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Effects of palmitoylethanolamide on release of mast cell peptidases and neurotrophic factors after spinal cord injury
Brain, Behavior, and Immunity 25 (2011) 1099–1112
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Effects of palmitoylethanolamide on release of mast cell peptidases and neurotrophic factors after spinal cord injury Emanuela Esposito a, Irene Paterniti a, Emanuela Mazzon b, Tiziana Genovese a, Rosanna Di Paola b, Maria Galuppo a, Salvatore Cuzzocrea a,b,⇑ a b
Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Italy IRCCS Centro Neurolesi ‘‘Bonino-Pulejo’’, Messina, Italy
a r t i c l e
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Article history: Received 15 November 2010 Received in revised form 14 February 2011 Accepted 14 February 2011 Available online 25 February 2011 Keywords: Mast cells GDNF NT-3 NGF CB2 Spinal cord injury
a b s t r a c t Spinal cord injury (SCI) has a significant impact on quality of life, expectancy, and economic burden, with considerable costs associated with primary care and loss of income. The complex pathophysiology of SCI may explain the difficulty in finding a suitable therapy for limiting neuronal injury and promoting regeneration. Although innovative medical care, advances in pharmacotherapy have been limited. The aim of the present study was to carefully investigate molecular pathways and subtypes of glial cells involved in the protective effect of PEA on inflammatory reaction associated with an experimental model of SCI. The compression model induced by applying an aneurysm clip to the spinal cord in mice is closer to the human situation, since it replicates the persistence of cord compression. Spinal cord trauma was induced in mice by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. Repeated PEA administration (10 mg/kg i.p., 6 and 12 h after SCI) significantly reduced the degree of the severity of spinal cord trauma through the reduction of mast cell infiltration and activation. Moreover, PEA treatment significantly reduced the activation of microglia and astrocytes expressing cannabinoid CB2 receptor after SCI. Importantly, the protective effect of PEA involved changes in the expression of neurotrophic factors, and in spinal cord dopaminergic function. Our results enhance our understanding about mechanisms related to the anti-inflammatory property of the PEA suggesting that this N-acylethanolamine may represent a crucial therapeutic intervention both diminishing the immune/inflammatory response and promoting the initiation of neurotrophic substance after SCI. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Spinal cord injury (SCI) has a significant impact on quality and expectancy of life, and economic burden, with considerable costs associated with primary care and loss of income. During the first 24 h post-injury, the synthesis and release of the potent proinflammatory mediators are markedly elevated at the lesion site where they play a major role in the development of secondary tissue degeneration after SCI in animals and in humans. Neurons continue to die for hours after SCI due to several mechanisms including exitotoxicity, vascular abnormalities and inflammatory response that can contribute to evolution of SCI. Historically, administration of high-dose methylprednisolone (MP), acutely after SCI has been considered the standard of care ⇑ Corresponding author. Address: Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi, 98100 Messina, Italy. Fax: +39 090 2213300. E-mail address: [email protected] (S. Cuzzocrea). 0889-1591/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2011.02.006
in the United States. Although clinical results were initially promising, there have been growing concerns that the modest neurological improvements seen with high-dose MP treatment in injured patients are not worth the associated risks. Therefore, there is a critical need to develop new pharmacologic therapies for treatment of SCI. Regeneration and compensatory sprouting of axons are very limited after injury to the adult SC. This deficiency has been attributed to a lack of growth promoting factors and the presence of inhibitory molecules and physical barriers to axonal regeneration (Schwab, 2002). Resident microglia and macrophages originating from blood are two key cell types related to the occurrence of neuronal degeneration in CNS after traumatic injury. In particular, when SCI occurs, microglia in parenchyma is activated and macrophages in circulation cross the blood–brain barrier to act as intrinsic spinal phagocytes. Therefore, these cells can release various neurotrophic peptides, which are excellent substrates for neurite outgrowth. In addition, various study have clearly demonstrated that mast cells (MCs) have long been known to participate in the inflammatory process, in fact MCs are present and recruited to
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all inflammatory sites (Andoh et al., 2006; Mican and Metcalfe, 1990). However, it has only recently become clear that MCs play an important role in orchestrating the whole inflammatory process from initiation events to chronic activation (Pejler et al., 2007). MCs are the first immune cells to considerably stimulate the inflammatory process due to a rapid release of pro-inflammatory and vasoactive mediators. MCs make and secrete an abundance of peptidases, which are stored in such large amounts in granules. The main peptidases (tryptases, chymases, carboxypeptidase A3, and dipeptidylpeptidase I) were best known as markers of degranulation, they are released locally in response to MC stimulation and can be distributed systemically and detected in blood (Trivedi and Caughey, 2010). During the last years, the endocannabinoid system has attracted the attention of researchers working in neural damage and repair and is being considered a promising target for the development of new therapies (Di Marzo, 2008; Pacher et al., 2006). Moreover, some components of the endocannabinoid system, that are constitutively expressed in the normal spinal cord such as diacylglycerol (DAG) lipases, fatty acid amide hydrolase and CB1 receptor (Bisogno et al., 2003; Cravatt et al., 2004; Romero et al., 2002; Tsou et al., 1998a,b) are modulated after neurodegenerative diseases or after peripheral nerve lesions (Bilsland et al., 2006; Lim et al., 2003;
Petrosino et al., 2007; Shoemaker et al., 2007; Witting et al., 2004; Wotherspoon et al., 2005; Zhang et al., 2003). Furthermore, recently have been reported that the endocannabinoid system is activated in a clinically relevant model of traumatic SCI in rats (Garcia-Ovejero et al., 2009). The endogenous fatty acid palmitoylethanolamide (PEA) is one of the members of N-acyl-ethanolamines family. In addition to the hypothesis that PEA has potent immunoregulatory properties (Aloe et al., 1993; Berdyshev, 2000; Berdyshev et al., 1997; Facci et al., 1995; Mazzari et al., 1996; Ross et al., 2000; Scarampella et al., 2001), recent data have demonstrated that PEA may also play a key role in the regulation of complex systems involved in the inflammatory response, pruritus, neurogenic and neuropathic pain (Di Marzo et al., 2000). Recently, we have also clearly demonstrated that the treatment with PEA significantly reduced the inflammation associated with experimental SCI in mice (Genovese et al., 2008). In particular, PEA protected against locomotor dysfunction following SCI. However, the basic molecular mechanisms as well as cellular types accounting for the anti-inflammatory properties of PEA in SCI have not yet been carefully investigated. Thus, the aim of the present study is to carefully investigate molecular pathways and subtypes of glial cells involved in the
Fig. 1. PEA reduces the severity of spinal cord trauma. The low-magnification images of spinal cord after compression injury show tissue disorganization, white matter alteration, and inflammation in the perilesional area at 24 h after injury (panels b, b1). A significant protection from the SCI was evident in the tissue collected from PEAtreated mice (c). The histological score was made by an independent observer (d). ⁄⁄⁄p<0.001 vs. Sham and ##p<0.01 vs. SCI. Values shown are means ± SE mean of 10 mice for each group.
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protective effect of PEA in SCI, and to identify in PEA a crucial therapeutic intervention after SCI.
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2.3. Experimental groups and treatments Mice were randomly allocated into the following groups:
2. Methods 2.1. Animals Male adult CD1 mice (25–30 g, Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986). 2.2. SCI Mice were anaesthetized using chloral hydrate (400 mg/kg body weight). We used the clip compression model described by Rivlin and Tator (1978) and produced SCI by extradural compression of a section of the SC exposed via a four-level T5–T8 laminectomy, in which the prominent spinous process of T5 was used as a surgical guide. With the aneurysm clip applicator oriented in the bilateral direction, an aneurysm clip with a closing force of 24 g was applied extradurally at T5–T8 level. The clip was then rapidly released with the clip applicator, which caused SC compression. In the injured groups, the cord was compressed for 1 min and then the clips were removed. Following surgery, 1.0 cc of saline was administered subcutaneously in order to replace the blood volume lost during the surgery. During recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. During this time period, the animals’ bladders were manually voided twice a day until the mice were able to regain normal bladder function. Sham injured animals were only subjected to laminectomy.
(i) Sham + vehicle group. Mice were subjected to laminectomy but the aneurysm clip was not applied, and treated intraperitoneally (i.p.) with vehicle (10% polyethylene glycol and 5% Tween 80 sterile distilled water) (N = 30). (ii) Sham + PEA group. Identical to Sham + vehicle group except for the administration of PEA (10 mg/kg) 6 and 12 h after laminectomy (N = 30). (iii) SCI + vehicle group. Mice were subjected to SCI and were administered vehicle (10% polyethylene glycol and 5% Tween 80 sterile distilled water, i.p. bolus) at 6 and 12 h after SCI (N = 30). (iv) SCI + PEA group. Mice were subjected to SCI and administered PEA (10 mg/kg) at 6 and 12 h after SCI (N = 30). PEA was purchased from Tocris bioscience (UK); The doses of PEA (10 mg/kg) used here were based on previous in vivo study (Genovese et al., 2008). In a separate set of experiments to investigate the motor score, additional animals were observed until 20 days after SCI. PEA (10 mg/kg) was administered 6 and 12 h after SCI and daily until day 19. 2.4. Tissue processing At the end of the experimental period (24 h), animals were deeply anesthetized with sodium pentobarbital and then perfused transcardially with cold phosphate-buffered saline (PBS, 0.1 M) followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Spinal cord tissues were removed under magnified vision. Tissue segments containing the lesion (1 cm on each side of the lesion) were paraffin embedded, cut into longitudinal sections for posterior area
Fig. 2. Staining of MCs. The slides stained with acidified Toluidine blue were also shown to have dark lilac blue granules, which identifies the cells as mast cells. Many of the mast cells are arranged separately in concentric rings around small blood-vessels. Different gradients of staining intensity were evident in the granulated and degranulated mast cells. In the spinal cord tissues collected at 24 h after SCI there is the presence of MCs (panels b, b1) mainly localized in the perivascular area. On the contrary, PEA reduced MC infiltration in the spinal cord tissues after SCI (panel c). No granules were found in the spinal cord tissues from sham-operated mice (panel a).
