Annexin A2 in primary afferents contributes to neuropathic pain associated with tissue type plasminogen activator

Neuroscience 314 (2016) 189–199

ANNEXIN A2 IN PRIMARY AFFERENTS CONTRIBUTES TO NEUROPATHIC PAIN ASSOCIATED WITH TISSUE TYPE PLASMINOGEN ACTIVATORI H. YAMANAKA, * K. KOBAYASHI, M. OKUBO AND K. NOGUCHI

INTRODUCTION Neuropathic pain is a chronic pain state that is usually accompanied by nerve injury. The impact of an axonal injury includes changes in nerve functions both at the site of injury and central terminals. The damaged nerve fibers convey incorrect signals that trigger hyperexcitability of the spinal dorsal horn neurons, which is often referred to as central sensitization (Stucky et al., 2001; Ji et al., 2003; Salter, 2005; Woolf and Ma, 2007; Costigan et al., 2009). Primary afferent terminals of the injured axon are thought to facilitate synaptic transmission leading to central sensitization of dorsal horn neurons (Campbell and Meyer, 2006). Although the development of genetic and newer pharmacological techniques has explored the underlying mechanisms of central sensitization, the key mechanisms that control its induction and maintenance remain unclear. A growing body of evidence suggests that there is an important role for extracellular proteolysis in synaptic plasticity. Tissue-type plasminogen activator (tPA) is an extracellular serine protease, initially described as an enzyme that converts plasminogen into the broad-spectrum substrate serine protease plasmin which is involved in thrombolysis. Enzymatic activity of tPA regulates extracellular matrix proteins and is an important factor to induce long-term potentiation (LTP) in the hippocampus by modulating NMDA-R or synaptic morphology (Qian et al., 1993; Chen and Strickland, 1997; Baranes et al., 1998). Our previous study found that proteolytic activity of tPA was involved in neuropathic pain, and that tPA was induced in dorsal root ganglia (DRG) neurons and astrocytes by nerve injury (Yamanaka et al., 2004; Kozai et al., 2007). Annexin A2 (ANX2) is a pleiotropic calcium- and anionic phospholipid-binding protein which exists as a monomer and as a heterotetrameric complex protein with p11. ANX2 is also known as a cell-surface coreceptor for tPA and plasminogen (Kim and Hajjar, 2002). By binding to ANX2, tPA increases the catalytic efficiency by
60-fold (Hajjar and Menell, 1997). We hypothesize that injury-induced tPA and ANX2 exert painful outcomes following peripheral nerve injury. We herein investigated the expression of ANX2 in primary afferents and examined the involvement of ANX2 in pain behavior in relation to tPA using established neuropathic pain models.

Department of Anatomy and Neuroscience, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan

Abstract—Annexin A2 (ANX2) is a calcium (Ca2+)-binding protein that binds to acidic phospholipids and is known to play a crucial role in many cellular regulatory processes. In particular, ANX2 has been described as a crucial receptor for thrombolysis by the tissue-type plasminogen activator (tPA) and plasmin system. In the nervous system, tPA is involved in processes of neuronal plasticity such as hippocampal long-term potentiation (LTP) and in the dorsal horn pain in several pain models. We investigated detailed changes in expression of ANX2 after nerve injury and evaluated the interaction with tPA using the rat spared nerve injury (SNI) model. SNI-induced the expression of ANX2 in L4/5 dorsal root ganglia (DRG) neurons. In the spinal cord, constitutive ANX2-immunoreactivity was expressed in laminae I–II. Peripheral nerve injury increased the ANX2 immunoreactive terminals mainly in laminae I–V of the dorsal horn on the side ipsilateral to the nerve injury. Double-labeling analysis revealed the co-localization of ANX2 with tPA in the axons of primary afferents in the dorsal horn. Experimental inhibition of ANX2 and tPA interaction by intrathecal administration of homocysteine significantly prevented and reversed SNI-induced mechanical allodynia. Thus, alterations of ANX2 may be involved in tPA-dependent plasticity after peripheral nerve injury and have an important role in neuropathic pain. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: annexin A2, tissue type plasminogen activator (tPA), neuropathic pain, dorsal root ganglia, dorsal horn, plasticity.

q This work was supported in part by Grants-in-Aid for Scientific Research (ID 25290016), and a Pain Research Group Grant, Hyogo College of Medicine, both from the Japanese Ministry of Education, Science, and Culture. *Corresponding author. Tel: +81-0798-45-6416; fax: +81-0798-456417. E-mail address: [email protected] (H. Yamanaka). Abbreviations: ANX2, annexin A2; DRG, dorsal root ganglia; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IHC, immunohistochemistry; ir, immunoreactivity; ISH, in situ hybridization; LTP, long-term potentiation; PB, phosphate buffer; PBS, phosphatebuffered saline; SNI, spared nerve injury; tPA, tissue-type plasminogen activator.

http://dx.doi.org/10.1016/j.neuroscience.2015.11.058 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 189

190

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

EXPERIMENTAL PROCEDURES Animal treatment

Reverse transcription-polymerase chain reaction (RT-PCR)

A total of 136 Male Sprague–Dawley rats (Nihon Dobutsu, Osaka, Japan) weighing 200–250 g were used. All animal experimental procedures were approved by the Hyogo College of Medicine Committee on Animal Research and were carried out in accordance with the guidelines of the National Institutes of Health on animal care. Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the tibial and common peroneal nerves were transected, while the sural nerve was left intact (spared nerve injury; SNI model). The wounds were then closed and the rats were allowed to recover. In the sham operation, the procedures were the same except that the nerves were only exposed but not transected. At several time points (1, 3, 7 and 14 days) following the surgery, groups of rats were processed for analysis. Every effort was made to minimize animal suffering and reduce the number of animals used.

For the RT-PCR, the rats were decapitated under deep anesthesia with sodium pentobarbital (125 mg/kg body weight, i.p.) at 0, 1, 3, 7 and 14 days after surgery, and the left L4, 5 DRG were removed and rapidly frozen with powdered dry ice and stored at 80 °C until ready for use (n = 4 each time points). The procedure for extraction of total RNA using an RNA extraction reagent ISOGEN (Nippon Gene, Tokyo, Japan) was described in our previous study (Yamanaka et al., 2004). PCR primers for ANX2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA were designed as follows: ANX2 primers (accession number L13039), sense 50 -CCCCCAAGTGCCTATGGGTC-30 and antisense 50 -CTCCAGATCGGTCTTGTACATTT-30 ; GAPDH primers (accession number M17701), sense 50 -CCAGC CTTCTCCATGGTGGT-30 and antisense 50 -CCAGCCTT CTCCATGGTGGT-30 . The subsequent PCR reaction was performed using a standard method (Yamanaka et al., 2004).

