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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Department of Clinical and Experimental Medicine and Pharmacology, Torre Biologica, Policlinico Universitario, Messina, Italy (S.C., T.G., Em.M., C.C., R.D.P., C.M.); Istituto di Ricovero e Cura a Carattere Scientifico Centro Neurolesi "Bonino-Pulejo," Messina, Italy (S.C., T.G., Em.M., P.B.); International Agency for Research on Cancer, Lyon, France (W.M., J.-H.L., Z.-Q.W.); Department of Experimental Pharmacology, University of Naples "Federico II," Napoli, Italy (E.E); Guilford Pharmaceuticals Inc., Baltimore, Maryland (W.X., J.Z.); and Lilium Pharmaceuticals, Cockeysville, Maryland (Ed.M.)
Received for publication
May 18, 2006
Accepted
July 5, 2006.
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and interleukin-1
), and apoptosis. In a separate experiment, we have clearly demonstrated that PARG inhibition significantly ameliorated the recovery of limb function. Taken together, our results indicate that PARG activity modulates the inflammatory response and tissue injury events associated with spinal cord trauma and participate in target organ damage under these conditions.
). The cardinal features of inflammation, namely infiltration of inflammatory cells, release of inflammatory mediators, and activation of endothelial cells leading to increased vascular permeability, edema formation, and tissue destruction have been well and extensively characterized in animal SCI models (Popovich et al., 1996; Genovese et al., 2005). The production of reactive oxygen species (ROS) also contributes to the tissue injury observed during posttraumatic inflammatory reaction after SCI (Liu et al., 1997; Xu et al., 2001). Molecularly, ROS also cause DNA damage (Cuzzocrea et al., 1998a,b; Szabò and Dawson, 1998), which results in the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1, depletion of NAD+ and ATP, and ultimately cell death (Cuzzocrea et al., 1998a; Szabò, 1998). PARP-1 is a member of the PARP enzyme family consisting of PARP-1 and several recently identified novel poly(ADP-ribosylating) enzymes. Despite its function in DNA repair, overactivation of PARP-1 has long been recognized to induce cell death under certain conditions (Berger, 1985). PARP-1 inhibitors and PARP-1 gene disruption can reduce cell death resulted from oxidative stress (Schraufstatter et al., 1986), radiation (Piela-Smith et al., 1992), nitric oxide, peroxynitrite (Szabò et al., 1998), and other agents that damage DNA (Ha and Snyder, 1999). In vivo, genetic or pharmacological inhibition of PARP-1 reduces ischemic cell death (Szabò and Dawson, 1998) and prevents neuronal death (Pieper et al., 1999). Poly(ADP-ribose) glycohydrolase (PARG; EC 3.2.1.143
[EC]
) is responsible to degrade poly(ADP-ribose) polymer. Within minutes after its synthesis by PARP-1, the poly(ADP-ribose) is hydrolyzed by PARG to ADP-ribose (Lu et al., 2003). The half-life of poly-(ADP-ribose) in cells is less than 5 min, and the NAD level can drop to less than 20% of the normal level within 30 min after severe DNA damage that activates PARP-1. PARG cleaves the ribose-ribose bonds of linear and branched portion of polymer, specifically the glycosidic and glycosidic linkages of PAR. The final products of the reaction are mono-ADP-ribosyl protein and ADP-ribose. ADP-ribose is known to be a weak PARG inhibitor with an IC of 0.1 mM (Slama et al., 1995). Because PARP-1 is inhibited by extensive auto-poly-(ADP-ribosyl)ation, PARG inhibitors could thereby indirectly inhibit PARP-1 activity. Previous work has shown that the PARG inhibitor gallotannin can markedly reduce death of astrocytes after oxidative stress (Ying and Swanson, 2000). However, the first generation of PARG inhibitors are often associated with low potency, poor specificity or scarce availability of the compounds, low penetration of the blood-brain barrier, and intercalates into DNA (Slama et al., 1995). To overcome these limitations, a series of novel small-molecule PARG inhibitors with increased potency and reduced mol. wt. (Li et al., 2000) has been developed as potential therapeutic agents. One such novel PARG inhibitor, GPI 16552 (IC 5.5 µM, mol. wt. 503), a nontannin small molecule, reduced infarct volume in an in vivo model of brain ischemia/reperfusion injury. The result of neuroprotection by GPI 16552 treatment supports the notion that PARG could potentially be an alternative therapeutic target to regulate the poly-(ADP-ribose) pathway. Recently, Patel et al. (2005) have generated mutant mice lacking the 110-kDa isoform of the PARG protein [PARG110KO mice (KO)], which are viable and fertile. However, these KO mice were hypersensitive to genotoxic and endotoxic treatment, most probably due to down-regulation of PARP-1 automodification activity. On the contrary, recently it has been clearly demonstrated that inflammatory process associated with ischemia and reperfusion (kidney and intestine) is significantly reduced in these KO mice (Cuzzocrea et al., 2005; Patel et al., 2005). Thus, the KO mice represent a useful system to study the function of PARG in rodent model of diseases and validate the pathways that may be target for pharmaceutics application/intervention.