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of the spinal cord, and processed for different immunohistochemical procedures. Identification of mast cells was carried out following the application of Toluidine blue staining protocols described previously by Michaloudi and Papadopoulos (1999). For immunoblotting analysis, mice (N = 10) were anaesthetized as described above and exsanguinated via cardiac puncture. Spinal cords were dissected, and cleaned from meninges and nerve roots. One centimeter of the cord centered at the injury site was dissected and homogenized for preparing cytosolic and nuclear extracts. 2.5. Immunohistochemical localization of chymase, tryptase and dopamine transporter (DAT) At the 24 h after SCI, the tissues were fixed in PBS-buffered formaldehyde and 8 lm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60%
(v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated overnight with anti-chymase (purified human skin chymase) polyclonal antibody (1:1000), or anti-tryptase (mast cell tryptase isolated from human lung) polyclonal antibody (1:1000), antiDAT antibody (1:500). Sections were washed and incubated with secondary antibody. Specific labeling were detected with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vector Lab. Inc., Burlingame, CA). To verify the binding specificity for chymase, tryptase and DAT, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). Immunocytochemistry photographs (n = 5 photos from each samples collected from all mice in each experimental group) were assessed by densitometry as previously described (Cuzzocrea
Fig. 3. PEA reduces chymase expression. There was no staining for chymase in spinal cord tissues obtained from the sham-operated mice (panel a). A substantial increase in serine peptidases chymase expression was found mainly localized in MCs in the spinal cord tissues collected at 24 h after SCI (panel b). Chymase levels were attenuated in mice treated with PEA (panel c). This figure is representative of at least three experiments performed on different experimental days. To verify the binding specificity for chymase, some sections were also incubated with only the secondary antibody (no primary). In these situations no positive staining was found in the sections (d) indicating that the immunoreactions were positive in all the experiments carried out. ⁄⁄⁄p < 0.001 vs. Sham + vehicle and ###p < 0.001 vs. SCI. Values shown are means ± SE mean of 10 mice for each group.
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et al., 2001; Shea, 1994) by using Optilab Graftek software on a Macintosh personal computer. 2.6. Immunofluorescence After deparaffinization and rehydration, detection of CB2, Neu and CD11b was carried out after boiling in 0.01 M citrate buffer
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for 4 min. Endogenous peroxidase was quenched with 0.3% (vol/ vol) hydrogen peroxide in 60% (vol/vol) methanol for 30 min. Non-specific adsorption was minimized by incubating the section in 2% (vol/vol) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated with mouse monoclonal anti-mast Cell Chymase (1:100,
Fig. 4. PEA reduces tryptase expression. There was no staining for tryptase in spinal cord tissues obtained from the sham-operated mice (panel a). A substantial increase in expression was found mainly localized in mast cells in the spinal cord tissues collected at 24 h after SCI (panel b). Tryptase levels were attenuated in mice treated with PEA (panel c). This figure is representative of at least three experiments performed on different experimental days. To verify the binding specificity for chymase, some sections were also incubated with only the secondary antibody (no primary). In these situations no positive staining was found in the sections (d) indicating that the immunoreactions were positive in all the experiments carried out. ⁄⁄⁄p < 0.001 vs. Sham + vehicle and ###p < 0.001 vs. SCI. Values shown are means ± SE mean of 10 mice for each group.
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v/vol) (Thermo Scientific), or with polyclonal rabbit anti-CB2 (mapping at the C-terminus of CB2 of human origin 1:100, v/v) (Santa Cruz, Biotechnology), or with rabbit anti-Neu (raised again peptide mapping at the C-terminus of Neu of human 1:100, v/v) (Santa Cruz Biotechnology), or with mouse monoclonal anti-CD11b (mouse anti bovine 1:100, v/v) (Serotec), or with mouse monoclonal anti-GFAP (1:100, v/v) (Santa Cruz Biotechnology) antibody in a humidified chamber for 1 h at 37 °C. Sections were washed with PBS and were incubated with secondary antibody FITC-conjugated anti-mouse antibody (1:100 in PBS, v/v) (Jackson, West Grove, Pennsylvania), and with TEXAS RED-conjugated anti-rabbit antibody (1:100 in PBS, v/v) (Jackson, West Grove, Pennsylvania) for 1 h at 37 °C. Sections were washed and for nuclear staining 40 ,60 diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt; Germany) 0.5 lg/ml in PBS was added. Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels. Optical sections of fluorescence specimens were obtained using a HeNe laser (543 nm), a laser UV (361–365 nm) and an argon laser (458 nm) at a 1-min, 2-s scanning speed with up to 8 averages; 1.5-lm sections were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly labeled pixels and applying settings that allowed clear visualization of structural details while keeping the highest pixel intensities close to 200. The same settings were used for all images obtained from the other samples that had been processed in parallel. Digital images were cropped and figure montages prepared using Adobe Photoshop 7.0 (Adobe Systems; Palo Alto, CA).
2.7. Staining of mast cells Spinal cord sections were cut 5 lm thick and stained with 0.25% Toluidine blue, pH 2.5, for 45 min at room temperature (Michaloudi and Papadopoulos, 1999). The sections were then dehydrated and mounted in xylene-based medium for viewing. Three non-sequential sections were chosen from one random block from each spinal cord for examination. All sections were evaluated at 200, while some sections were photographed at 400 using a Nikon inverted microscope. 2.8. Light microscopy Tissue segments containing the lesion (1 cm on each side of the lesion) were paraffin embedded and cut into 5-lm-thick sections. Tissue sections were deparaffinized with xylene, stained with Haematoxylin/Eosin (H&E) and studied using light microscopy (Dialux 22 Leitz). The segments of each spinal cord contained the lesion (1 cm on each side of the lesion), were evaluated by an experienced histopathologist. Damaged neurons were counted and the histopathologic changes of the gray matter were scored on a 6-point scale (Sirin et al., 2002): 0, no lesion observed, 1, gray matter contained 1–5 eosinophilic neurons; 2, gray matter contained 5–10 eosinophilic neurons; 3, gray matter contained more than 10 eosinophilic neurons; 4, small infarction (less than one third of the gray matter area); 5, moderate infarction; (one third to one half of the gray matter area); 6, large infarction (more than half of the gray matter area). The scores from all the sections from each spinal cord were averaged to give a final score for individual
Fig. 5. Colocalization of CB2 and chymase in SCI. Cell nuclei were stained in blue with DAPI (a–c). Cells were double stained with antibodies against CB2 receptors (red; a2, b2, c2) and chymase (green; a1, b1, c1). The yellow spots indicate that chymase and CB2 colocalize (b3). Reported images are representative of triplicate experiments. Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels.
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mice. All the histological studies were performed in a blinded fashion. 2.9. Grading of motor disturbance The motor function of mice subjected to compression trauma was assessed once a day for 20 days after injury. Recovery from motor disturbance was graded using the Basso Mouse Scale (BMS) open-field score (Basso et al., 2006), since the BMS has been shown to be a valid locomotor rating scale for mice. The evaluations were made by two blind observers for all analyzed groups. Briefly, the BMS is a nine-point scale that provides a gross indication of locomotor ability and determines the phases of locomotor recovery and features of locomotion. The BMS scale ranges from 0 (indicating complete paralysis) to 9 (indicating normal hindlimb function), rating locomotion on aspects of hindlimb function such as weight support, stepping ability, coordination, and toe clearance. The BMS score was determined for ten mice in each group. 2.10. Western blot analysis for NGF, GDNF, NT-3, Trk Cytosolic and nuclear extracts were prepared as previously described (Bethea et al., 1998) with slight modifications. The levels of NGF, GDNF, NT-3, and Trk were quantified in cytosolic fraction from spinal cord tissue collected after 24 h after SCI. The filters were blocked with 1 PBS, 5% (w/v) non fat dried milk (PM) for 40 min at room temperature and subsequently probed with specific Abs NGF (1:1000, Santa Cruz Biotechnology), or GDNF
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(1:1000, Santa Cruz Biotechnology), or anti-NT-3 (1:500, Santa Cruz Biotechnology), or anti-Trk (1:1000, Santa Cruz Biotechnology) in 1 PBS, 5% w/v non fat dried milk, 0.1% Tween-20 (PMT) at 4 °C, overnight. Membranes were incubated with peroxidaseconjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of proteic lysates, they were also incubated in the presence of the antibody against a-tubulin protein (1:10,000 Sigma–Aldrich Corp.). The relative expression of the protein bands was quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (GS-700, Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst, IBM), and values were normalized to a-tubulin.
2.11. Statistical evaluation All values in the figures and text are expressed as means ± standard error of the mean (SEM) of N observations. For the in vivo studies N represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments (histological or immunohistochemistry coloration) performed on different experimental days on the tissue sections collected from all the animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons.
Fig. 6. Colocalization of CB2 receptor and GFAP after SCI. Cells were double stained with antibodies against GFAP (green; a1, b1, c1) and CB2 receptors (red; a2, b2, c2). Reported images are representative of triplicate experiments. Spinal cord sections revealed increased astrogliosis (GFAP + cells) in the perilesioned area from SCI (panel b1). Colocalization of GFAP and CB2 was evident in panel b3. On the contrary, no GFAP positive cells were found in the spinal cord tissues after SCI collected from mice which have been treated with PEA (panel c1). Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels.