Intrathecal drug administration

Histological analysis

After the SNI, the L6 vertebra was laminectomized and a soft tube (SILASTIC laboratory tubing, Dow Corning Corporation, Midland, MI; outer diameter, 0.64 mm) filled with 5 ll of saline was inserted into the subarachnoid space for an
0.5 cm length. After the muscle incision was closed, the mini-osmotic pumps (model 2001, Alzet, Palo Alto, CA, USA) filled with phosphate-buffered saline (PBS) or homocysteine (Sigma, St. Louis, MO, USA) were connected to the tube. The concentrations of homocysteine were 0.5, 5 or 50 nmol/ll diluted in PBS (n = 8 for behavioral analysis at each drug condition). Then, the pump was laid under the skin and the incision was closed. The tube is held to the L6 spinous process and to back muscle using 4-0 nylon surgical sutures. The pump was implanted at least 24 h before the first testing and the connection between pump and spinal cord was confirmed at the end day of the behavioral analysis session. Naı¨ ve rats were used for the intrathecal tPA injection experiment. The concentration of tPA (Sigma, St. Louis, MO) was diluted in 10.4 ng/ll in PBS (adjusted 2 lg/day). tPA was administrated using the mini-osmotic pump (Alzet model 2001D, CA, USA). Homocysteine and ANX2 neutralizing antibody (BD Transduction Laboratories, Franklin Lakes, NJ, USA) were co-administrated with tPA. The concentration of homocysteine and anti ANX2 antibody in tPA solution was 6.25 nmol/ll: 1.2 lmol/day and 44.1 ng/ml: 10 lg/day, respectively. Since the antibody used for the detection of ANX2 in this study has neutralization activity against plasminogen activator (Lokman et al., 2013), the specificity of neutralizing antibody was confirmed by Western blot and immunohistochemistry (IHC) (Figs. 2 and 3). Purified mouse IgG (BD pharmingenTM, San Diego, CA, USA) was co-administrated with tPA for the control experiments in this study.

Rats that received SNI (0, 1, 3, 7, 14 and 30 days, n = 4 at each time point) were deeply anesthetized with sodium pentobarbital (125 mg/kg body weight, i.p.), and perfused transcardially with 100 ml of 1% paraformaldehyde in 0.1 M phosphate buffer (PB) pH 7.4, followed by 500 ml of 4% paraformaldehyde in 0.1 M PB. The L4/5 DRG and spinal cord were removed and post-fixed in the same fixative for 4 h at 4 °C, followed by immersion in 30% sucrose in 0.1 M PB at 4 °C overnight. The tissue was frozen in powdered dry ice, cut on a cryostat at a 25-lm thickness for the spinal cord and a 5-lm thickness for the DRG. The sections were processed for in situ hybridization (ISH) and IHC. ISH The protocol for ISH was described in detail in a previous paper (Yamanaka et al., 2004). A clone (p-GEM T-easy; Promega, Madison, MI, USA) containing a partial sequence corresponding to the coding regions of ANX2, (71-481, accession number L13039) was prepared and alpha-35S UTP-labeled antisense and sense cRNA probes were synthesized using the enzyme-digested clones. The 35 S-labeled probes in hybridization buffer were placed on the tissue sections on slides. The sections were incubated at 55 °C overnight, then washed and treated with 1 lg/ml RNase A. Next, the sections were air-dried. After the hybridization reaction, the slides were coated with NTB emulsion (Kodak, Rochester, NY USA) and exposed for 2–5 weeks. Once developed in D-19 (Kodak), the sections were stained with hematoxylin–eosin and coverslipped. IHC SNI model and naı¨ ve control rats were used for IHC. The following antibodies were used: mouse anti ANX2 monoclonal antiserum (1:500; BD Transduction

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

191

Fig. 1. Expression of ANX2 mRNA in the L4, 5 DRG after SNI. (A) The levels of ANX2 mRNA in the ipsilateral L4, 5 DRG were determined by RTPCR. Gel panels show PCR products from the L4, 5 DRG taken at 1, 3, 7 and 14 days after surgery (n = 4 at each time points). (B) Graph shows the mRNA levels of ANX2 expressed as percentages of the mRNA level in normal control ganglia (mean ± SEM). #indicates significance compared with the naive control (p < 0.05; ANOVA). (C and D) Dark field images of ISH showing ANX2 mRNA in the L4 DRG of a control rat (C) and 7 days after SNI (D). (E and F) Bright-field images of the ISH for ANX2 mRNA in the control (E) and ipsilateral DRG at 7 days after SNI (F). (G) Scatter plot diagrams of ANX2 mRNA expression in the injured (7 days) and control L4 and L5 DRG (total 609 neurons for control, total 634 neurons for SNI). Individual cell profiles were plotted according to the cross sectional area (along x-axis) and S/N ratio (along the y-axis). The sections were stained with hematoxylin–eosin (E and F). Scale bar = 250 lm (C, D), 25 lm (E, F).

Laboratories, NJ, USA) and rabbit anti tissue tPA polyclonal antiserum (1:2000; Molecular Innovations, Inc., MI, USA). In brief, DRG and spinal cord sections were incubated with a mixture of primary antibodies over night at 4 °C and followed by a mixture of Alexa

Fluor, 488 or 594 conjugated secondary antibodies (1:5000; Molecular Probes, Eugene, OR, USA) overnight at 4 °C. For single staining of ANX2, the sections were processed for IHC using the ABC method (Yamanaka et al., 2004).

192

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

Fig. 2. Induction of ANX2 protein in the DRG following SNI. (A) Western blots of ANX2 protein using the total protein extracted from the L4, 5 DRG taken at 1, 3, 7 and 14 days after surgery. (B) Graph shows the protein levels of ANX2 expressed as percentages of the protein level in the normal control ganglia (mean ± SEM; each time points n = 4,). # indicates p < 0.05 (ANOVA) compared to naı¨ ve control (day 0). (C–F), Low magnified photomicrographs show ANX2-ir in the DRG of normal control (C), 3 days (D), 7 days (E) and 14 days after SNI surgery (F). (G and H) Higher magnified images of naı¨ ve DRG neurons (G) and 14 days (H) after injury. These panels show subsets of low levels of cytoplasmic ANX2-ir in naı¨ ve DRG. A substantial increase of ANX2-ir was observed in the cytoplasm and plasma membrane of DRG neurons at 14 days after SNI. Scale bar = 100 lm (C–F), 25 lm (G and H).

