen peroxide. As an acute phase protein that scavenges haemoglobin in the event of intravascular or extravascular haemolysis, haptoglobin increases during inflammation, and this has been demonstrated in different pathological conditions such as parasitic, infectious and non-infectious diseases (diabetes, cardiovascular disease, and obesity) [41]. Furthermore, it seems that haptoglobin is involved in neurodegenerative diseases, such as Huntington's disease [42], whereas other studies demonstrated that a haptoglobin precursor might be a candidate biomarker for Alzheimer's disease [43]. Finally, there are evidences that in response to ischemic insult, haptoglobin can be synthesized by the astrocytes in an “in vivo” model of brain ischemia [44]. The mechanism of neuropathic pain involves the immune system activity during peripherals and central sensitization. After neuron injury, the activated macrophages, either resident or recruited from the blood by chemotactic cytokines, contribute to pain states by releasing many inflammatory mediators, such as cytokines (TNFa) and interleukin-1b, nerve growth factor, nitric oxide, and prostanoids [45]. Haptoglobin is expressed during neuron damage so that the macrophage could be recruited to the damaged region and undergo antioxidant and anti-inflammatory effects. On the other hand, the release of pro-inflammatory

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cytokine by macrophage could be a reason for neuroninflammation and long-term neuropathic pain. Spot O corresponds to transthyretin TTR (Fig. 3C), which is a protein synthesized and released into the blood circulation by the liver, the choroid plexus of the brain, and the endocrine pancreas. It is principally involved in the blood transport of thyroid hormones due to its ability to bind thyroxin (T4), triiodothyronine (T3) and, in association with retinol-binding protein, it can bind to retinol (vitamin A) [46,47]. It has been reported that 1e2% of blood transthyretin circulates bound to high-density lipoprotein (HDL) and that the association to the HDL vesicle occurs through the binding to apolipoprotein A1 [48]. TTR is a tetrameric protein of 14kDa subunits with a concentration in human serum of 160e380 mg/L. Only a small amount of TTR in the monomeric form is present in vivo in normal individuals [49]. There is also an evidence showing that transthyretin is associated with neuropathic amyloidosis which is a clinical disorder caused by extracellular deposition of insoluble abnormal protein fibrils derived by aggregation of misfolded, normally soluble, precursor [50,51]. Other authors revealed that TTR is involved in nerve fibre regeneration, axonal growth enhancement during peripheral nervous system regeneration, and neuroprotection in Alzheimer's disease [52,53]. It is well known from the literature that transthyretin serum concentration decreases under inflammation condition [54]. This correlates with the results obtained in this study, since the expression of TTR decreased, though not significantly, in the serum of the CCIoperated mice with respect to the controls. Interestingly, in the serum of CCI-GABA mice, the level of transthyretin increased significantly with respect to the CCI-operated mice (P < 0.01) (Fig. 3C) and this may be related to the regeneration process that could be induced by the gabapentin. To support this hypothesis there is the fact that some authors found increased levels of TTR in the serum of ligated rats 5 weeks post-surgery which was related to nerve regeneration processes [33]. In addition, other authors suggested a positive role of gabapentin on the nerve morphology after a chronic constriction of the sciatic nerve by the modulation of the nerve myelin basic protein that stabilizes the myelin structure by scaffolding the lipid components [55]. Alpha-2-macroglobulin (a2M), concentration showed a slight and not significant decrease in the serum of CCI-operated mice if compared to the control mice, while it increased significantly (P < 0.05) in the serum of CCI-GABA mice if compared to the CCI-

Fig. 4. Prediction of epsilon amino groups of lysines glycation in mouse haptoglobin (Accession: AAI38873) (NetGlycate 1.0 [25]).

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operated mice serum (spot Q; Fig. 3D). Differently to human a2M which is composed of a 185 kDa subunit, the mouse a2M consists of two subunits of 165.0 kDa and 35.0 kDa and another subunit of 185.0 kDa. The 165.0 and 35.0 kDa subunits are present in an equimolar amount and derive from proteolytic processes involving the 185.0 kDa subunit. Despite this difference in the subunit structure, the murine protein behaves identically to human a2M as regard to its function. a2M is a plasma proteinase inhibitor acting against several classes of proteinase such as serine-, cysteine-, aspartic-, and metalloproteinases and is able to bind several growth factors and cytokines, regulating their activity thanks to a proteininteraction site, identified in the protein structure [56]. Furthermore, a2M, through the binding to its receptor, the low-density lipoprotein receptor-related protein (LRP-1) and to the 78.0 kDa glucose-regulated protein (GRP78) is involved in cell signalling and therefore may have a role in innate immunity, in the control of inflammation, and may regulate cell physiology [57e59]. a2M undergoes conformational changes, induced by proteases or small primary amines, that control the functionality of the protein. In plasma, it is mainly present a2M in its native conformation, since once completely transformed, the protein is cleared after LRP-1 binding [60]. Arandjelovic et al. [56] found that a derivative of a2M, activated for cytokine binding (MAC) is able to regulate the response to peripheral nerve injury. The cytokines that bind to MAC are TNF-a, IL-6, and IL-18. In particular, these authors showed that MAC inhibits local TNF-a expression, decreases axonal degeneration and limits inflammation in mice with sciatic nerve crush injury or CCI. Some changes observed in the injured nerves are promoted by TNF-a, therefore MAC can counteract these changes [61]. In this work some proteins related to the inflammation processes were found significantly altered in the serum of CCIoperated mice, one-week after surgery, with respect to the control mice. Notably, we did not observe alterations in the expression levels of proteins related to nerve regeneration processes such as apolipoproteins A1 and apolipoprotein E, involved in the remyelination processes, vitamin D-binding protein, prostaglandin-D H2isomerase, which have been found by other authors in the serum of rat five weeks after ligation [33,62]. This can be due either to the short time interval from sciatic nerve ligation, that can affect the nerve degeneration/regeneration processes [33] or to the fact that their amount in mice serum is below the limit of detection of our method. 5. Conclusions In this work, the serum of a surgical mice model with a sciatic nerve chronic constriction injury (CCI-operated), used as an NP model, was analysed by a proteomic approach in order to identify significantly altered proteins compared to a control mice serum (sham-operated) one-week after surgery. The results obtained were also compared to CCI-operated mice treated with gabapentin (CCI-GABA), a drug used to treat neuropathic pain. The proteomic studies performed on the serum samples correlate with the behavioural data, in fact the nerve-ligated animals (CCI-operated), which resulted strongly allodynic, showed in the serum the presence of four overexpressed haptoglobin isoforms and a decreased concentration of TTR and a2M with respect to the sham-operated group (controls). The treatment of the ligated mice with gabapentin (CCI-GABA mice) exerted an antinociceptive effect at the administered dose; it reversed the expression levels of the abovementioned proteins which returned to the values observed in the control mice serum. From the results obtained, it seems interesting the different behaviour of TTR in the serum of the three analysed mice group, and in this regard it could be hypothesized that the observed decrease of this protein in the serum of the nerve-ligated

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