To investigate the mechanism that leads to various forms of human SCI, several experimental models have been developed (Beattie et al., 2002). The most commonly used model is the compression model; in this model, injury is induced by applying either a weight or an aneurysm clip to the spinal cord. This model aims to add to that of the contusion model by replicating the persistence of cord compression that is commonly observed in human SCI (Tator, 1995).
Apoptosis is an important mediator of secondary damage after SCI (Beattie et al., 2002). It incurs its affects through at least two phases: an initial phase, in which apoptosis accompanies necrosis in the degeneration of multiple cell types and a later phase, which is predominantly confined to white matter and involves oligodendrocytes and microglia (Merrill et al., 1993). Generation of free radicals and nitric oxide by activated macrophages has been implicated in causing oligodendrocyte apoptosis (Merrill et al., 1993). Chronologically, apoptosis initially occurs 6 h postinjury at the lesion center and lasts for several days associated with the steadily increased number of apoptotic cells in this region. An important intracellular signal transduction pathway that leads to apoptosis after SCI involves activation of the caspases, in particular, caspase-3 (Janicke et al., 1998). This protease is required for DNA fragmentation and morphologic changes associated with apoptotic cell death (Janicke et al., 1998), and PARP-1 is a major substrate of activated caspase-3 (West et al., 2005). However, the role of PARP-1 autopoly(ADP-ribosyl)ation during apoptosis is still unclear and disputed. In this regard, we have demonstrated recently that PARP inhibitors inhibits in vivo apoptotic cell death in spinal cord tissue from mice subjected to trauma (Genovese et al., 2005), suggesting that inhibition of PARP directly protects cells by preventing the activation of the apoptosis pathway. Moreover, it is well known that Bax, a proapoptotic gene, plays an important role in developmental cell death (Chittenden et al., 1995) and in central nervous system injury (Bar-Peled et al., 1999). Likewise, it has been shown that the administration of Bcl-xL fusion protein (Bcl-xL FP) (Bcl-2 is the most expressed antiapoptotic molecule in adult central nervous system) into injured spinal cords significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death (Nesic-Taylor et al., 2005). The objective of this article is to evaluate the molecular pathways of SCI mechanisms and provide information for possible treatments. In the present study, using this experimental model of SCI, we have investigated the role of PARG activation in the pathogenesis of SCI and evaluate the therapeutic effects of GPI 16552.
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) in the 129/Sv/Ola background were kept in the specific pathogen-free facility of the International Agency for Research on Cancer (Lyon, France). Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) and with the EEC regulations (Official Journal of the European Communities L 358/1, December 18, 1986). Experimental Designs. Mice (male, 8-10 weeks old, 20-22 g) were randomly divided eight groups: sham (WT) group, mice were subjected to identical surgical procedures except for SCI and were maintained under anesthesia for the duration of the experiment; SCI (WT) group mice, mice were subjected to SCI; sham PARG-/- group, mice were subjected to identical surgical procedures except for SCI and were maintained under anesthesia for the duration of the experiment; SCI PARG-/- group, mice were subjected to SCI; sham (WT) + GPI 16552 group, identical to sham-operated mice except for the administration of GPI 16552; SCI (WT) + GPI 16552 group, mice were subjected to SCI and administered GPI 16552 (40 mg/kg i.p.) 30 min after SCI; sham-PARG-/- + GPI 16552 group, identical to sham-operated mice except for the administration of GPI 16552; and SCI-PARG-/- + GPI 16552 group, mice were subjected to SCI and administered GPI 16552 (40 mg/kg i.p.) 30 min after SCI. At different time points (see Fig. 1), the animals (n = 10) from each group for each time point were sacrificed to evaluate the various parameter as described below.
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SCI. Mice were anesthetized using chloral hydrate (400 mg/kg body weight). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles. These muscles were dissected away, exposing T5 to T8 vertebrae. The spinal cord was exposed via a two-level T6 to T7 laminectomy, and SCI was produced by extradural compression of the spinal cord using an aneurysm clip with a closing force of 24 g. After surgery, 1.0 ml of saline was administered subcutaneously 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. The mice were singly housed in a temperature-controlled room at 27°C for a survival period of 24 h. Food and water were provided to the mice ad libitum. During this time period, the animals' bladders were manually voided. In all injured groups, the spinal cord was compressed for 1 min. Sham-injured animals were only subjected to laminectomy.