W analysisB For the Western blot analysis, the rats were decapitated under deep anesthesia at 0, 1, 3, 7 and 14 days after SNI (n = 4 each time points) and the ipsilateral L4/5 DRG and spinal cord were removed and rapidly frozen with powdered dry ice. The frozen spinal cord was homogenized (NS-310, Microtec Nition CO. LTD, Chiba, Japan Nikon, Tokyo, Japan) at 10% (w/v) in a modified buffer containing 20 mM Tris–HCl, pH 7.4, 10% sucrose, and protease inhibitors (Protease inhibitor cocktail, 1:100; Nakarai, Kyoto, Japan). Homogenates were mixed for 60 min with intervening cooling and centrifuged for 60 min at 14,000 rpm at 4 °C to recover the supernatant. Proteins were resolved using 10% SDS–polyacrylamide gel electrophoresis, and 15 lg of

Fig. 3. Increases in ANX2-ir in dorsal horn of SNI model rats. (A–C) Immunostaining of annexin-A2 in the spinal cord showing increase in ANX2-ir after nerve injury. Dorsal horn in a control naı¨ ve rat (A), 7 days (B), and 14 days after SNI surgery (C). (D) Quantification of the ANX2-ir levels in the laminae I–II and III–IV of dorsal horn after SNI (mean ± SEM; each time points n = 4). # indicates p < 0.05 (ANOVA) compared to naı¨ ve control (day 0). Scale bar = 100 lm (A–C).

protein was applied to each lane. After electrophoresis, proteins were transferred onto PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA) in 25 mM Tris/200 mM glycine for 100 min at 1 mA/cm2. Blots were blocked for 1 h in 10% Blocking One P (Nakarai) in 0.1 M Tris-buffered saline containing 0.05% Tween 20. Incubations with primary antibodies were performed

193

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

overnight at 4 °C. Secondary antibodies, IgG conjugated to alkaline phosphatase, were incubated for 1 h at room temperature. Signal was detected by chemiluminescence using CPD-Star ready-to-use reagent (Roche, Indianapolis, IN, USA). Films were scanned and quantified using the NIH Image system, version 1.61.

Measurement Measurement of ISH was performed as described before (Yamanaka et al., 2004). Any slices within the two non-serial L4/5 DRG sections that contained at least 400 neurons with visible nuclei were used for measurement of signal intensity. These two non-serial sections were separately corrected from one DRG at the distance of more than 200 lm. For image analysis of the sections processed for ISH, we analyzed the density of silver grains over all neuronal profiles containing nuclei in the selected sections using a computerized image analysis system (NIH Image version 1.61). At a magnification of 200 and with bright-field illumination, the upper and lower thresholds of gray level density were set such that only silver grains were accurately discriminated from the background in the outlined cell or tissue profile and read by the computer pixel-by-pixel. Next, the area of discriminated pixels was measured and divided by the area of the outlined profile, giving a grain density for each cell or tissue profile. To reduce the risk of biased sampling of the data owing to varying emulsion thickness, the percentage of grain-occupied area of each neuronal profile was divided by the background grain density giving a signal/ noise (S/N) ratio. The S/N ratio of an individual neuron and its cross-sectioned area, which was computed from the outlined profile, are plotted in Fig. 1G. Bright-field images of spinal ANX2 IHC were used for the quantification. At a magnification of 100, ANX2 immunoreactive profiles in substantia gelatinosa (laminae I/II) and deep layer (laminae III–IV) were measured using a computerized image analysis system (NIH Image version 1.61). Signal area values were normalized to control. Immunohistochemistry of Iba1 and GFAP was used for quantification of spinal microglia and astrocytes. Confocal images of Iba1 and GFAP in L4/5 spinal segments were captured at a magnification of 200 and immunoreactive profiles were quantified on an image analyzer (Image J, NIH). The values were normalized to control immunoreactive levels.

Photomicrographs All images from the ISH and double labeling of ISH and immunohistochemistry with DAB staining were digitized with a Nikon DIAPHOT-300 microscope (Nikon, Japan) connected to a Nikon DXM-1200 digital camera (Nikon, Japan). Double staining 2D images were acquired using a confocal laser scanning microscope (model LSM 510 version 2.8; Carl Zeiss Microimaging GmbH, Jena, Germany) with the Plan-Neofular 10 and 40 objective lens. We used Adobe Photoshop CS2 (Adobe Systems, Mountain View, CA, USA) to optimize the images and to make all figures.

Behavioral tests All SNI rats were tested for mechanical allodynia and hyperalgesia of the plantar surface of the hindpaw 1 day before surgery and 0, 3, 5, 7, 9, 12 and 14 days after surgery. Mechanical allodynia was assessed with a dynamic plantar esthesiometer (Ugo Basile, Comerio, Italy), which is an automated von Frey-type system (Kalmar et al., 2003; Lever et al., 2003). To measure mechanical thresholds of the hindpaw, rats were placed in a plastic cage with a wire mesh floor and allowed to acclimate for 15 min before each test session. A pawflick response was elicited by applying an increasing force (measured in grams) using a plastic filament (0.5 mm diameter) focused on the lateral of the plantar surface of the ipsilateral hindpaw (sural nerve area). The force applied was initially below the detection threshold, then increased from 1 to 50 g in 1 g steps over 20 s, and was then held at 50 g for an additional 10 s. The rate of force increase was 2.5 gm/s. The force required to elicit a reflex removal of the ipsilateral hindpaw was monitored. This amount of force was defined as the mean of three measurements made at 5-min intervals. Data are expressed as mean ± SEM. Differences of values over time of each group were tested using a one-way ANOVA, followed by individual post hoc comparisons (Fisher’s PLSD). Pairwise comparisons (Student’s t test) were used to assess the effect of the delayed administration of homocysteine, intrathecal tPA injection and on the control experiments (Fig. 5B, D and Fig. 6). A difference was accepted as significant if p < 0.05.