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3. Results 3.1. PEA reduces the severity of spinal cord trauma and MCs infiltration In order to better study the severity of the trauma at the level of the perilesional area, the spinal cord tissues were stained 24 h after injury by Haematoxylin/Eosin (H&E). In agreement with our previous observation (Genovese et al., 2008), histology showed prominent and thickened blood vessels, ischemic changes and gliosis in the cord parenchyma (Fig. 1b). Infiltration of the parenchyma by numerous macrophages, scattered lymphocytes, and occasional neutrophils was also evident (panels b1 and b2). In this study we have confirmed that the treatment with PEA significantly reduced the spinal cord injury damage at 24 h after trauma (Fig. 1c, see histological score Fig. 1d). The histological pattern of SCI described above appeared to be correlated with cellular changes into the spinal cord. In particular, a presence of MCs was observed in the spinal cord tissues collected at 24 h after SCI (Fig. 2, panel b) mainly localized in the perivascular area (see particles panel b1). Please note that in panels b and b1 of Fig. 2 the arrow indicate the presence of mast cell and arrowhead showed the presence of MCs in degranulation phase. Cells showing to have metachromatic granules when stained with acidified Toluidine blue were identified as mast cells. On the contrary, significant less MCs density and degranulation were observed in the spinal cord tissues after SCI collected from mice which have been treated with PEA (Fig. 2, panel c). No granules were found in the spinal cord tissues from sham-operated mice (Fig. 2, panel a).
3.2. PEA administration reduces chymase and tryptase expression during SCI In order to test whether PEA treatment may modulate and direct the inflammatory response through the regulation of the serine peptidases, we analyzed by immunohistochemistry the spinal cord expression of chymase and tryptase. There was no staining for chymase and tryptase in the spinal cord tissues obtained from the sham-operated mice (Figs. 3a and 4a). A substantial increase in chymase and tryptase expression was found mainly localized in MCs in spinal cord tissues collected at 24 h after SCI (Figs. 3b and 4b). Spinal cord expression of chymase and tryptase were attenuated in the spinal cord from mice that have received PEA treatment (Figs. 3c and 4c). 3.3. PEA treatment reduces the increased CB2 cannabinoid receptor and chymase expression after SCI To address the potential involvement of CB2 cannabinoid receptors in SCI and the cells implicated in the effects of PEA, we evaluated in spinal cord cross sections the co-localization of CB2 with chymase, a marker for mast cells (Fig. 5). The CB2 receptor (panels a2–b2–c2) and chymase (panels a1–b1–c1) was highly expressed in the spinal cord tissues collected at 24 h after SCI (panels b1 and b2). Colocalization of chymase and CB2 by confocal microscopy was evident in panel b3. The yellow spots indicate that chymase and CB2 colocalize, and are present mainly around the vessels and in inflammatory cells (panels b3). On the contrary, no positive staining for CB2 (panel c2) and chymase (panel c1) was observed in
Fig. 7. Localization of CB2 receptor and CD11b after SCI. Cells were double stained with antibodies against CD11b (green; a1, b1, c1) and CB2 receptors (red; a2, b2, c2). Microglial cells (CD11b-positive cells) expressed CB2 receptor as shown in panel b3. Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels.
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Fig. 8. Effect of PEA on NGF, GDNF and NT3 expression. NGF, GDNF and NT-3 expression was evaluated by immunohistochemistry (A–C). SCI reduced neurotrophic factors expression (Ab, Bb and Cb, see score panels d), while PEA treatment restored NGF, GDNF and NT-3 expression (Ac, Bc and Cc, see score panels d). By Western Blot analysis, a basal level of NGF, GDNF, and NT-3 was detected in the spinal cord from sham-operated animals (Ae, Be and Ce, respectively), whereas NGF, GDNF and NT-3 levels were reduced in SCI mice. PEA treatment prevented the SCI-induced neurotrophic factors degradation (Ae, Be and Ce, respectively). a-Tubulin was used as internal control. A representative blot of three independent experiments is shown, and densitometry analysis of all animals is reported (n = 5 mice from each group). ⁄⁄p < 0.01, and ⁄⁄⁄p < 0.001 vs. Sham + vehicle; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. SCI.
the spinal cord tissues collected from mice that have received PEA treatment (Fig. 5). Please note that in uninjured spinal cords we found no CB2 (panel a2) and chymase (panel a1) immunoreactivity. 3.4. PEA treatment reduces astrocyte activation and increased CB2 cannabinoid receptor expression after SCI Further analysis was performed to elucidate the nature of cells targeting by PEA. Spinal cord sections revealed increased astrogliosis (GFAP + cells) in the perilesional area after SCI (Fig. 6, panel b1). CB2 expression was induced in spinal cord tissue following trauma (Fig. 5, panel b2); moreover, there was evident colocalization of GFAP and CB2 (Fig. 6, panel b3). On the contrary, a significant number of non-GFAP positive cells were found in the spinal cord of PEA-treated mice (Fig. 6, panel c1).
3.5. PEA treatment reduces microglial activation and increased CB2 cannabinoid receptor expression after SCI Microglial cells were activated following SCI as shown by increased CD11b-positive staining (Fig. 7, panel b1). Moreover, microglial cells expressed CB2 receptor as shown in Fig. 7, panel b3. 3.5.1. Effect of PEA on neurotrophic factors levels after SCI To test whether PEA modulates the inflammatory process through regulation of the neutrophic factors levels, we have examined NGF, GDNF and NT-3 levels in the perilesioned zone both by immunohistochemistry and western blot analysis (Fig. 8A–C). In the spinal cord tissues collected at 24 h after the trauma, neurotrophic factors expression levels were significantly reduced in comparison to sham animals (Fig. 8A panels b and d, B panels b
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Fig. 8 (continued)
and d, and C panels b and d). Particularly, in the Western blot analysis it was evident that reduction in NGF and GDNF was about 65% and 67%, whereas for NT-3 the trend of decrease was 20% (Fig. 8A panel e, B panel e, C panel e). The treatment with PEA significantly restore the levels of three neurotrophic factors up to that of uninjured mice (Fig. 8A, panels d and e, B panel d and e, C panels d and e). 3.5.2. Effect of PEA on TrkA expression after SCI To investigate the mechanisms by which treatment with PEA might modulate the expression of neurotrophins, we also evaluated the expression of tyrosine protein kinase, TrkA, by western blot in spinal cord tissue homogenates after SCI. A significant increase in TrkA expression levels were observed in SCI mice (Fig. 9A). Treatment of mice with PEA significantly reduced the expression of Trk (Fig. 9A, ⁄p < 0.05 vs. Sham and ##p < 0.01 vs. SCI + vehicle). 3.5.3. Effect of PEA treatment on motor function and DAT expression As previously demonstrated (Genovese et al., 2008), the above described inflammatory process is associated with loss of motor
function (Fig. 9B). In particular, while motor function was only slightly impaired in sham-operated mice (data not shown), mice subjected to SCI displayed significant deficits in hind limb movement (Fig. 9B). PEA treatment significantly ameliorated the functional deficits induced by SCI. Moreover, to test whether PEA treatment reduce the motor function alteration through regulation of the expression DAT, we have examined the DAT expression in the perilesioned zone by immunohistochemistry. In the spinal cord tissues collected at 24 h after the trauma, the expression of DAT were reduced in comparison to sham animals (Fig. 9C, panel b). The treatment with PEA restored DAT expression up to that of uninjured mice (Fig. 9C, panel c). 4. Discussion Primary injury to the adult spinal cord is irreversible, whereas secondary degeneration is delayed and therefore amenable to intervention. Accordingly, several studies have shown that therapies targeting various factors involved in the secondary degeneration cascade lead to tissue sparing and improved behavioral outcomes in spinal cord-injured animals (Bao et al., 2003;
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Fig. 8 (continued)
Cuzzocrea et al., 2006; Glaser et al., 2006). Strategies have been used to focus on the prevention of further neuronal death or to induce the regeneration of these cells in SCI animal models. For instance, GDNF has been shown to have a potent survival-promoting effect on various neuronal populations, including dopaminergic (Sariola and Saarma, 2003), noradrenergic (Saarma, 2000), cortical (Pezeshki et al., 2003), retinal ganglion (Klocker et al., 1997), sensory (Del Fiacco et al., 2002; Laurikainen et al., 2000), and motor neurons (Shneider et al., 2009). In addition, GDNF induced the growth of motor and sensory axons and remyelination in laboratory rats with partial and complete spinal cord transections (Blesch and Tuszynski, 2003). In this study, protein analysis indicated that the levels of GDNF, NT-3 and NGF were down-regulated by spinal cord compression in mice, while for the first time an local and sustained increase in the NGF, NT-3 and GDNF expression in the perilesioned tissue following intraperitoneally administration of PEA was shown. Neurotrophins signal through high-affinity receptor tyrosine kinases, TrkA–C, and/or the low-affinity receptor p75 (Bibel and Barde, 2000). All changes in TrkA expression are correlated with inflammation, in fact the up-regulation of TrkA may be dependent
on some inflammatory mediators such as IL-1b, which is known to be engaged in the NGF production. Interestingly, administration of PEA significantly reduced TrkA expression in spinal cord tissue. Here, we confirm that PEA treatment exerts beneficial effects in a mice model of SCI, as previously reported (Genovese et al., 2008). In accordance with previous data (Genovese et al., 2008), in this study the authors administered PEA at 6 and 12 h after SCI. In this study, we extend our results by showing that PEA reduces (1) mast cell infiltration, (2) chymase/tryptase expression, (3) the loss of DAT level, (4) astrogliosis, (5) microglial activation, (6) the loss of NGF, GDNF and NT-3 expression, and (7) Trk expression. PEA has been shown to be effective in several experimental models of inflammation, both of immunogenic and neurogenic origin (Conti et al., 2002; Mazzari et al., 1996). Despite its various described pharmacological properties, the cellular/receptor mechanism responsible for the actions of PEA is still debated. The first hypothesis on the mechanism of action of PEA was formulated when Aloe et al. (1993) introduced the ALIA acronym (Autacoid Local Inflammation Antagonism) to indicate that some endogenous N-acylethanolamines, like PEA, exerted a local antagonism on
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Fig. 9. Effect of PEA on TrkA, DAT expression, and hind limb motor disturbance after spinal cord injury. Western blot analysis demonstrated increased expression of tyrosine kinase receptor (TrkA) after SCI (A). PEA treatment restored TrkA expression (A). Mice were scored in an open-field environment by two individuals who were blinded to experimental conditions. Individual scores from each animal were pooled and averaged, for a maximum of 9 points for the BMS score (B). Treatment with PEA reduces the motor disturbance after SCI. DAT immunoreactivity was performed 24 h after SCI (C). Significant reduction in spinal cord sections was observed after injury (C, panel b). PEA treatment returned DAT almost to the Sham level (Cc). A representative blot of three independent experiments is shown. Values shown are means ± SEM of 10 mice for each group. ⁄p < 0.05 and ⁄⁄⁄p < 0.001 vs. Sham + vehicle; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. SCI + vehicle.
inflammation. The effect was first attributed to MC activity control (Aloe et al., 1993).