Measurement of Myeloperoxidase Activity. Myeloperoxidase (MPO) activity, which was used as an indicator of polymorphonuclear cell infiltration, was measured as described previously at 4 and 24 h after SCI according to precious studies (Carlson et al., 1998).
Annexin-V Evaluation. The binding of annexin V-fluorescein isothiocyanate to externalized phosphatidylserine was used as a measurement of apoptosis in spinal cord tissue section 24 h after SCI with an annexin-V-propidium iodide (PI) apoptosis detection kit (Santa Cruz, Milan, Italy) according to the manufacturer's instructions. In brief, normal viable cells will stain negative for annexin V FITC and negative for PI. Cells that are induced to undergo apoptosis will stain positive for annexin V FITC and negative for PI as early as 1 h after stimulation (Schutte et al., 1998). Both cells in later stages of apoptosis and necrotic cells will stain positive for annexin V FITC and PI. Sections were washed as before, mounted with 90% glycerol in phosphate-buffered saline (PBS), and observed with an LSM 510 Zeiss laser confocal microscope equipped with a 40x oil objective (Carl Zeiss MicroImaging GmbH, Jena, Germany).
Immunohistochemical Localization of Tumor Necrosis Factor-, Interleukin 1-, MPO, Bax, and Bcl-2. At 24 h after SCI, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde, and 8-mm 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. Nonspecific 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 (DBA-ITALIA Srl, Milan, Italy), respectively. Sections were incubated overnight with anti-tumor necrosis factor (TNF)- (1:500 in PBS, v/v), anti-interleukin (IL)-1 polyclonal antibody (1:500 in PBS, v/v), anti-MPO (1:1000), anti-Bax rabbit polyclonal antibody (1:500 in PBS, v/v), or with anti-Bcl-2 polyclonal antibody rat. Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA). To verify the binding specificity for MPO, TNF-, IL-1, Bax, and Bcl-2, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreactions were positive in all of the experiments carried out. Immunohistochemical photographs (n = 5 photos from each samples collected from all mice in each experimental group) were assessed by densitometry using Optilab Graftek software (Image Analysis System; Graftek, Villanterio, Paria, Italy) on a Macintosh personal computer (CPU G3-266; Apple Inc., Cupertino, CA).
Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling Assay. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was conducted by using a TUNEL detection kit according to the manufacturer's instruction (HRP kit DBA; Apotag, Milan, Italy). In brief, sections were incubated with 15 µg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase buffer containing deoxynucleotidyl transferase and biotinylated dUTP in terminal deoxynucleotidyltransferase buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.
Light Microscopy. Spinal cord biopsies were taken at 24 h after the trauma. The biopsies were fixed for 24 h in para-formaldehyde solution (4% in 0.1 M phosphate-buffered saline) at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (5-µm thickness) were deparaffinized with xylene, stained with hematoxylin and eosin and Luxol Fast Blue staining (used to assess demyelization), and studied using light microscopy (Dialux 22; Leitz, Wetzlar, Germany). All of the histological studies were performed in a blinded fashion.
Materials. Unless stated otherwise, all reagents and compounds used were obtained from Sigma Chemical Co. (Milan, Italy).
Data Analysis. In the experiments involving histology the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way analysis of variance followed by a Bonferroni's post hoc test for multiple comparisons. p < 0.05 was considered significant.
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Myelin structure was observed by Luxol fast blue staining. In sham animals (Fig. 2E), myelin structure was clearly stained by Luxol fast blue in both lateral and dorsal funiculi of the spinal cord. At 24 h after the injury in SCI-operated WT mice (Fig. 2F), a significant loss of myelin in lateral and dorsal funiculi was observed. In contrast, in KO mice (Fig. 2G) and in WT mice treated with GPI 16552 (Fig. 2H), the myelin degradation was attenuated in the central part of lateral and dorsal funiculi. To evaluate if histological damage to the spinal cord was associated with a loss of motor function, the modified Basso, Beattie, and Bresnahan Locomotor Rating Scale hind limb locomotor rating scale score was evaluated. Although motor function was only slightly impaired in sham mice groups, SCI-operated WT mice developed a significant deficits in hind limb movement (Fig. 3). In contrast, a significant amelioration of hind limb motor disturbances was observed in KO mice as well as in WT mice treated with GPI 16552 (Fig. 3).