RESULTS Expression of mRNA in the DRGANX2 To examine whether the ANX2 mRNA level is upregulated in response to the sciatic nerve injury, we performed RT-PCR using the total RNA extracted from L4 and L5 DRGs on the side ipsilateral to the injury (Fig. 1A, B). The intensity of the amplified band of ANX2 was significantly increased as early as 3 days (190.6 ± 26.4%, p < 0.05, n = 4) and rose to double of the constitutive expression levels at 7 days following the sciatic nerve injury, (211.5 ± 32.7%, p < 0.05, n = 4). The increase in expression remained significant for 2 weeks following the peripheral nerve injury. Expression of ANX2 mRNA at the cellular level was examined by ISH. In the control, most of the DRG neurons expressed ANX2 mRNA at low levels (S/N ratio > 5) (Fig. 1C, E and G). SNI clearly increased the ANX2 mRNA signals in the ipsilateral DRG (Fig. 1D, F). Increased hybridization signals were accumulated in substantial population of neurons in the ipsilateral DRG (Fig 1F). Indeed, quantification of the silver grains on neuronal somata revealed that nerve injury facilitated ANX2 transcription in every size DRG neurons (Fig. 1G). Expression of protein in the DRGANX2 Expression of ANX2 protein was confirmed by Western blot analysis of the DRG using antibody against ANX2. As was expected from the mRNA analysis (Fig. 1), the

194

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

the localization of ANX2 protein in the DRG. In contrast to the mRNA signals in Fig. 1, ANX2 protein was expressed in a variety of sizes of DRG neurons. In the control DRG, cytoplasmic ANX2-ir was mainly seen in small-sized neurons (Fig. 2C). Control DRG neurons that lacked cytoplasmic ANX2-ir expressed ANX2-ir in the plasma membrane. These neurons appeared as ring-like ANX2-ir (Fig. 2C, G). After nerve injury, the ANX2-ir was increased both in cytoplasm and plasma membrane of DRG neurons (Fig. 2D–F and H). The increase of ANX2 was evident at day 3 (Fig. 2D) and continued for at least 14 days after nerve injury (Fig. 2F, H). Expression of in the spinal cord following SNIANX2

Fig. 4. Co-localization analysis of ANX2 and tPA in the dorsal horn after SNI. Double labeling of ANX2 (A) and tPA (B) in the dorsal horn at 7 days after SNI. Nerve injury increased ANX2 and tPA-ir terminal in ipsilateral dorsal horn. ANX2 and tPA-ir signals showed overlap in the ipsilateral laminae I–II of the dorsal horn (C). (D, E), Higher magnification images of double labeling showing partial co-localization of ANX2 with tPA immunoreactive terminals. Scale bar = 100 lm (A–C), 20 lm (D, E).

protein levels of ANX2 in the DRG were dramatically up-regulated after peripheral nerve injury. Western blot analysis showed an increase of the 40-kDa single ANX2-immunoreactivities (ir) band following SNI (Fig. 2A). 3 days after SNI, ANX2 protein levels were significantly increased and continued to be elevated until day 14 (Fig. 2A, B, n = 4 each time point). We used immunohistochemistry to examine the effect of SNI on

As was seen in the DRG, nerve injury affected the expression of ANX2-ir in the dorsal horn. In control rats, ANX2-ir in the spinal cord was predominantly localized in laminae I–II but low levels were observed in the deep layer showing the primary afferents terminal-like staining pattern (Fig. 3A). Immunohistochemistry of ANX2 revealed that SNI increased the ANX2-ir profiles not only in laminae I–II but also in deep layers of the L4-5 spinal cord ipsilateral to the injury (Fig. 3A–C). In contrast to the DRG, spinal ANX2 showed delayed induction in the terminal of the primary afferent. The SNI significantly increased ANX2 protein from the 7th day and remained elevated for at least 30 days after injury in the L4-5 spinal cord (Fig. 3D). It has been reported that ANX2 forms a complex with tPA that accelerates catalytic activity (Cesarman et al., 1994; Kang et al., 1999). In order to elucidate the interactions of these molecules in dorsal horn, we examined the double labeling of ANX2 with tPA in the spinal cord following SNI using confocal images. The dorsal horn of SNI model rats at day 7 showed a substantial increase of ir for ANX2 and tPA compared with the contra-lateral side (Fig. 4A, B). Double-labeling confocal images revealed that ANX2-ir was mainly co-localized with tPA-ir in laminae I and II after SNI (Figs. 4A–C). In contrast to laminae I–II, the majority of ANX2-ir did not overlap with tPA in laminae III–IV. Higher magnified double-labeling images showed a considerable amount of ANX2-ir terminals that co-expressed tPA in laminae I–II (Fig. 4D, E). However, we detected some tPA-ir profiles did not co-label with ANX2 (Fig. 4E). Prevention of neuropathic pain and tPA-dependent mechanical hypersensitivity by intrathecal infusion of homocysteine To investigate whether the ANX2–tPA complex is involved in the development of neuropathic pain, we examined the effect of the intrathecal administration of homocysteine, a sulfhydryl-containing amino acid which blocks t-PA binding to annexin II (Hajjar and Jacovina, 1998) in pain behaviors (n = 8, each group). SNI models treated with vehicle showed significant mechanical allodynia which appeared on day 3 after surgery and was maintained for at least 2 weeks (Fig. 5A). We administrated the homocysteine from day 2 to day 9. The homocysteine at

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

195

Fig. 5. Effects of intrathecal chronic administration of homocysteine on neuropathic pain behaviors. Mechanical sensitivity was determined with a Dynamic Plantar Esthesiometer (A), the effects of chronic intrathecal administration of homocysteine (HC) on mechanical allodynia in the SNI model. The highest concentration of homocysteine (1.2 lmol/day) administration reversed the mechanical allodynia for up to 9 days after surgery. The lower concentration of homocysteine (120 nmol/day) had a significant effect from 3 to 7 days. The lowest concentration of homocysteine administration(12 nmol/day) had no effect on mechanical hyperalgesia (B). Intrathecal homocysteine (1.2 lmol/day) did not change the basal mechanical sensitivity of sham operated rats. (C) Post-treatment with homocysteine reversed nerve injury-induced tactile allodynia. Homocysteine was delivered intrathecally via an osmotic pump starting on post-nerve injury day 8. (D) Mechanical threshold value of the contralateral side (C). Intrathecal delayed administration of homocysteine (1.2 lmol/day) did not change the mechanical sensitivity of the contralateral hindpaw. Lines on the graph indicate the period of intrathecal administration of homocysteine. # indicates p < 0.05 compared to the PBS-treated group. In all graphs, values are represented as mean ± SEM (n = 8 for each group of neuropathic pain model and n = 6 for basal sensitivity analysis).

