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Department of Clinical and Experimental Medicine and Pharmacology, Torre Biologica, Policlinico Universitario, Messina, Italy;
* International Agency for Research on Cancer, Lyon, France;
Guilford Pharmaceuticals Inc., Baltimore, Maryland, USA; and
Lilium Pharmaceuticals, Cockeysville, Maryland, USA
1Correspondence: 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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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-). Reperfused ileum tissue sections from SAO-shocked WT mice and rats showed positive staining for P-selectin and ICAM-1 localized mainly in the vascular endothelial cells. Genetic disruption of the PARG gene in mice or pharmacological inhibition of PARG by PARG inhibitors significantly improved the histological status of the reperfused tissues associated with reduced expression of P-selectin and ICAM-1, neutrophil infiltration into the reperfused intestine, and TNF- production. These results suggest that PARG activity modulates the inflammatory response in ischemia/reperfusion and participates in end (target) organ damage under these conditions.—Cuzzocrea, S., Di Paola, R., Mazzon, E., Cortes, U., Genovese, T., Muià, C., Li, W., Xu, W., Li, J.-H., Zhang, J., Wang, Z.-Q. PARG activity mediates intestinal injury induced by splanchnic artery occlusion and reperfusion.
Key Words: SAO shock • ischemia and reperfusion • PARG inhibitor • neutrophil infiltration • organ injury
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The half-life of pADPR in cells is <5 min. Poly(ADP-ribosylation) has been postulated to be involved in various DNA-related processes including chromatin decondensation, DNA replication, DNA repair, gene expression, cell death, and genomic stability (1
, 2)
.
Many studies aimed at clarifying the biological function of poly(ADP-ribosylation) have focused on the synthesizing enzyme PARP-1. PARP-1 inhibitors such us nicotinamide and 3-aminobenzamide have been demonstrated in both in vivo and in vitro studies to have beneficial effects in pathological conditions ranging from ischemia-reperfusion injury to inflammation (3)
. The degree of organ injury caused by ischemia and reperfusion is attenuated in mice in which the gene for PARP-1 has been disrupted (4)
as well as in mice treated with PARP-1 inhibitors (5
6
7)
.
Poly (ADP-ribose) glycohydrolase (PARG, EC 3.2.1.143) is responsible for degradation of pADPR. Within minutes after its synthesis by PARP-1, the pADPR is hydrolyzed by PARG (8)
. PARG cleaves the ribose-ribose bonds of linear and branched portion of polymer, specifically the glycosidic and glycosidic linkages of pADPR. 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 IC50 of 0.1 mM (9)
. pADPR metabolism has been found in a variety of animal models of disease (10)
. Genetic and cellular studies have been conducted to understand the biological role of PARG in vitro and in vivo. PARG or poly(ADP-ribosylation) is involved in cell cycle progression (11)
, gene transcription (12)
, cell differentiation (13)
, apoptosis (14)
, and DNA repair (15)
. In addition, PARG can be cleaved by caspase-3 during apoptosis in human cells, concomitant with PARP-1 cleavage (16)
, indicating that PARG activity may be precisely regulated during apoptosis.
Until recently, the lack of specific, potent, and membrane-permeable PARG inhibitors has hindered in vivo animal testing to definitively establish PARG’s role in ischemia and reperfusion. Nevertheless, preliminary studies with PARG inhibitors in vitro revealed their cellular protective effects. For example, it has been documented that PARG inhibitors (e.g., 1,2,3,4,6-o-penta-ß-D-galloylglucose) prevent cell death induced by hydrogen peroxide toxicity (18)
. Ying and Swanson found that PARG inhibitors such as gallotannin were capable of preventing oxidative cell death (18
, 20)
. We recently developed several families of small-molecule PARG inhibitors with improved potency (21)
. One such novel PARG inhibitor, N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide (GPI 16552), a non-tannin small molecule related to the tilorone family of PARG inhibitors, reduces infarct volume in an in vivo model of brain ischemia/reperfusion injury (8)
. More recently we demonstrated that GPI 18214, another novel PARG inhibitor, exerts a protective effect against organ injury associated with non-septic shock (22)
. The IC50 for GPI 16552 and 18214 are 1.7 and 4.2 µM, respectively (10)
.