Effects of PARG Inhibition on Neutrophil Infiltration. The above-mentioned histological pattern of SCI seemed to be correlated with the influx of leukocytes into the spinal cord. Therefore, we investigated the role of inhibition of PARG on the neutrophil infiltration by measurement of MPO activity. MPO activity was significantly elevated at 4 (Fig. 4A) and 24 (Fig. 4B) h in SCI-operated WT mice compared with spinal cord tissue collected from sham-operated mice (Fig. 4). Spinal cord MPO activity was significantly attenuated in comparison with those of SCI-operated WT mice group at 4 (Fig. 4A) and 24 h (Fig. 4B) in KO mice as well as in WT mice treated with GPI 16552. In addition, tissue sections obtained at 24 h from SCI-operated WT mice demonstrate positive staining for MPO mainly localized in the infiltrated inflammatory cells in injured area (Figs. 5B and 6). In KO mice (Figs. 5C and 6) as well as in WT mice treated with GPI 16552 (Figs. 5D and 6), the staining for MPO was visibly and significantly reduced in comparison with the SCI-operated WT mice. There was no staining for MPO in spinal cord tissues obtained from the sham group of mice (Figs. 5A and 6).
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and IL-1. Immunohistological analysis of the spinal cord after SCI was performed to determine whether PARG inhibition may modulate the secondary inflammatory reaction also through the regulation of the secretion of cytokines. A substantial increase of IL-1 (Figs. 6 and 7B) and TNF- (Figs. 6 and 7F) formation was found in spinal cord samples collected from SCI-operated WT mice at 24 h after SCI (Fig. 6). Spinal cord levels of IL-1 and TNF- were significantly attenuated in KO mice (Figs. 6 and 7, C and G, respectively) and in WT mice treated with GPI 16552 (Figs. 6 and 7, D and H, respectively). There was no staining for either IL-1 or TNF- in spinal cord obtained from the sham group of mice (Figs. 6 and 7, A and E, respectively).
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Effect of PARG Inhibition on Bax and Bcl-2 Expression. Spinal cord tissues were taken at 24 h after SCI to determine the immunohistological staining for Bax and Bcl-2. Sections of spinal cord from sham-operated mice stained negative for Bax (Figs. 6 and 10A). Spinal cord sections obtained from SCI-operated WT mice exhibited positive staining for Bax (Figs. 6 and 10B). On the contrary, the degree of positive staining for Bax was significantly reduced in spinal cord tissues collected from KO mice (Figs. 6 and 10C) as well as from WT mice treated with GPI 16552 (Figs. 6 and 10D). In addition, sections of spinal cord from shamoperated mice demonstrated positive staining for Bcl-2 (Figs. 6 and 10E). Spinal cord sections obtained from SCI-operated WT mice exhibited significantly less staining for Bcl-2 (Figs. 6 and 10F). The loss of positive staining for Bcl-2 was significantly reduced in spinal cord tissues collected from KO mice (Figs. 6 and 10, G, G1) as well as in WT mice treated with GPI 16552 (Figs. 6 and 10H).
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; Xu et al., 2001). ROS and peroxynitrite also cause DNA damage (Cuzzocrea et al., 1998a; Szabò and Dawson, 1998), which results in the activation of the nuclear enzyme PARP-1. Our recent work clearly demonstrates that the PARP-1 inhibitors significantly reduced the experimental SCI in mice (Genovese et al., 2005). The current study demonstrates that PARG inhibition may be equally effective as PARP-1 inhibition for suppressing the NAD/ATP depletion. Therefore, it also supports the hypothesis that blocking the poly(ADP-ribose) pathway, which can be achieved by either PARG or PARP-1 inhibitors, is the common mechanism for anti-inflammatory effect.
Indeed, our data demonstrate that PARG activation plays a major role in SCI, characterized by histological damage, motor damage, neutrophil infiltration, cytokine expression (TNF- and IL-1), positive for annexin-V, increased apoptosis (TUNEL staining), and induction of Bax and down-regulation of Bcl-2. The PARG gene disruption and enzymatic inhibition of PARG reversed the above symptoms. What, then, is the mechanism by which PARG inhibition protects the spinal cord against injury and dysfunction?
Previously, PARG inhibitors have been evaluated in vitro against necrotic cell death. The PARG inhibitor, 1,2,3,4,6-o-inhibipenta-b-D-galloylglucose, prevented death of P388D1 macrophage cell line by hydrogen peroxide treatment (Li and Zhang, 2000). Gallotannin and nobotannin reduced the death of the astrocytes and neurons treated with hydrogen peroxide, N-methyl-N9-nitro-N-nitroguanidine or N-methyl-D-aspartate (Ying and Swanson, 2000; Ying et al., 2001). Moreover, Zhang et al. (1998) have demonstrated recently that the new PARG inhibitor GPI 16552 exerts significant neuroprotection in a cerebral focal ischemia model (Lu et al., 2003).