the highest dose administered (1.2 lmol/day) reversed injury-induced mechanical allodynia from day 3 to day 9 after injury (Fig. 5A). In groups with lower concentrations of ANX2 antibody (120 nmol/day), a significant reduction of mechanical allodynia was observed from day 3 to day 7 after injury (Fig. 5A). In contrast, the lowest concentration of homocysteine treatment had no effects on the neuropathic pain behavior (Fig. 5A). In sham-operated rats, the basal mechanical sensitivity was not affected by the homocysteine, nor did it show any difference from the vehicle treatment (Fig. 5B). We examined the administration of homocysteine from days 8 to 14 after nerve injury and found that homocysteine partially suppressed established mechanical hypersensitivity but did not alter contralateral mechanical sensitivity (Fig. 5C, D). It has been reported that intrathecal injection of tPA reduced the withdrawal threshold of mechanical stimuli to the hindpaw (Berta et al., 2013). Our previous studies also reported the involvement of tPA in the neuropathic pain behavior (Yamanaka et al., 2004). We then analyzed the involvement of ANX2 in tPA-induced mechanical hypersensitivity by co-administrating homocysteine and tPA into subarachnoid space using an osmotic pump for 24 h of continuous injection and then examined the mechanical withdrawal threshold of the hindpaw (n = 8, each group) (Fig. 6A). Intrathecal chronic injection of tPA (2 lg/day) reduced the withdrawal threshold to

mechanical stimuli in the hindpaw. This result was similar to the previous report (Berta et al., 2013). Intrathecal co-injection of homocysteine (1.2 lmol/day) with tPA (2 lg/day) prevented tPA-induced mechanical hypersensitivity (Fig. 6A). In order to provide additional evidence to show that the tPA-induced mechanical hypersensitivity is dependent on the spinal ANX2, we co-injected tPA (2 lg/day) and ANX2 neutralization antibody (10 lg/day) intrathecally and examined the withdrawal threshold of hindpaw (n = 6, each group). The neutralizing antibody for ANX2 injected into subarachnoidal space has been used to inhibit the conversion of plasminogen to plasmin (Sharma et al., 2006; Lokman et al., 2013). Intrathecal co-administration of ANX2 neutralization antibody with tPA suppressed mechanical hypersensitivity (Fig 6B). Homocysteine treatment did not affect the activation of spinal glial cells following peripheral nerve injury The ANX2–tPA complex was involved in the activation of microglia in kainic acid-induced hippocampus damage (Siao and Tsirka, 2002). Following nerve injury, activation of spinal glial cells has been considered as an important pathological component of neuropathic pain (Gao and Ji, 2010; Ji et al., 2013). We therefore examined the effects of intrathecal injection of homocysteine on the activation of glial cells in the dorsal horn of SNI model rats.

196

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

to laminae I–V of the spinal cord; (3) laminae I–II of ANX2-ir showed co-localization with tPA; (4) intrathecal chronic administration of homocysteine, which inhibits the formation of the tPA–ANX2 complex, suppressed mechanical allodynia following peripheral nerve injury. Functional diversity of the annexin family

Fig. 6. Chronic intrathecal injection of tPA (2 lg/day) reduced mechanical hypersensitivity. The reduction of the withdrawal threshold returned to normal level at 2 days. Intrathecal co-injection of tPA (2 lg/day) and homocysteine (1.2 lmol/day) prevented the emergence of mechanical hypersensitivity (A). Intrathecal co-administration of tPA (2 lg/day) and ANX2 neutralizing antibody (10 mg/day) also prevented the mechanical hypersensitivity (B). * indicates p < 0.05 compared to 0 day, ANOVA. # indicates p < 0.05 compared to tPA treated group, t-test, n = 8 in each group of homocysteine treatment experiments and n = 6 in each group of ANX2 neutralizing antibody administration experiments.

In spite of the structural similarity of annexin family, functional diversity within the annexin family has been reported (Gerke and Moss, 2002). Among the annexin family, annexin A1 (ANX1) is well characterized as an endogenous anti-inflammatory molecule. ANX1 has been known as a cellular mediator of anti-inflammatory glucocorticoids since its expression and secretion are regulated by glucocorticoids (Comera and Russo-Marie, 1995; Philip et al., 1998) and since administration of ANX1 produced anti-inflammatory activities in several types of inflammation (Flower and Rothwell, 1994). In the inflammatory tissue neutrophil-derived ANX1 inhibits the expression of cytokines and cyclooxygenase via the specific receptor formyl-peptide-receptor-like 1(FPR2/ ALX) (Ayoub et al., 2008; Chen et al., 2014). ANX1 plays an anti-nociceptive role not only in the peripheral tissue but also in the nervous system (Chen et al., 2014). Pei et al., have shown that ANX1 suppressed nociception in DRG levels of rat in the inflammatory pain model (Pei et al., 2011). Therefore ANX1 in peripheral tissue and primary afferent has been considered as an endogenous anti-nociceptive molecule. In contrast to the ANX1, ANX2 plays an important role in the excitability and pronociceptive activity of DRG neuron (discussed below). Expression of in DRG neurons after nerve injuryANX2

Iba1 immunostaining was employed to assess microglia activation patterns, both in PBS- and homocysteinetreated rats. Homocysteine was administered into the subarachnoidal space in the dose that reduced neuropathic pain behavior. In PBS-injected controls, there were indeed characteristic injury-induced changes in number and morphology of microglia (Fig. 7A left). Staining pattern of Iba1 in the homocysteine treatment group also showed the typical activation of microglia in dorsal horn (Fig. 7A, right). Quantification of Iba1 immunoreactive area in the dorsal horn revealed that there was no suppression of activation of microglia in the homocysteine treatment group. Astrocyte morphology was examined by the immunostaining of GFAP. Hypertrophy of astrocytes in the dorsal horn of SNI rats showed no differences in PBS and homocysteine treatment (Fig. 7C). Measurement of GFAP-labeling profiles in dorsal horn showed that homocysteine treatment did not affect the increase of GFAP after nerve injury (Fig 7D).