Cortes and colleagues have (17)
generated viable and fertile mutant mice by a specific deletion of the 110 kDa isoform of the PARG protein (PARG110KO mice). However, these PARG110KO mice were hypersensitive to genotoxic and endotoxic treatment most likely due to down-regulation of PARP-1 automodification activity. PARG110KO mice therefore provide a useful system with which to study the function of PARG in rodent model of diseases and to validate pathways that may be targets for pharmaceutical application/intervention. Taken together, in vitro and in vivo results support the notion that PARG could potentially be an alternative therapeutic target in the poly(ADP-ribose) pathway.
In this study, we investigated the role of PARG in a model of splanchnic artery occlusion shock (SAO) ischemia and reperfusion shock in rats and in PARG110KO and PARG wild-type (WT) mice. To characterize the role of PARG in the SAO shock model, we determined the following end points of the shock response: 1) cytokine production, 2) neutrophil infiltration, 3) adhesion molecule expression, 4) organ injury, and 5) mortality in PARG110KO mice as well as in WT mice and rats treated with GPI 18214 or GPI 16552.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(8- to 10-wk-old, 20–22 g) in 129/Sv/Ola background were kept in the pathogen-free facility of the International Agency for Research on Cancer Lyon. Animal care complied with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) and with EEC regulations (O.J. of E.C. L 358/1 12/18/1986).
Experimental design
Rats were randomly divided into four groups
Upon completion of surgical procedures, rats were randomly allocated to the following: 1) I/R + saline group: rats were subjected to SAO shock (45 min) followed by reperfusion (60 min) (n=10); 2) I/R + GPI 16552 group: rats were subjected to identical surgical procedures as above and administered GPI 16552 (40 mg/kg, i.p.) 30 min before ischemia (n=10); 3) I/R + GPI 18214 group: rats were subjected to identical surgical procedures as above and administered GPI 18214 (40 mg/kg, i.p.) 30 min before ischemia (n=10); 4) sham + dimethyl sulfoxide group (sham-operated): rats were subjected to identical surgical procedures except for SAO shock and were maintained under anesthesia for the duration of the experiment (n=10); 5) sham + GPI 16552 group: identical to sham-operated rats except for the administration of GPI 16552 (n=10); 6) sham + GPI 18214 group: identical to sham-operated rats except for administration of GPI 18214. (n=10). Treatment with 40 mg/kg i.p. GPI 16552 afforded neuroprotection in the middle cerebral artery occlusion model (8)
.
Mice were randomly divided into 8 groups
Surgical procedures
Rats
Male Sprague-Dawley rats weighing 250–300 g were allowed access to food and water ad libitum. After anesthesia, a catheter was placed in the jugular vein as described (23)
. Rats were anesthetized with sodium pentobarbital (45 mg/kg, i.p.). After midline laparotomy, the celiac and superior mesenteric arteries were isolated near their aortic origins. During this procedure, the intestinal tract was maintained at 37°C by placing it between gauze pads soaked with warmed 0.9% NaCl solution. Rats were observed for 30 min stabilization before splanchnic ischemia or sham ischemia. The superior mesenteric artery and the celiac trunk were clamped, resulting in total occlusion of these arteries for 45 min to induce SAO shock. After occlusion, the clamps were removed. The various groups of animals were killed at 60 min for histological examination of the bowel and for biochemical studies, described below.
Mice
Male PARG110KO and WT mice were allowed access to food and water ad libitum (yes). The mice were anesthetized with sodium urethane (45 mg/kg, i.p.). After midline laparotomy, celiac and superior mesenteric arteries were isolated near their aortic origins. During this procedure, the intestinal tract was maintained at 37°C by placing it between gauze pads soaked with warmed 0.9% NaCl solution. Mice were observed for 30 min of stabilization before splanchnic ischemia or sham ischemia. SAO shock was induced by clamping the superior mesenteric artery and the celiac trunk, resulting in a total occlusion of these arteries for 45 min. After this period of occlusion, the clamps were removed. In one study, rats were killed at 60 min for histological examination of the bowel and for biochemical studies, as described below.