Although PEA does not bind with high affinity to CB1 or CB2 receptors, it still maintains CB-like anti-inflammatory actions in
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several mast cells-mediated experimental models of inflammation. Moreover, various studies have hypothesized that PEA may act via indirect interaction with CB2 receptors (Farquhar-Smith et al., 2002; Lambert and Di Marzo, 1999; Onaivi et al., 2008). In fact, it has been showed that the use of SR144528, a CB2 specific receptor antagonist, eliminated the antinociceptive effects of PEA (Calignano et al., 1998, 2001; Conti et al., 2002; Farquhar-Smith and Rice, 2003). Then, it was found that PEA could potentiate the effect of anandamide on CB or vanilloid receptor (VR1) (De Petrocellis et al., 2001; Hansen, 2010), the so-called ‘‘entourage effect’’. Considering the known changes of endocannabinoid system in the lesioned spinal cord (increases in 2-AG and CB2, decreases in CB1), PEA may indirectly regulate CB receptors expression through the increase in endocannabinoid tone (Costa et al., 2008) or the modulation of pro-inflammatory cytokines. Indeed, CB2 receptors would play a crucial role in limiting the spreading of this neuroinflammatory process. Pro-inflammatory cytokines present in the CNS could potentially regulate the expression of the CB2 receptor on microglial cells, astrocytes, and MCs, whose expression level is likely to be important in the regulation of inflammation in the CNS during autoimmunity (Maresz et al., 2005). In this study, we have clearly showed that CB2 receptor expression is increased following spinal cord trauma, coinciding with the appearance of microglial cells, astrocytes, and MCs; PEA treatment reduced the expression of CB2 receptor on these cells in the perilesioned zone. Future studies are needed in order to clarify these observations, as well as the involvement of peroxisome proliferator-activated receptors (PPARs) expression. PEA, in fact, is considered an endogenous activator of PPAR-a, interacting with this receptor to inhibit inflammatory response with a potency comparable to that of the synthetic PPAR-a agonist GW7647 (Lo Verme et al., 2005). We report here that spinal cord trauma caused a significant infiltration (identified morphologically by Toluidine blue staining) and activation (evaluated as chymase and tryptase expression) of MCs in the spinal cord tissues at 24 h, whereas treatment with PEA reduced both the infiltration and the activation. These observations are in agreement with other studies in which have been shown that PEA treatment is an effective tool to naturally control mast cells hyperactivity, which occurs not only in inflammation, but also in inflammatory hyperalgesia (Mazzari et al., 1996; Rice et al., 2002) and neuropathic hyperalgesia (Theodosiou et al., 1999; Zuo et al., 2003). MCs possess several biological mediators that are released from cytoplasmic granules primarily due to stimulus-induced degranulation, including vasoactive amines such as histamine, proteoglycans (mainly heparin and chondroitin sulphate), neutral serine proteases, such as tryptase and chymase, cytokines and growth factors, such as vascular endothelial growth factor, basic fibroblast growth factor, NGF, transforming growth factor-b and tumor necrosis factor (TNF)-a (Gordon and Galli, 1991; Kownatzki, 1982; Raposo et al., 1997). The spinal cord is known to receive dopaminergic projections originating from brain areas. All subtypes of dopamine receptors, including the D1 subtype, are present in the mammalian spinal cord (e.g. rats, mice, cats and monkeys). Moreover, Lapointe and collaborators (Lapointe et al., 2009) suggested that the spinal dopaminergic system may play a role in mediating locomotor-like movements in SCI subjects as well as normal locomotor behaviours in intact vertebrate species. In this contest, DAT, that terminates the signal of the neurotransmitter, could play a central role. In our model PEA reduced the loss of functional dopamine transporters induced by compression of spinal cord. In summary, the effect of PEA against SCI may be mediated by the inhibition of neutrophil accumulation (Farquhar-Smith and Rice, 2003; Rice et al., 2002), as well as by the ability of PEA of negatively modulating the secretion of mediators from MCs, the
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activation of microglia and astrocytes all expressing CB2 receptors, independently from CB receptor activation, via a so called ‘ALIA’ mechanism or ‘entourage effect’. However, these hypothesis identify in PEA a crucial therapeutic intervention diminishing the immune/inflammatory response and promoting the initiation of neurotrophic substance after SCI. 5. Conflicts of Interest The authors declare no conflicts of interest in relationship to performing this study. Acknowledgment The authors thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study. References Aloe, L., Leon, A., Levi-Montalcini, R., 1993. A proposed autacoid mechanism controlling mastocyte behaviour. Agents Actions 39 (Spec. No.), C145–C147. Andoh, A., Deguchi, Y., Inatomi, O., Yagi, Y., Bamba, S., Tsujikawa, T., Fujiyama, Y., 2006. Immunohistochemical study of chymase-positive mast cells in inflammatory bowel disease. Oncol. Rep. 16, 103–107. Bao, F., DeWitt, D.S., Prough, D.S., Liu, D., 2003. Peroxynitrite generated in the rat spinal cord induces oxidation and nitration of proteins: reduction by Mn (III) tetrakis (4-benzoic acid) porphyrin. J. Neurosci. Res. 71, 220–227. Basso, D.M., Fisher, L.C., Anderson, A.J., Jakeman, L.B., McTigue, D.M., Popovich, P.G., 2006. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J. Neurotrauma 23, 635–659. Berdyshev, E.V., 2000. Cannabinoid receptors and the regulation of immune response. Chem. Phys. Lipids 108, 169–190. Berdyshev, E.V., Boichot, E., Germain, N., Allain, N., Anger, J.P., Lagente, V., 1997. Influence of fatty acid ethanolamides and delta9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells. Eur. J. Pharmacol. 330, 231–240. Bethea, J.R., Castro, M., Keane, R.W., Lee, T.T., Dietrich, W.D., Yezierski, R.P., 1998. Traumatic spinal cord injury induces nuclear factor-kappaB activation. J. Neurosci. 18, 3251–3260. Bilsland, L.G., Dick, J.R., Pryce, G., Petrosino, S., Di Marzo, V., Baker, D., Greensmith, L., 2006. Increasing cannabinoid levels by pharmacological and genetic manipulation delay disease progression in SOD1 mice. FASEB J. 20, 1003–1005. Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M.G., Ligresti, A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E.J., Gangadharan, U., Hobbs, C., Di Marzo, V., Doherty, P., 2003. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J. Cell Biol. 163, 463–468. Blesch, A., Tuszynski, M.H., 2003. Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination. J. Comp. Neurol. 467, 403–417. Calignano, A., La Rana, G., Giuffrida, A., Piomelli, D., 1998. Control of pain initiation by endogenous cannabinoids. Nature 394, 277–281. Calignano, A., La Rana, G., Piomelli, D., 2001. Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide. Eur. J. Pharmacol. 419, 191–198. Conti, S., Costa, B., Colleoni, M., Parolaro, D., Giagnoni, G., 2002. Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat. Br. J. Pharmacol. 135, 181–187. Costa, B., Comelli, F., Bettoni, I., Colleoni, M., Giagnoni, G., 2008. The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and antihyperalgesic effects in a murine model of neuropathic pain: involvement of CB(1), TRPV1 and PPARgamma receptors and neurotrophic factors. Pain 139, 541–550. Cravatt, B.F., Saghatelian, A., Hawkins, E.G., Clement, A.B., Bracey, M.H., Lichtman, A.H., 2004. Functional disassociation of the central and peripheral fatty acid amide signaling systems. Proc. Natl. Acad. Sci. USA 101, 10821–10826. Cuzzocrea, S., Genovese, T., Mazzon, E., Crisafulli, C., Min, W., Di Paola, R., Muia, C., Li, J.H., Esposito, E., Bramanti, P., Xu, W., Massuda, E., Zhang, J., Wang, Z.Q., 2006. Poly(ADP-ribose) glycohydrolase activity mediates post-traumatic inflammatory reaction after experimental spinal cord trauma. J. Pharmacol. Exp. Ther. 319, 127–138. Cuzzocrea, S., Riley, D.P., Caputi, A.P., Salvemini, D., 2001. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/ reperfusion injury. Pharmacol. Rev. 53, 135–159. De Petrocellis, L., Davis, J.B., Di Marzo, V., 2001. Palmitoylethanolamide enhances anandamide stimulation of human vanilloid VR1 receptors. FEBS Lett. 506, 253– 256. Del Fiacco, M., Quartu, M., Serra, M.P., Follesa, P., Lai, M.L., Bachis, A., 2002. Topographical localization of glial cell line-derived neurotrophic factor in the
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Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi
Effects of palmitoylethanolamide on release of mast cell peptidases and neurotrophic factors after spinal cord injury Emanuela Esposito a, Irene Paterniti a, Emanuela Mazzon b, Tiziana Genovese a, Rosanna Di Paola b, Maria Galuppo a, Salvatore Cuzzocrea a,b,⇑ a b
Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Italy IRCCS Centro Neurolesi ‘‘Bonino-Pulejo’’, Messina, Italy
a r t i c l e
i n f o
Article history: Received 15 November 2010 Received in revised form 14 February 2011 Accepted 14 February 2011 Available online 25 February 2011 Keywords: Mast cells GDNF NT-3 NGF CB2 Spinal cord injury