DISCUSSION The present study demonstrates the contribution of ANX2 to neuropathic pain by regulating tPA at the spinal level. The principal findings are (1) peripheral nerve injury upregulated ANX2 in a subpopulation of injured DRG neurons; (2) the induced ANX2 protein was transported

Expression and possible roles of ANX2 in the nervous system have been reported. The expression of ANX2 is induced under pathological conditions, including various brain tumors (Reeves et al., 1992; Roseman et al., 1994; Nygaard et al., 1998), ischemic injury, seizures, and Alzheimer’s disease (Eberhard et al., 1994). Therefore, ANX2 is considered to be a brain pathologyassociated protein. In contrast, previous studies have shown that DRG neurons expressed substantial amounts of ANX2 (Naciff et al., 1996; Cesarman-Maus and Hajjar, 2005; Avenali et al., 2014). Immunostaining results from our present study seemed identical to the results of Avenali et. al. However, the results of the previous studies presented different immunostaining patterns of ANX2 in DRG, and our present ISH and IHC studies showed different expression patterns of ANX2 in DRG. These discrepancies may be due to the sensitivity of the antibodies or the threshold value. Despite the divergence of observations, our present study and previous reports strongly suggest a function of ANX2 in DRG neurons. It has been reported that membrane-associated ANX2 is present on the endothelial cell surface as a heterotetramer with the S100 family protein p11 (Cesarman-Maus and Hajjar, 2005). The protein p11 is involved in the trafficking of several types of channel proteins such as voltage-gated sodium channel Nav1.8, ligand-gated ion channels acid-sensing ion channel 1a

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

197

Fig. 7. Intrathecal injection of homocysteine (1.2 lmol/day) from 3 to 7 days did not affect the nerve injury-induced glial activation in the dorsal horn of the spinal cord. Immunofluorescence images of Iba1 in the ipsilateral dorsal horn of SNI model rats treated with PBS (A left) and homocysteine (A right). Quantification of Iba1 immunoreactive area in the dorsal horn of naı¨ ve and SNI model rats treated with PBS or homocysteine (B). Immunofluorescence images of GFAP in the ipsilateral dorsal horn of PBS (C left) and homocysteine treatment (C right). Quantification of GFAP immunoreactive area in the dorsal horn of naı¨ ve and SNI model rats treated with PBS or homocysteine (D). Scale bar = 100 lm.

(ASIC1a) and the TWIK-related acid-sensitive K+ channe1 (TASK-1) (Okuse et al., 2002; Donier et al., 2005; Renigunta et al., 2006). A previous study showed that the deletion of p11 from nociceptive DRG neurons represented a phenotype with deficits in the expression of Nav1.8 (Foulkes et al., 2006). Therefore, ANX2 in DRG may play a significant role in the regulation of channel protein expression via its interaction with p11. Indeed, selective deletion of the p11 gene in nociceptive DRG neuron showed a substantial reduction of mechanical allodynia in a neuropathic pain model (Foulkes et al., 2006). Simultaneous up-regulation of p11 and ANX2 has been reported in proteomic analyses in peripheral nerve injury models (Zhang et al., 2008). Thus, injury-induced ANX2 may accelerate the recombination and transportation of many ion channels and contribute to the neuropathic pain. Interaction of and tPA in the dorsal horn during neuropathic pain stateANX2 Apart from the roles ANX2 has in molecular trafficking, ANX2 also functions as a co-activator of tPA on the membrane surface and markedly increases the tPAmediated conversion of plasminogen to plasmin (Cesarman et al., 1994; Kassam et al., 1998). ANX2 has been considered to play an important role in the modulation of catalytic activity of tPA and plasminogen in the fibrinolytic system. Interestingly, the complete deficiency of ANX2 in mice leads to a lack of tPA cofactor activity, accumulation of intravascular fibrin, and failure to clear arterial thrombi (Ling et al., 2004). Similarly, ANX2 plays a role in cell surface proteolysis (Hajjar, 1995; Choi et al., 2001; Kim and Hajjar, 2002), angiogenesis (Kwon

et al., 2002; Tuszynski et al., 2002), and neurite outgrowth (Fox et al., 1991; Jacovina et al., 2001). Given that ANX2 is a potent co-activator of tPA, it is of interest to investigate whether the ANX2–tPA complex has an important role to play in neuronal plasticity (Huang et al., 1996; Baranes et al., 1998; Calabresi et al., 2000; Pang et al., 2004). Neuronal plasticity processes, such as LTP and central sensitization, are thought to share similar mechanisms in terms of activation of NMDA-R, which is believed to be a molecular target of tPA (Calabresi et al., 2000; Ji et al., 2003; Noel et al., 2011; Ng et al., 2012). Involvement of tPA activity in pain behavior has been reported in several types of pain models, such as peripheral nerve injury, dorsal root compression, and chronic opioid-induced antinociceptive tolerance (Yamanaka et al., 2004; Kozai et al., 2007; Berta et al., 2013). Our present study showing the co-localization of ANX2 with tPA in the dorsal horn suggests that tPA activity was modulated in the primary afferent following nerve injury. Blockage of the tPA–ANX2 interaction by spinal injection of homocysteine reduced neuropathic pain behaviors in a dose-dependent manner. This inhibitory effect suggests that the regulatory function of the ANX2–tPA complex contributes to neuropathic pain in injured primary afferents in dorsal horn. Delayed administration of homocysteine reversed the established neuropathic pain behavior. Thus, the increased ANX2–tPA interaction may be involved both in the mechanisms of onset, and maintenance of neuropathic pain. Additionally, intrathecal co-injection of tPA and homocysteine or ANX2 neutralizing antibody suggested a role for spinal ANX2 in tPA-dependent mechanical hypersensitivity.

198

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199

Binding of tPA did not have an effect on the activation of glia cells–ANX2 Several studies support the hypothesis that tPA is involved in the initiation of microglial activation (Rogove et al., 1999; Pineda et al., 2012). This tPA cytokine-like activity is mediated by its finger domain as well as its interaction with ANX2 or low-density lipoprotein receptorrelated protein 1 in a catalytic independent manner (Siao and Tsirka, 2002; Zhang et al., 2009). Here, we have shown that ANX2 was expressed mainly in primary afferents, but not in the spinal glia cells. Thus, the spinal tPA could bind to primary afferents in an autocrine or paracrine manner, but not to glia cells via the ANX2 pathway. In the present study, we showed that the inhibition of tPA binding to ANX2 by intrathecal injection of homocysteine did not suppress the activation of microglia and astrocytes. Therefore, the effect of the tPA–ANX2 interaction can be ruled out from possible glia cell activation mechanisms in spinal cord following peripheral nerve injury.