Immunohistochemical analysis of P-selectin and ICAM-1
P-selectin and intracellular adhesion molecule-1 (ICAM-1) localization was detected as described (7)
in ileum sections by immunohistochemistry. After reperfusion, tissues were fixed in 10% buffered formalin, then sections were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 20 min and incubated in 2% normal serum for 60 min. Sections were incubated for 12 h at 4°C with specific antibodies directed at the above-mentioned adhesion molecules. Controls included nonspecific purified glycoprotein immunoglobulin G (IgG). After blocking endogenous avidin and biotin (Vector Laboratories), specific labeling of antigen-antibody complex was detected with the chromogen diaminobenzidine.
Myeloperoxidase activity
Myeloperoxidase activity (MPO), an index of polymorphonuclear leukocyte (PMN) accumulation, was determined as described (24)
. Intestinal and lung tissues, collected 60 min after reperfusion, were homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 x g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured by a spectrophotometer at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol of peroxide minutes at 37°C and was expressed in µ units/g weight of wet tissue.
Measurement of cytokines
Tumor necrosis factor- (TNF-) levels were evaluated in plasma samples 60 min after reperfusion. The assay was carried out using a colorimetric commercial kit (Calbiochem-Novabiochem Corporation, San Diego, CA, USA). The ELISA had a lower detection of 30 pg/mL.
Light microscopy
For histopathological examination, biopsies of small intestine were taken 60 min after reperfusion. Tissues were fixed in Dietric solution (14.25% ethanol, 1.85% formaldehyde, 1% acetic acid) for 1 wk at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ, USA). From each biopsy, 7 µm sections were obtained with hematoxylin and eosin to evaluate intestine morphology. The tissue slices were observed with a Dialux 22 Leitz microscope. For quantitative estimation of damage caused by ischemia/reperfusion, sections (n=6 for each animal) were scored by two independent observers blinded to the experimental protocol. The following morphological criteria were used for scoring: 0, no damage; 1, focal epithelial edema and necrosis (mild); 2, diffuse swelling and necrosis of the villi (moderate), 3, necrosis with evidence of neutrophil infiltration in the submucosa (severe), 4, widespread necrosis with massive neutrophil infiltration and evidence of hemorrhage (major).
Reagents
Biotin blocking kit, biotin-conjugated goat anti-rabbit IgG, and avidin-biotin peroxidase complex were obtained from Vector Laboratories (Burlingame, CA, USA). Primary anti-P-Selectin and anti-ICAM-1 were purchased from Santa Cruz (DBA, Milan, Italy). All other reagents and compounds were purchased from Sigma Chemical Company (Sigma, St. Louis, MO, USA).
Data analysis
All values in the figures and text are expressed as mean ± standard error of the mean of n observations, where n represents the number of animals studied. In experiments involving histology or immunohistochemistry, the figures are representative of at least three experiments performed on different experimental days. Data sets were examined by 1- and 2-way ANOVA; individual group means were then compared with Student’s unpaired t test. Nonparametric data were analyzed with the Fisher’s exact test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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a). Upon release of the occlusion, there was a gradual fall in mean arterial blood pressure in vehicle-treated rats (Fig. 1a
). Animals were killed after 60 min reperfusion to collect blood and tissues for biochemical analysis. Reperfusion of the ischemic splanchnic circulation led to a substantial increase in plasma levels of TNF- at the end of reperfusion (Fig. 1b
) and profound neutrophil infiltration into intestinal tissues from SAO-shocked rats (Fig. 1c
). These inflammatory reactions were triggered by the reperfusion, since no such changes were observed when blood or tissues were removed right after the period of ischemia (data not shown). Either of the two PARG inhibitors, GPI 18214 or GPI 16552 (40 mg/kg, i.p. 30 min before ischemia), reduced the fall in MABP observed during reperfusion (Fig. 1a
) and significantly suppressed the increase of plasma levels of TNF- (Fig. 1b
) and neutrophil infiltration into the ileum (Fig. 1c
).