a b s t r a c t Spinal cord injury (SCI) has a significant impact on quality of life, expectancy, and economic burden, with considerable costs associated with primary care and loss of income. The complex pathophysiology of SCI may explain the difficulty in finding a suitable therapy for limiting neuronal injury and promoting regeneration. Although innovative medical care, advances in pharmacotherapy have been limited. The aim of the present study was to carefully investigate molecular pathways and subtypes of glial cells involved in the protective effect of PEA on inflammatory reaction associated with an experimental model of SCI. The compression model induced by applying an aneurysm clip to the spinal cord in mice is closer to the human situation, since it replicates the persistence of cord compression. Spinal cord trauma was induced in mice by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. Repeated PEA administration (10 mg/kg i.p., 6 and 12 h after SCI) significantly reduced the degree of the severity of spinal cord trauma through the reduction of mast cell infiltration and activation. Moreover, PEA treatment significantly reduced the activation of microglia and astrocytes expressing cannabinoid CB2 receptor after SCI. Importantly, the protective effect of PEA involved changes in the expression of neurotrophic factors, and in spinal cord dopaminergic function. Our results enhance our understanding about mechanisms related to the anti-inflammatory property of the PEA suggesting that this N-acylethanolamine may represent a crucial therapeutic intervention both diminishing the immune/inflammatory response and promoting the initiation of neurotrophic substance after SCI. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Spinal cord injury (SCI) has a significant impact on quality and expectancy of life, and economic burden, with considerable costs associated with primary care and loss of income. During the first 24 h post-injury, the synthesis and release of the potent proinflammatory mediators are markedly elevated at the lesion site where they play a major role in the development of secondary tissue degeneration after SCI in animals and in humans. Neurons continue to die for hours after SCI due to several mechanisms including exitotoxicity, vascular abnormalities and inflammatory response that can contribute to evolution of SCI. Historically, administration of high-dose methylprednisolone (MP), acutely after SCI has been considered the standard of care ⇑ Corresponding author. Address: Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi, 98100 Messina, Italy. Fax: +39 090 2213300. E-mail address: [email protected] (S. Cuzzocrea). 0889-1591/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2011.02.006
in the United States. Although clinical results were initially promising, there have been growing concerns that the modest neurological improvements seen with high-dose MP treatment in injured patients are not worth the associated risks. Therefore, there is a critical need to develop new pharmacologic therapies for treatment of SCI. Regeneration and compensatory sprouting of axons are very limited after injury to the adult SC. This deficiency has been attributed to a lack of growth promoting factors and the presence of inhibitory molecules and physical barriers to axonal regeneration (Schwab, 2002). Resident microglia and macrophages originating from blood are two key cell types related to the occurrence of neuronal degeneration in CNS after traumatic injury. In particular, when SCI occurs, microglia in parenchyma is activated and macrophages in circulation cross the blood–brain barrier to act as intrinsic spinal phagocytes. Therefore, these cells can release various neurotrophic peptides, which are excellent substrates for neurite outgrowth. In addition, various study have clearly demonstrated that mast cells (MCs) have long been known to participate in the inflammatory process, in fact MCs are present and recruited to
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all inflammatory sites (Andoh et al., 2006; Mican and Metcalfe, 1990). However, it has only recently become clear that MCs play an important role in orchestrating the whole inflammatory process from initiation events to chronic activation (Pejler et al., 2007). MCs are the first immune cells to considerably stimulate the inflammatory process due to a rapid release of pro-inflammatory and vasoactive mediators. MCs make and secrete an abundance of peptidases, which are stored in such large amounts in granules. The main peptidases (tryptases, chymases, carboxypeptidase A3, and dipeptidylpeptidase I) were best known as markers of degranulation, they are released locally in response to MC stimulation and can be distributed systemically and detected in blood (Trivedi and Caughey, 2010). During the last years, the endocannabinoid system has attracted the attention of researchers working in neural damage and repair and is being considered a promising target for the development of new therapies (Di Marzo, 2008; Pacher et al., 2006). Moreover, some components of the endocannabinoid system, that are constitutively expressed in the normal spinal cord such as diacylglycerol (DAG) lipases, fatty acid amide hydrolase and CB1 receptor (Bisogno et al., 2003; Cravatt et al., 2004; Romero et al., 2002; Tsou et al., 1998a,b) are modulated after neurodegenerative diseases or after peripheral nerve lesions (Bilsland et al., 2006; Lim et al., 2003;
Petrosino et al., 2007; Shoemaker et al., 2007; Witting et al., 2004; Wotherspoon et al., 2005; Zhang et al., 2003). Furthermore, recently have been reported that the endocannabinoid system is activated in a clinically relevant model of traumatic SCI in rats (Garcia-Ovejero et al., 2009). The endogenous fatty acid palmitoylethanolamide (PEA) is one of the members of N-acyl-ethanolamines family. In addition to the hypothesis that PEA has potent immunoregulatory properties (Aloe et al., 1993; Berdyshev, 2000; Berdyshev et al., 1997; Facci et al., 1995; Mazzari et al., 1996; Ross et al., 2000; Scarampella et al., 2001), recent data have demonstrated that PEA may also play a key role in the regulation of complex systems involved in the inflammatory response, pruritus, neurogenic and neuropathic pain (Di Marzo et al., 2000). Recently, we have also clearly demonstrated that the treatment with PEA significantly reduced the inflammation associated with experimental SCI in mice (Genovese et al., 2008). In particular, PEA protected against locomotor dysfunction following SCI. However, the basic molecular mechanisms as well as cellular types accounting for the anti-inflammatory properties of PEA in SCI have not yet been carefully investigated. Thus, the aim of the present study is to carefully investigate molecular pathways and subtypes of glial cells involved in the
Fig. 1. PEA reduces the severity of spinal cord trauma. The low-magnification images of spinal cord after compression injury show tissue disorganization, white matter alteration, and inflammation in the perilesional area at 24 h after injury (panels b, b1). A significant protection from the SCI was evident in the tissue collected from PEAtreated mice (c). The histological score was made by an independent observer (d). ⁄⁄⁄p<0.001 vs. Sham and ##p<0.01 vs. SCI. Values shown are means ± SE mean of 10 mice for each group.
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protective effect of PEA in SCI, and to identify in PEA a crucial therapeutic intervention after SCI.
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2.3. Experimental groups and treatments Mice were randomly allocated into the following groups:
2. Methods 2.1. Animals Male adult CD1 mice (25–30 g, Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986). 2.2. SCI Mice were anaesthetized using chloral hydrate (400 mg/kg body weight). We used the clip compression model described by Rivlin and Tator (1978) and produced SCI by extradural compression of a section of the SC exposed via a four-level T5–T8 laminectomy, in which the prominent spinous process of T5 was used as a surgical guide. With the aneurysm clip applicator oriented in the bilateral direction, an aneurysm clip with a closing force of 24 g was applied extradurally at T5–T8 level. The clip was then rapidly released with the clip applicator, which caused SC compression. In the injured groups, the cord was compressed for 1 min and then the clips were removed. Following surgery, 1.0 cc of saline was administered subcutaneously in order to replace the blood volume lost during the surgery. During recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. During this time period, the animals’ bladders were manually voided twice a day until the mice were able to regain normal bladder function. Sham injured animals were only subjected to laminectomy.
(i) Sham + vehicle group. Mice were subjected to laminectomy but the aneurysm clip was not applied, and treated intraperitoneally (i.p.) with vehicle (10% polyethylene glycol and 5% Tween 80 sterile distilled water) (N = 30). (ii) Sham + PEA group. Identical to Sham + vehicle group except for the administration of PEA (10 mg/kg) 6 and 12 h after laminectomy (N = 30). (iii) SCI + vehicle group. Mice were subjected to SCI and were administered vehicle (10% polyethylene glycol and 5% Tween 80 sterile distilled water, i.p. bolus) at 6 and 12 h after SCI (N = 30). (iv) SCI + PEA group. Mice were subjected to SCI and administered PEA (10 mg/kg) at 6 and 12 h after SCI (N = 30). PEA was purchased from Tocris bioscience (UK); The doses of PEA (10 mg/kg) used here were based on previous in vivo study (Genovese et al., 2008). In a separate set of experiments to investigate the motor score, additional animals were observed until 20 days after SCI. PEA (10 mg/kg) was administered 6 and 12 h after SCI and daily until day 19. 2.4. Tissue processing At the end of the experimental period (24 h), animals were deeply anesthetized with sodium pentobarbital and then perfused transcardially with cold phosphate-buffered saline (PBS, 0.1 M) followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Spinal cord tissues were removed under magnified vision. Tissue segments containing the lesion (1 cm on each side of the lesion) were paraffin embedded, cut into longitudinal sections for posterior area
Fig. 2. Staining of MCs. The slides stained with acidified Toluidine blue were also shown to have dark lilac blue granules, which identifies the cells as mast cells. Many of the mast cells are arranged separately in concentric rings around small blood-vessels. Different gradients of staining intensity were evident in the granulated and degranulated mast cells. In the spinal cord tissues collected at 24 h after SCI there is the presence of MCs (panels b, b1) mainly localized in the perivascular area. On the contrary, PEA reduced MC infiltration in the spinal cord tissues after SCI (panel c). No granules were found in the spinal cord tissues from sham-operated mice (panel a).