CONCLUSION In this study, we provide new evidence about the molecular mechanism of ANX2 in primary afferent neurons, which contributes to the development of neuropathic pain. A large body of research suggests that an injured primary terminal can stimulate the dorsal horn neurons which leads to neuropathic pain. We have also shown that the levels of ANX2 and tPA were locally up-regulated in the terminals of injured primary afferents. Identification of ANX2 as a terminal tPA receptor and characterization of the role of their complex in pain-producing pathways would allow a potentially relevant therapeutic approach to neuropathic pain. Acknowledgments—This work was supported in part by Grantsin-Aid for Scientific Research, and Pain Research Group Grant, Hyogo College of Medicine, both from the Japanese Ministry of Education, Science, and Culture. We thank Yu Wadazumi and Noriko Kusumoto for technical assistance. We thank D.A. Thomas for correcting the English usage.

REFERENCES Avenali L, Narayanan P, Rouwette T, Cervellini I, Sereda M, GomezVarela D, Schmidt M (2014) Annexin A2 regulates TRPA1dependent nociception. J Neurosci 34:14506–14516. Ayoub SS, Yazid S, Flower RJ (2008) Increased susceptibility of annexin-A1 null mice to nociceptive pain is indicative of a spinal antinociceptive action of annexin-A1. Br J Pharmacol 154:1135–1142. Baranes D, Lederfein D, Huang YY, Chen M, Bailey CH, Kandel ER (1998) Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway. Neuron 21:813–825. Berta T, Liu YC, Xu ZZ, Ji RR (2013) Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1beta and ERK signaling in the spinal cord of mice. Neuroscience 247:376–385. Calabresi P, Napolitano M, Centonze D, Marfia GA, Gubellini P, Teule MA, Berretta N, Bernardi G, Frati L, Tolu M, Gulino A (2000)

Tissue plasminogen activator controls multiple forms of synaptic plasticity and memory. Eur J Neurosci 12:1002–1012. Campbell JN, Meyer RA (2006) Mechanisms of neuropathic pain. Neuron 52:77–92. Cesarman GM, Guevara CA, Hajjar KA (1994) An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA). II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation. J Biol Chem 269:21198–21203. Cesarman-Maus G, Hajjar KA (2005) Molecular mechanisms of fibrinolysis. Br J Haematol 129:307–321. Chen ZL, Strickland S (1997) Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 91:917–925. Chen L, Lv F, Pei L (2014) Annexin 1: a glucocorticoid-inducible protein that modulates inflammatory pain. Eur J Pain 18:338–347. Choi KS, Fitzpatrick SL, Filipenko NR, Fogg DK, Kassam G, Magliocco AM, Waisman DM (2001) Regulation of plasmindependent fibrin clot lysis by annexin II heterotetramer. J Biol Chem 276:25212–25221. Comera C, Russo-Marie F (1995) Glucocorticoid-induced annexin 1 secretion by monocytes and peritoneal leukocytes. Br J Pharmacol 115:1043–1047. Costigan M, Scholz J, Woolf CJ (2009) Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci 32:1–32. Donier E, Rugiero F, Okuse K, Wood JN (2005) Annexin II light chain p11 promotes functional expression of acid-sensing ion channel ASIC1a. J Biol Chem 280:38666–38672. Eberhard DA, Brown MD, VandenBerg SR (1994) Alterations of annexin expression in pathological neuronal and glial reactions. Immunohistochemical localization of annexins I, II (p36 and p11 subunits), IV, and VI in the human hippocampus. Am J Pathol 145:640–649. Flower RJ, Rothwell NJ (1994) Lipocortin-1: cellular mechanisms and clinical relevance. Trends Pharmacol Sci 15:71–76. Foulkes T, Nassar MA, Lane T, Matthews EA, Baker MD, Gerke V, Okuse K, Dickenson AH, Wood JN (2006) Deletion of annexin 2 light chain p11 in nociceptors causes deficits in somatosensory coding and pain behavior. J Neurosci 26:10499–10507. Fox MT, Prentice DA, Hughes JP (1991) Increases in p11 and annexin II proteins correlate with differentiation in the PC12 pheochromocytoma. Biochem Biophys Res Commun 177: 1188–1193. Gao YJ, Ji RR (2010) Targeting astrocyte signaling for chronic pain. Neurotherapeutics 7:482–493. Gerke V, Moss SE (2002) Annexins: from structure to function. Physiol Rev 82:331–371. Hajjar KA (1995) Cellular receptors in the regulation of plasmin generation. Thromb Haemost 74:294–301. Hajjar KA, Jacovina AT (1998) Modulation of annexin II by homocysteine: implications for atherothrombosis. J Investig Med 46:364–369. Hajjar KA, Menell JS (1997) Annexin II: a novel mediator of cell surface plasmin generation. Ann N Y Acad Sci 811:337–349. Huang YY, Bach ME, Lipp HP, Zhuo M, Wolfer DP, Hawkins RD, Schoonjans L, Kandel ER, Godfraind JM, Mulligan R, Collen D, Carmeliet P (1996) Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with latephase long-term potentiation in both Schaffer collateral and mossy fiber pathways. Proc Natl Acad Sci U S A 93:8699–8704. Jacovina AT, Zhong F, Khazanova E, Lev E, Deora AB, Hajjar KA (2001) Neuritogenesis and the nerve growth factor-induced differentiation of PC-12 cells requires annexin II-mediated plasmin generation. J Biol Chem 276:49350–49358. Ji RR, Kohno T, Moore KA, Woolf CJ (2003) Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci 26:696–705. Ji RR, Berta T, Nedergaard M (2013) Glia and pain: is chronic pain a gliopathy? Pain 154(Suppl 1):S10–28. Kalmar B, Greensmith L, Malcangio M, McMahon SB, Csermely P, Burnstock G (2003) The effect of treatment with BRX-220, a