The injured environment during the acute phase of SCI is dominated by the presence of the proinflammatory cytokines TNF, IL-1, and IL-6 (Tyor et al., 2002). Direct evidence that TNF- and IL-1 play a role in the outcome of the damage after injury to the spinal cord has been obtained in animal models in which exogenous local administration of proinflammatory cytokines to mice after SCI which may influence these intrinsic processes (Klusman and Schwab, 1997). We confirm in the present study that the model of SCI used here leads to a substantial increase of the levels of TNF- and IL-1 in the spinal cord. Interestingly, the levels of this proinflammatory cytokines are significantly lower in the tissues obtained from KO mice as well as WT mice treated with GPI 16552. These findings, therefore, suggest that inhibition of PARG reduced the activation and the subsequent expression of proinflammatory genes.
We demonstrated in the present study that the genetic or pharmacological inhibition of PARG reduces the inflammatory cell infiltration as assessed by the specific granulocyte enzyme myeloperoxidase at different time points. The reduced neutrophil recruitment represents another mechanism for the protective anti-inflammatory effects. Consistent with this notion/hypothesis, Taoka et al. (1997) have demonstrated that activated neutrophils are involved in compression trauma-induced SCI in rats. Neutrophils recruited into the tissue can contribute to tissue destruction by the production of reactive oxygen metabolites (Tator, 1995; Carlson et al., 1998), granule enzymes, and cytokines that further amplify the inflammatory response.
We have identified proapoptotic transcriptional changes, including up-regulation of proapoptotic Bax and down-regulation of antiapoptotic Bcl-2, by immunohistochemical staining. We report in the present study for the first time that the genetic or pharmacological inhibition of PARG in SCI experimental model documents features of apoptotic cell death after SCI, suggesting that protection from apoptosis may be a prerequisite for regenerative approaches to SCI. In particular, we demonstrated that the genetic or pharmacological inhibition of PARG reduced Bax expression, whereas Bcl-2 expressed much more in KO mice as well as WT mice treated with GPI 16552. This result suggests that PARG inhibition prevents the loss of the antiapoptotic way and reduces the proapoptotic pathway activation with a mechanism still to be discovered.
Finally, in the mice spinal cord, two early morphologic changes occur in the myelin sheath after SCI (Zhang et al., 1998). Paranodal myelin breakdown with associated nodal widening and random Wallerian-type degeneration of nerve fibers in the white matter are observed. In this study, the histological examination of the spinal cord from SCI-operated mice revealed that microcystic cavitation and degenerating axons are significantly ameliorated in the SCI-treated KO mice as well as SCI-WT mice treated with GPI 16552.
It is interesting to note that KO mutant mice are hypersensitive to lipopolysaccharide-induced septic shock (Cortes et al., 2004) as well as to postischemic brain damage (Cozzi et al., 2005). Because PARP-1 can still be activated in KO mutant mice but with a low degree of automodification, DNA damage-induced cell death pathway may become prominent in this model.
The involvement of poly(ADP-ribose) pathway in pathological conditions is well documented by numerous studies of chemical PARP-1 inhibition and PARP-1 gene deletion (Szabò et al., 1998; Genovese et al., 2005). The data presented in the present study, together with other reports (Slama et al., 1995; Ying and Swanson, 2000), demonstrate that homeostasis of poly(ADP-ribose) modulated by PARP-1/PARG either directly or indirectly regulates the inflammation response. The mechanism of these actions clearly requires further investigation. Further experiments with multiple approaches can be expected to unlock the full potential of intervening the poly(ADP-ribose) pathway for therapeutic purposes through PARP-1 and/or PARG inhibition.
In conclusion, we have demonstrated for the first time in vivo that genetic and pharmacological inhibition of PARG attenuates SCI. Therefore, our results provide further important experimental evidence that PARG may be a novel target by therapeutic applications for treating shock and inflammation.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SCI, spinal cord injury; ROS, reactive oxygen species; PARP, poly(ADP-ribose) polymerase; PARG, poly(ADP-ribose) glycohydrolase; GPI 16552, N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide; KO, knockout; WT, wild type; MPO, myeloperoxidase; PI, propidium iodide; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; IL, interleukin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; i.p. intraperitoneally.
Address correspondence to: Dr. Salvatore Cuzzocrea, Institute of Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi, 98100 Messina, Italy. E-mail: salvator{at}unime.it
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