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of the spinal cord, and processed for different immunohistochemical procedures. Identification of mast cells was carried out following the application of Toluidine blue staining protocols described previously by Michaloudi and Papadopoulos (1999). For immunoblotting analysis, mice (N = 10) were anaesthetized as described above and exsanguinated via cardiac puncture. Spinal cords were dissected, and cleaned from meninges and nerve roots. One centimeter of the cord centered at the injury site was dissected and homogenized for preparing cytosolic and nuclear extracts. 2.5. Immunohistochemical localization of chymase, tryptase and dopamine transporter (DAT) At the 24 h after SCI, the tissues were fixed in PBS-buffered formaldehyde and 8 lm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60%
(v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated overnight with anti-chymase (purified human skin chymase) polyclonal antibody (1:1000), or anti-tryptase (mast cell tryptase isolated from human lung) polyclonal antibody (1:1000), antiDAT antibody (1:500). Sections were washed and incubated with secondary antibody. Specific labeling were detected with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vector Lab. Inc., Burlingame, CA). To verify the binding specificity for chymase, tryptase and DAT, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). Immunocytochemistry photographs (n = 5 photos from each samples collected from all mice in each experimental group) were assessed by densitometry as previously described (Cuzzocrea
Fig. 3. PEA reduces chymase expression. There was no staining for chymase in spinal cord tissues obtained from the sham-operated mice (panel a). A substantial increase in serine peptidases chymase expression was found mainly localized in MCs in the spinal cord tissues collected at 24 h after SCI (panel b). Chymase levels were attenuated in mice treated with PEA (panel c). This figure is representative of at least three experiments performed on different experimental days. To verify the binding specificity for chymase, some sections were also incubated with only the secondary antibody (no primary). In these situations no positive staining was found in the sections (d) indicating that the immunoreactions were positive in all the experiments carried out. ⁄⁄⁄p < 0.001 vs. Sham + vehicle and ###p < 0.001 vs. SCI. Values shown are means ± SE mean of 10 mice for each group.
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et al., 2001; Shea, 1994) by using Optilab Graftek software on a Macintosh personal computer. 2.6. Immunofluorescence After deparaffinization and rehydration, detection of CB2, Neu and CD11b was carried out after boiling in 0.01 M citrate buffer
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for 4 min. Endogenous peroxidase was quenched with 0.3% (vol/ vol) hydrogen peroxide in 60% (vol/vol) methanol for 30 min. Non-specific adsorption was minimized by incubating the section in 2% (vol/vol) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated with mouse monoclonal anti-mast Cell Chymase (1:100,
Fig. 4. PEA reduces tryptase expression. There was no staining for tryptase in spinal cord tissues obtained from the sham-operated mice (panel a). A substantial increase in expression was found mainly localized in mast cells in the spinal cord tissues collected at 24 h after SCI (panel b). Tryptase levels were attenuated in mice treated with PEA (panel c). This figure is representative of at least three experiments performed on different experimental days. To verify the binding specificity for chymase, some sections were also incubated with only the secondary antibody (no primary). In these situations no positive staining was found in the sections (d) indicating that the immunoreactions were positive in all the experiments carried out. ⁄⁄⁄p < 0.001 vs. Sham + vehicle and ###p < 0.001 vs. SCI. Values shown are means ± SE mean of 10 mice for each group.
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v/vol) (Thermo Scientific), or with polyclonal rabbit anti-CB2 (mapping at the C-terminus of CB2 of human origin 1:100, v/v) (Santa Cruz, Biotechnology), or with rabbit anti-Neu (raised again peptide mapping at the C-terminus of Neu of human 1:100, v/v) (Santa Cruz Biotechnology), or with mouse monoclonal anti-CD11b (mouse anti bovine 1:100, v/v) (Serotec), or with mouse monoclonal anti-GFAP (1:100, v/v) (Santa Cruz Biotechnology) antibody in a humidified chamber for 1 h at 37 °C. Sections were washed with PBS and were incubated with secondary antibody FITC-conjugated anti-mouse antibody (1:100 in PBS, v/v) (Jackson, West Grove, Pennsylvania), and with TEXAS RED-conjugated anti-rabbit antibody (1:100 in PBS, v/v) (Jackson, West Grove, Pennsylvania) for 1 h at 37 °C. Sections were washed and for nuclear staining 40 ,60 diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt; Germany) 0.5 lg/ml in PBS was added. Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels. Optical sections of fluorescence specimens were obtained using a HeNe laser (543 nm), a laser UV (361–365 nm) and an argon laser (458 nm) at a 1-min, 2-s scanning speed with up to 8 averages; 1.5-lm sections were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly labeled pixels and applying settings that allowed clear visualization of structural details while keeping the highest pixel intensities close to 200. The same settings were used for all images obtained from the other samples that had been processed in parallel. Digital images were cropped and figure montages prepared using Adobe Photoshop 7.0 (Adobe Systems; Palo Alto, CA).
2.7. Staining of mast cells Spinal cord sections were cut 5 lm thick and stained with 0.25% Toluidine blue, pH 2.5, for 45 min at room temperature (Michaloudi and Papadopoulos, 1999). The sections were then dehydrated and mounted in xylene-based medium for viewing. Three non-sequential sections were chosen from one random block from each spinal cord for examination. All sections were evaluated at 200, while some sections were photographed at 400 using a Nikon inverted microscope. 2.8. Light microscopy Tissue segments containing the lesion (1 cm on each side of the lesion) were paraffin embedded and cut into 5-lm-thick sections. Tissue sections were deparaffinized with xylene, stained with Haematoxylin/Eosin (H&E) and studied using light microscopy (Dialux 22 Leitz). The segments of each spinal cord contained the lesion (1 cm on each side of the lesion), were evaluated by an experienced histopathologist. Damaged neurons were counted and the histopathologic changes of the gray matter were scored on a 6-point scale (Sirin et al., 2002): 0, no lesion observed, 1, gray matter contained 1–5 eosinophilic neurons; 2, gray matter contained 5–10 eosinophilic neurons; 3, gray matter contained more than 10 eosinophilic neurons; 4, small infarction (less than one third of the gray matter area); 5, moderate infarction; (one third to one half of the gray matter area); 6, large infarction (more than half of the gray matter area). The scores from all the sections from each spinal cord were averaged to give a final score for individual
Fig. 5. Colocalization of CB2 and chymase in SCI. Cell nuclei were stained in blue with DAPI (a–c). Cells were double stained with antibodies against CB2 receptors (red; a2, b2, c2) and chymase (green; a1, b1, c1). The yellow spots indicate that chymase and CB2 colocalize (b3). Reported images are representative of triplicate experiments. Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels.
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mice. All the histological studies were performed in a blinded fashion. 2.9. Grading of motor disturbance The motor function of mice subjected to compression trauma was assessed once a day for 20 days after injury. Recovery from motor disturbance was graded using the Basso Mouse Scale (BMS) open-field score (Basso et al., 2006), since the BMS has been shown to be a valid locomotor rating scale for mice. The evaluations were made by two blind observers for all analyzed groups. Briefly, the BMS is a nine-point scale that provides a gross indication of locomotor ability and determines the phases of locomotor recovery and features of locomotion. The BMS scale ranges from 0 (indicating complete paralysis) to 9 (indicating normal hindlimb function), rating locomotion on aspects of hindlimb function such as weight support, stepping ability, coordination, and toe clearance. The BMS score was determined for ten mice in each group. 2.10. Western blot analysis for NGF, GDNF, NT-3, Trk Cytosolic and nuclear extracts were prepared as previously described (Bethea et al., 1998) with slight modifications. The levels of NGF, GDNF, NT-3, and Trk were quantified in cytosolic fraction from spinal cord tissue collected after 24 h after SCI. The filters were blocked with 1 PBS, 5% (w/v) non fat dried milk (PM) for 40 min at room temperature and subsequently probed with specific Abs NGF (1:1000, Santa Cruz Biotechnology), or GDNF
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(1:1000, Santa Cruz Biotechnology), or anti-NT-3 (1:500, Santa Cruz Biotechnology), or anti-Trk (1:1000, Santa Cruz Biotechnology) in 1 PBS, 5% w/v non fat dried milk, 0.1% Tween-20 (PMT) at 4 °C, overnight. Membranes were incubated with peroxidaseconjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of proteic lysates, they were also incubated in the presence of the antibody against a-tubulin protein (1:10,000 Sigma–Aldrich Corp.). The relative expression of the protein bands was quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (GS-700, Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst, IBM), and values were normalized to a-tubulin.
2.11. Statistical evaluation All values in the figures and text are expressed as means ± standard error of the mean (SEM) of N observations. For the in vivo studies N represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments (histological or immunohistochemistry coloration) performed on different experimental days on the tissue sections collected from all the animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons.
Fig. 6. Colocalization of CB2 receptor and GFAP after SCI. Cells were double stained with antibodies against GFAP (green; a1, b1, c1) and CB2 receptors (red; a2, b2, c2). Reported images are representative of triplicate experiments. Spinal cord sections revealed increased astrogliosis (GFAP + cells) in the perilesioned area from SCI (panel b1). Colocalization of GFAP and CB2 was evident in panel b3. On the contrary, no GFAP positive cells were found in the spinal cord tissues after SCI collected from mice which have been treated with PEA (panel c1). Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels.