H. Yamanaka et al. / Neuroscience 314 (2016) 189–199 co-inducer of heat shock proteins, on sensory fibers of the rat following peripheral nerve injury. Exp Neurol 184:636–647. Kang HM, Choi KS, Kassam G, Fitzpatrick SL, Kwon M, Waisman DM (1999) Role of annexin II tetramer in plasminogen activation. Trends Cardiovasc Med 9:92–102. Kassam G, Choi KS, Ghuman J, Kang HM, Fitzpatrick SL, Zackson T, Zackson S, Toba M, Shinomiya A, Waisman DM (1998) The role of annexin II tetramer in the activation of plasminogen. J Biol Chem 273:4790–4799. Kim J, Hajjar KA (2002) Annexin II: a plasminogen-plasminogen activator co-receptor. Front Biosci 7:d341–348. Kozai T, Yamanaka H, Dai Y, Obata K, Kobayashi K, Mashimo T, Noguchi K (2007) Tissue type plasminogen activator induced in rat dorsal horn astrocytes contributes to mechanical hypersensitivity following dorsal root injury. Glia 55:595–603. Kwon M, Caplan JF, Filipenko NR, Choi KS, Fitzpatrick SL, Zhang L, Waisman DM (2002) Identification of annexin II heterotetramer as a plasmin reductase. J Biol Chem 277:10903–10911. Lever I, Cunningham J, Grist J, Yip PK, Malcangio M (2003) Release of BDNF and GABA in the dorsal horn of neuropathic rats. Eur J Neurosci 18:1169–1174. Ling Q, Jacovina AT, Deora A, Febbraio M, Simantov R, Silverstein RL, Hempstead B, Mark WH, Hajjar KA (2004) Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo. J Clin Invest 113:38–48. Lokman NA, Elder AS, Ween MP, Pyragius CE, Hoffmann P, Oehler MK, Ricciardelli C (2013) Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis. Oncotarget 4:1199–1211. Naciff JM, Kaetzel MA, Behbehani MM, Dedman JR (1996) Differential expression of annexins I-VI in the rat dorsal root ganglia and spinal cord. J Comp Neurol 368:356–370. Ng KS, Leung HW, Wong PT, Low CM (2012) Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor. J Biol Chem 287:25520–25529. Noel M, Norris EH, Strickland S (2011) Tissue plasminogen activator is required for the development of fetal alcohol syndrome in mice. Proc Natl Acad Sci U S A 108:5069–5074. Nygaard SJ, Haugland HK, Kristoffersen EK, Lund-Johansen M, Laerum OD, Tysnes OB (1998) Expression of annexin II in glioma cell lines and in brain tumor biopsies. J Neurooncol 38:11–18. Okuse K, Malik-Hall M, Baker MD, Poon WY, Kong H, Chao MV, Wood JN (2002) Annexin II light chain regulates sensory neuronspecific sodium channel expression. Nature 417:653–656. Pang PT, Teng HK, Zaitsev E, Woo NT, Sakata K, Zhen S, Teng KK, Yung WH, Hempstead BL, Lu B (2004) Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306:487–491. Pei L, Zhang J, Zhao F, Su T, Wei H, Tian J, Li M, Shi J (2011) Annexin 1 exerts anti-nociceptive effects after peripheral inflammatory pain through formyl-peptide-receptor-like 1 in rat dorsal root ganglion. Br J Anaesth 107:948–958. Philip JG, Flower RJ, Buckingham JC (1998) Blockade of the classical pathway of protein secretion does not affect the cellular exportation of lipocortin 1. Regul Pept 73:133–139. Pineda D, Ampurdanes C, Medina MG, Serratosa J, Tusell JM, Saura J, Planas AM, Navarro P (2012) Tissue plasminogen activator

199

induces microglial inflammation via a noncatalytic molecular mechanism involving activation of mitogen-activated protein kinases and Akt signaling pathways and AnnexinA2 and Galectin-1 receptors. Glia 60:526–540. Qian Z, Gilbert ME, Colicos MA, Kandel ER, Kuhl D (1993) Tissueplasminogen activator is induced as an immediate-early gene during seizure, kindling and long-term potentiation. Nature 361:453–457. Reeves SA, Chavez-Kappel C, Davis R, Rosenblum M, Israel MA (1992) Developmental regulation of annexin II (Lipocortin 2) in human brain and expression in high grade glioma. Cancer Res 52:6871–6876. Renigunta V, Yuan H, Zuzarte M, Rinne S, Koch A, Wischmeyer E, Schlichthorl G, Gao Y, Karschin A, Jacob R, Schwappach B, Daut J, Preisig-Muller R (2006) The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1. Traffic 7:168–181. Rogove AD, Siao C, Keyt B, Strickland S, Tsirka SE (1999) Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system. J Cell Sci 112(Pt 22):4007–4016. Roseman BJ, Bollen A, Hsu J, Lamborn K, Israel MA (1994) Annexin II marks astrocytic brain tumors of high histologic grade. Oncol Res 6:561–567. Salter MW (2005) Cellular signalling pathways of spinal pain neuroplasticity as targets for analgesic development. Curr Top Med Chem 5:557–567. Sharma MR, Koltowski L, Ownbey RT, Tuszynski GP, Sharma MC (2006) Angiogenesis-associated protein annexin II in breast cancer: selective expression in invasive breast cancer and contribution to tumor invasion and progression. Exp Mol Pathol 81:146–156. Siao CJ, Tsirka SE (2002) Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II. J Neurosci 22:3352–3358. Stucky CL, Gold MS, Zhang X (2001) Mechanisms of pain. Proc Natl Acad Sci U S A 98:11845–11846. Tuszynski GP, Sharma MR, Rothman VL, Sharma MC (2002) Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc Res 64:448–462. Woolf CJ, Ma Q (2007) Nociceptors–noxious stimulus detectors. Neuron 55:353–364. Yamanaka H, Obata K, Fukuoka T, Dai Y, Kobayashi K, Tokunaga A, Noguchi K (2004) Tissue plasminogen activator in primary afferents induces dorsal horn excitability and pain response after peripheral nerve injury. Eur J Neurosci 19:93–102. Zhang Y, Wang YH, Zhang XH, Ge HY, Arendt-Nielsen L, Shao JM, Yue SW (2008) Proteomic analysis of differential proteins related to the neuropathic pain and neuroprotection in the dorsal root ganglion following its chronic compression in rats. Exp Brain Res 189:199–209. Zhang C, An J, Haile WB, Echeverry R, Strickland DK, Yepes M (2009) Microglial low-density lipoprotein receptor-related protein 1 mediates the effect of tissue-type plasminogen activator on matrix metalloproteinase-9 activity in the ischemic brain. J Cereb Blood Flow Metab 29:1946–1954.

(Accepted 25 November 2015) (Available online 28 November 2015)