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3. Results 3.1. PEA reduces the severity of spinal cord trauma and MCs infiltration In order to better study the severity of the trauma at the level of the perilesional area, the spinal cord tissues were stained 24 h after injury by Haematoxylin/Eosin (H&E). In agreement with our previous observation (Genovese et al., 2008), histology showed prominent and thickened blood vessels, ischemic changes and gliosis in the cord parenchyma (Fig. 1b). Infiltration of the parenchyma by numerous macrophages, scattered lymphocytes, and occasional neutrophils was also evident (panels b1 and b2). In this study we have confirmed that the treatment with PEA significantly reduced the spinal cord injury damage at 24 h after trauma (Fig. 1c, see histological score Fig. 1d). The histological pattern of SCI described above appeared to be correlated with cellular changes into the spinal cord. In particular, a presence of MCs was observed in the spinal cord tissues collected at 24 h after SCI (Fig. 2, panel b) mainly localized in the perivascular area (see particles panel b1). Please note that in panels b and b1 of Fig. 2 the arrow indicate the presence of mast cell and arrowhead showed the presence of MCs in degranulation phase. Cells showing to have metachromatic granules when stained with acidified Toluidine blue were identified as mast cells. On the contrary, significant less MCs density and degranulation were observed in the spinal cord tissues after SCI collected from mice which have been treated with PEA (Fig. 2, panel c). No granules were found in the spinal cord tissues from sham-operated mice (Fig. 2, panel a).
3.2. PEA administration reduces chymase and tryptase expression during SCI In order to test whether PEA treatment may modulate and direct the inflammatory response through the regulation of the serine peptidases, we analyzed by immunohistochemistry the spinal cord expression of chymase and tryptase. There was no staining for chymase and tryptase in the spinal cord tissues obtained from the sham-operated mice (Figs. 3a and 4a). A substantial increase in chymase and tryptase expression was found mainly localized in MCs in spinal cord tissues collected at 24 h after SCI (Figs. 3b and 4b). Spinal cord expression of chymase and tryptase were attenuated in the spinal cord from mice that have received PEA treatment (Figs. 3c and 4c). 3.3. PEA treatment reduces the increased CB2 cannabinoid receptor and chymase expression after SCI To address the potential involvement of CB2 cannabinoid receptors in SCI and the cells implicated in the effects of PEA, we evaluated in spinal cord cross sections the co-localization of CB2 with chymase, a marker for mast cells (Fig. 5). The CB2 receptor (panels a2–b2–c2) and chymase (panels a1–b1–c1) was highly expressed in the spinal cord tissues collected at 24 h after SCI (panels b1 and b2). Colocalization of chymase and CB2 by confocal microscopy was evident in panel b3. The yellow spots indicate that chymase and CB2 colocalize, and are present mainly around the vessels and in inflammatory cells (panels b3). On the contrary, no positive staining for CB2 (panel c2) and chymase (panel c1) was observed in
Fig. 7. Localization of CB2 receptor and CD11b after SCI. Cells were double stained with antibodies against CD11b (green; a1, b1, c1) and CB2 receptors (red; a2, b2, c2). Microglial cells (CD11b-positive cells) expressed CB2 receptor as shown in panel b3. Sections were observed and photographed using a Zeiss LSM 5 Duo laser scanning microscope (Carl Zeiss; Jena, Germany). All images were digitalized at a resolution of 8 bits into an array of 2048 2048 pixels.
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Fig. 8. Effect of PEA on NGF, GDNF and NT3 expression. NGF, GDNF and NT-3 expression was evaluated by immunohistochemistry (A–C). SCI reduced neurotrophic factors expression (Ab, Bb and Cb, see score panels d), while PEA treatment restored NGF, GDNF and NT-3 expression (Ac, Bc and Cc, see score panels d). By Western Blot analysis, a basal level of NGF, GDNF, and NT-3 was detected in the spinal cord from sham-operated animals (Ae, Be and Ce, respectively), whereas NGF, GDNF and NT-3 levels were reduced in SCI mice. PEA treatment prevented the SCI-induced neurotrophic factors degradation (Ae, Be and Ce, respectively). a-Tubulin was used as internal control. A representative blot of three independent experiments is shown, and densitometry analysis of all animals is reported (n = 5 mice from each group). ⁄⁄p < 0.01, and ⁄⁄⁄p < 0.001 vs. Sham + vehicle; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. SCI.
the spinal cord tissues collected from mice that have received PEA treatment (Fig. 5). Please note that in uninjured spinal cords we found no CB2 (panel a2) and chymase (panel a1) immunoreactivity. 3.4. PEA treatment reduces astrocyte activation and increased CB2 cannabinoid receptor expression after SCI Further analysis was performed to elucidate the nature of cells targeting by PEA. Spinal cord sections revealed increased astrogliosis (GFAP + cells) in the perilesional area after SCI (Fig. 6, panel b1). CB2 expression was induced in spinal cord tissue following trauma (Fig. 5, panel b2); moreover, there was evident colocalization of GFAP and CB2 (Fig. 6, panel b3). On the contrary, a significant number of non-GFAP positive cells were found in the spinal cord of PEA-treated mice (Fig. 6, panel c1).
3.5. PEA treatment reduces microglial activation and increased CB2 cannabinoid receptor expression after SCI Microglial cells were activated following SCI as shown by increased CD11b-positive staining (Fig. 7, panel b1). Moreover, microglial cells expressed CB2 receptor as shown in Fig. 7, panel b3. 3.5.1. Effect of PEA on neurotrophic factors levels after SCI To test whether PEA modulates the inflammatory process through regulation of the neutrophic factors levels, we have examined NGF, GDNF and NT-3 levels in the perilesioned zone both by immunohistochemistry and western blot analysis (Fig. 8A–C). In the spinal cord tissues collected at 24 h after the trauma, neurotrophic factors expression levels were significantly reduced in comparison to sham animals (Fig. 8A panels b and d, B panels b
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Fig. 8 (continued)
and d, and C panels b and d). Particularly, in the Western blot analysis it was evident that reduction in NGF and GDNF was about 65% and 67%, whereas for NT-3 the trend of decrease was 20% (Fig. 8A panel e, B panel e, C panel e). The treatment with PEA significantly restore the levels of three neurotrophic factors up to that of uninjured mice (Fig. 8A, panels d and e, B panel d and e, C panels d and e). 3.5.2. Effect of PEA on TrkA expression after SCI To investigate the mechanisms by which treatment with PEA might modulate the expression of neurotrophins, we also evaluated the expression of tyrosine protein kinase, TrkA, by western blot in spinal cord tissue homogenates after SCI. A significant increase in TrkA expression levels were observed in SCI mice (Fig. 9A). Treatment of mice with PEA significantly reduced the expression of Trk (Fig. 9A, ⁄p < 0.05 vs. Sham and ##p < 0.01 vs. SCI + vehicle). 3.5.3. Effect of PEA treatment on motor function and DAT expression As previously demonstrated (Genovese et al., 2008), the above described inflammatory process is associated with loss of motor
function (Fig. 9B). In particular, while motor function was only slightly impaired in sham-operated mice (data not shown), mice subjected to SCI displayed significant deficits in hind limb movement (Fig. 9B). PEA treatment significantly ameliorated the functional deficits induced by SCI. Moreover, to test whether PEA treatment reduce the motor function alteration through regulation of the expression DAT, we have examined the DAT expression in the perilesioned zone by immunohistochemistry. In the spinal cord tissues collected at 24 h after the trauma, the expression of DAT were reduced in comparison to sham animals (Fig. 9C, panel b). The treatment with PEA restored DAT expression up to that of uninjured mice (Fig. 9C, panel c). 4. Discussion Primary injury to the adult spinal cord is irreversible, whereas secondary degeneration is delayed and therefore amenable to intervention. Accordingly, several studies have shown that therapies targeting various factors involved in the secondary degeneration cascade lead to tissue sparing and improved behavioral outcomes in spinal cord-injured animals (Bao et al., 2003;
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Fig. 8 (continued)
Cuzzocrea et al., 2006; Glaser et al., 2006). Strategies have been used to focus on the prevention of further neuronal death or to induce the regeneration of these cells in SCI animal models. For instance, GDNF has been shown to have a potent survival-promoting effect on various neuronal populations, including dopaminergic (Sariola and Saarma, 2003), noradrenergic (Saarma, 2000), cortical (Pezeshki et al., 2003), retinal ganglion (Klocker et al., 1997), sensory (Del Fiacco et al., 2002; Laurikainen et al., 2000), and motor neurons (Shneider et al., 2009). In addition, GDNF induced the growth of motor and sensory axons and remyelination in laboratory rats with partial and complete spinal cord transections (Blesch and Tuszynski, 2003). In this study, protein analysis indicated that the levels of GDNF, NT-3 and NGF were down-regulated by spinal cord compression in mice, while for the first time an local and sustained increase in the NGF, NT-3 and GDNF expression in the perilesioned tissue following intraperitoneally administration of PEA was shown. Neurotrophins signal through high-affinity receptor tyrosine kinases, TrkA–C, and/or the low-affinity receptor p75 (Bibel and Barde, 2000). All changes in TrkA expression are correlated with inflammation, in fact the up-regulation of TrkA may be dependent
on some inflammatory mediators such as IL-1b, which is known to be engaged in the NGF production. Interestingly, administration of PEA significantly reduced TrkA expression in spinal cord tissue. Here, we confirm that PEA treatment exerts beneficial effects in a mice model of SCI, as previously reported (Genovese et al., 2008). In accordance with previous data (Genovese et al., 2008), in this study the authors administered PEA at 6 and 12 h after SCI. In this study, we extend our results by showing that PEA reduces (1) mast cell infiltration, (2) chymase/tryptase expression, (3) the loss of DAT level, (4) astrogliosis, (5) microglial activation, (6) the loss of NGF, GDNF and NT-3 expression, and (7) Trk expression. PEA has been shown to be effective in several experimental models of inflammation, both of immunogenic and neurogenic origin (Conti et al., 2002; Mazzari et al., 1996). Despite its various described pharmacological properties, the cellular/receptor mechanism responsible for the actions of PEA is still debated. The first hypothesis on the mechanism of action of PEA was formulated when Aloe et al. (1993) introduced the ALIA acronym (Autacoid Local Inflammation Antagonism) to indicate that some endogenous N-acylethanolamines, like PEA, exerted a local antagonism on
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Fig. 9. Effect of PEA on TrkA, DAT expression, and hind limb motor disturbance after spinal cord injury. Western blot analysis demonstrated increased expression of tyrosine kinase receptor (TrkA) after SCI (A). PEA treatment restored TrkA expression (A). Mice were scored in an open-field environment by two individuals who were blinded to experimental conditions. Individual scores from each animal were pooled and averaged, for a maximum of 9 points for the BMS score (B). Treatment with PEA reduces the motor disturbance after SCI. DAT immunoreactivity was performed 24 h after SCI (C). Significant reduction in spinal cord sections was observed after injury (C, panel b). PEA treatment returned DAT almost to the Sham level (Cc). A representative blot of three independent experiments is shown. Values shown are means ± SEM of 10 mice for each group. ⁄p < 0.05 and ⁄⁄⁄p < 0.001 vs. Sham + vehicle; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. SCI + vehicle.
inflammation. The effect was first attributed to MC activity control (Aloe et al., 1993).
Although PEA does not bind with high affinity to CB1 or CB2 receptors, it still maintains CB-like anti-inflammatory actions in
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several mast cells-mediated experimental models of inflammation. Moreover, various studies have hypothesized that PEA may act via indirect interaction with CB2 receptors (Farquhar-Smith et al., 2002; Lambert and Di Marzo, 1999; Onaivi et al., 2008). In fact, it has been showed that the use of SR144528, a CB2 specific receptor antagonist, eliminated the antinociceptive effects of PEA (Calignano et al., 1998, 2001; Conti et al., 2002; Farquhar-Smith and Rice, 2003). Then, it was found that PEA could potentiate the effect of anandamide on CB or vanilloid receptor (VR1) (De Petrocellis et al., 2001; Hansen, 2010), the so-called ‘‘entourage effect’’. Considering the known changes of endocannabinoid system in the lesioned spinal cord (increases in 2-AG and CB2, decreases in CB1), PEA may indirectly regulate CB receptors expression through the increase in endocannabinoid tone (Costa et al., 2008) or the modulation of pro-inflammatory cytokines. Indeed, CB2 receptors would play a crucial role in limiting the spreading of this neuroinflammatory process. Pro-inflammatory cytokines present in the CNS could potentially regulate the expression of the CB2 receptor on microglial cells, astrocytes, and MCs, whose expression level is likely to be important in the regulation of inflammation in the CNS during autoimmunity (Maresz et al., 2005). In this study, we have clearly showed that CB2 receptor expression is increased following spinal cord trauma, coinciding with the appearance of microglial cells, astrocytes, and MCs; PEA treatment reduced the expression of CB2 receptor on these cells in the perilesioned zone. Future studies are needed in order to clarify these observations, as well as the involvement of peroxisome proliferator-activated receptors (PPARs) expression. PEA, in fact, is considered an endogenous activator of PPAR-a, interacting with this receptor to inhibit inflammatory response with a potency comparable to that of the synthetic PPAR-a agonist GW7647 (Lo Verme et al., 2005). We report here that spinal cord trauma caused a significant infiltration (identified morphologically by Toluidine blue staining) and activation (evaluated as chymase and tryptase expression) of MCs in the spinal cord tissues at 24 h, whereas treatment with PEA reduced both the infiltration and the activation. These observations are in agreement with other studies in which have been shown that PEA treatment is an effective tool to naturally control mast cells hyperactivity, which occurs not only in inflammation, but also in inflammatory hyperalgesia (Mazzari et al., 1996; Rice et al., 2002) and neuropathic hyperalgesia (Theodosiou et al., 1999; Zuo et al., 2003). MCs possess several biological mediators that are released from cytoplasmic granules primarily due to stimulus-induced degranulation, including vasoactive amines such as histamine, proteoglycans (mainly heparin and chondroitin sulphate), neutral serine proteases, such as tryptase and chymase, cytokines and growth factors, such as vascular endothelial growth factor, basic fibroblast growth factor, NGF, transforming growth factor-b and tumor necrosis factor (TNF)-a (Gordon and Galli, 1991; Kownatzki, 1982; Raposo et al., 1997). The spinal cord is known to receive dopaminergic projections originating from brain areas. All subtypes of dopamine receptors, including the D1 subtype, are present in the mammalian spinal cord (e.g. rats, mice, cats and monkeys). Moreover, Lapointe and collaborators (Lapointe et al., 2009) suggested that the spinal dopaminergic system may play a role in mediating locomotor-like movements in SCI subjects as well as normal locomotor behaviours in intact vertebrate species. In this contest, DAT, that terminates the signal of the neurotransmitter, could play a central role. In our model PEA reduced the loss of functional dopamine transporters induced by compression of spinal cord. In summary, the effect of PEA against SCI may be mediated by the inhibition of neutrophil accumulation (Farquhar-Smith and Rice, 2003; Rice et al., 2002), as well as by the ability of PEA of negatively modulating the secretion of mediators from MCs, the
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activation of microglia and astrocytes all expressing CB2 receptors, independently from CB receptor activation, via a so called ‘ALIA’ mechanism or ‘entourage effect’. However, these hypothesis identify in PEA a crucial therapeutic intervention diminishing the immune/inflammatory response and promoting the initiation of neurotrophic substance after SCI. 5. Conflicts of Interest The authors declare no conflicts of interest in relationship to performing this study. Acknowledgment The authors thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study. References Aloe, L., Leon, A., Levi-Montalcini, R., 1993. A proposed autacoid mechanism controlling mastocyte behaviour. Agents Actions 39 (Spec. No.), C145–C147. Andoh, A., Deguchi, Y., Inatomi, O., Yagi, Y., Bamba, S., Tsujikawa, T., Fujiyama, Y., 2006. Immunohistochemical study of chymase-positive mast cells in inflammatory bowel disease. Oncol. Rep. 16, 103–107. Bao, F., DeWitt, D.S., Prough, D.S., Liu, D., 2003. Peroxynitrite generated in the rat spinal cord induces oxidation and nitration of proteins: reduction by Mn (III) tetrakis (4-benzoic acid) porphyrin. J. Neurosci. Res. 71, 220–227. Basso, D.M., Fisher, L.C., Anderson, A.J., Jakeman, L.B., McTigue, D.M., Popovich, P.G., 2006. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J. Neurotrauma 23, 635–659. Berdyshev, E.V., 2000. Cannabinoid receptors and the regulation of immune response. Chem. Phys. Lipids 108, 169–190. Berdyshev, E.V., Boichot, E., Germain, N., Allain, N., Anger, J.P., Lagente, V., 1997. Influence of fatty acid ethanolamides and delta9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells. Eur. J. Pharmacol. 330, 231–240. Bethea, J.R., Castro, M., Keane, R.W., Lee, T.T., Dietrich, W.D., Yezierski, R.P., 1998. Traumatic spinal cord injury induces nuclear factor-kappaB activation. J. Neurosci. 18, 3251–3260. Bilsland, L.G., Dick, J.R., Pryce, G., Petrosino, S., Di Marzo, V., Baker, D., Greensmith, L., 2006. Increasing cannabinoid levels by pharmacological and genetic manipulation delay disease progression in SOD1 mice. FASEB J. 20, 1003–1005. Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M.G., Ligresti, A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E.J., Gangadharan, U., Hobbs, C., Di Marzo, V., Doherty, P., 2003. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J. Cell Biol. 163, 463–468. Blesch, A., Tuszynski, M.H., 2003. Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination. J. Comp. Neurol. 467, 403–417. Calignano, A., La Rana, G., Giuffrida, A., Piomelli, D., 1998. Control of pain initiation by endogenous cannabinoids. Nature 394, 277–281. Calignano, A., La Rana, G., Piomelli, D., 2001. Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide. Eur. J. Pharmacol. 419, 191–198. Conti, S., Costa, B., Colleoni, M., Parolaro, D., Giagnoni, G., 2002. Antiinflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat. Br. J. Pharmacol. 135, 181–187. Costa, B., Comelli, F., Bettoni, I., Colleoni, M., Giagnoni, G., 2008. The endogenous fatty acid amide, palmitoylethanolamide, has anti-allodynic and antihyperalgesic effects in a murine model of neuropathic pain: involvement of CB(1), TRPV1 and PPARgamma receptors and neurotrophic factors. Pain 139, 541–550. Cravatt, B.F., Saghatelian, A., Hawkins, E.G., Clement, A.B., Bracey, M.H., Lichtman, A.H., 2004. Functional disassociation of the central and peripheral fatty acid amide signaling systems. Proc. Natl. Acad. Sci. USA 101, 10821–10826. Cuzzocrea, S., Genovese, T., Mazzon, E., Crisafulli, C., Min, W., Di Paola, R., Muia, C., Li, J.H., Esposito, E., Bramanti, P., Xu, W., Massuda, E., Zhang, J., Wang, Z.Q., 2006. Poly(ADP-ribose) glycohydrolase activity mediates post-traumatic inflammatory reaction after experimental spinal cord trauma. J. Pharmacol. Exp. Ther. 319, 127–138. Cuzzocrea, S., Riley, D.P., Caputi, A.P., Salvemini, D., 2001. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/ reperfusion injury. Pharmacol. Rev. 53, 135–159. De Petrocellis, L., Davis, J.B., Di Marzo, V., 2001. Palmitoylethanolamide enhances anandamide stimulation of human vanilloid VR1 receptors. FEBS Lett. 506, 253– 256. Del Fiacco, M., Quartu, M., Serra, M.P., Follesa, P., Lai, M.L., Bachis, A., 2002. Topographical localization of glial cell line-derived neurotrophic factor in the
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