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Neutrophil infiltration in the intestine was associated with expression of intracellular adhesion molecules (ICAM). Immunohistochemistry of ileum tissue sections obtained from sham-operated rats with the anti-ICAM-1 antibody showed a specific but low level of staining in the endothelium along the vascular wall, demonstrating that ICAM-1 is constitutively and moderately expressed (Fig. 2
a). At 60 min after reperfusion, strong staining for ICAM-1 was observed in the intestine localized mainly around the vessels (Fig. 2b
, arrows) in SAO-shocked rats. Such an up-regulation of the constitutive ICAM-1 was not observed in sections from GPI 18214- (Fig. 2c
) and GPI 16552- (Fig. 2d
) treated rats. We also performed immunohistochemistry on P-selectin. Although no staining was observed in sham-operated rats (Fig. 3
a) after 60 min reperfusion, an intense staining was obtained from SAO-shocked rats for P-selectin localized in the vessels (Fig. 3b
, arrows). In contrast, rats pretreated with GPI 18214 (Fig. 3c
) and GPI 16552 (Fig. 3d
) did not increase P-selectin expression 60 min after reperfusion.
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No histological alteration was observed in the intestinal sections from sham-operated rats (see Fig. 6a
). Histological examination of the small intestine at 60 min of reperfusion (Fig. 4
) revealed pathologic changes in SAO rats. Ileum sections from SAO-shocked rats (Fig. 4b
) showed mucosa injury characterized by epithelial layer damage and inflammatory cell infiltration extending through the wall and concentrated below the epithelial layer. GPI 18214- (Fig. 4c
) and GPI 16552- (Fig. 4d
) treated rats showed a great reduction in organ injury.
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Intestinal damage induced by SAO shock is reduced in PARG110KO mice
To ascertain the role of PARG in mesenteric infarction, PARG110KO and WT mice were subjected to 45 min occlusion, followed by 60 min reperfusion of the SAO. In sham-treated WT mice, the histological structure of the gastrointestinal tract was typical of normal architecture (Fig. 5
a). In WT mice, splanchnic ischemia/reperfusion resulted in tissue injury mainly localized to the small intestine. Further histological examination demonstrated that the damage was localized to the villi and associated with infiltration of the inflammatory cells into the mucosa and tissue hemorrhage (Fig. 5b
). The degree of the tissue injury was 3.14 ± 0.1 (on a score ranging from 0 to 4; see Materials and Methods). The damage score for PARG110KO mice was significantly lower (1.33±0.09) than that obtained from WT mice (P<0.001) or from histological observation (Fig. 5c
). Treatment of WT mice with the PARG inhibitor GPI 18214 (40 mg/kg, i.p.) 30 min before ischemia resulted in a significant reduction of the damage score (1.41±0.07) and a reduction in the histological signs of tissue injury (Fig. 5d
). Splanchnic ischemia/reperfusion results in up-regulation of proinflammatory cascades in the intestine and other organs (25)
. TNF- plasma levels were significantly increased in WT mice subjected to SAO shock compared with sham-operated animals (Fig. 6
a). This production was significantly reduced in SAO-shocked PARG110KO and WT mice treated with GPI 18214 (Fig. 6a
).
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Assessment of neutrophil infiltration into the ileum was performed by measuring the activity of MPO, an enzyme is contained in and specific for PMN lysosomes. MPO activity was significantly elevated after splanchnic ischemia/reperfusion in WT mice (Fig. 6b
). In WT mice treated with GPI 18214 and PARG110KO mice, tissue MPO activity (Fig. 6b
) was markedly reduced compared with that of WT animals subjected to SAO. The elevation in MPO activity was associated with an increase of immunohistochemical staining for P-selectin and ICAM-1 in the injured splanchnic tissue. Although almost no staining for P-selectin was observed in sham-operated WT mice (Fig. 7
a) after 60 min reperfusion, an intense staining was obtained from SAO-shocked WT mice (Fig. 7b
). Similar to sham-operated mice, almost no positive (greatly reduced) staining for P-selectin was found in the intestine of PARG110KO (Fig. 7c
) or WT mice pretreated with GPI 18214 subjected to splanchnic ischemia/reperfusion (Fig. 7d
). Moreover, constitutive staining for ICAM-1 was observed in intestinal tissue sections obtained from sham-operated WT mice (Fig. 8
a). By contrast, at 60 min of reperfusion a significant increase of positive staining for ICAM-1 was observed in tissue section from SAO-shocked WT intestine (Fig. 8b
). Significantly less positive staining for ICAM-1 was observed in the intestine of SAO-shocked PARG110KO mice (Fig. 8c
) and SAO-shocked WT mice treated with GPI 18214 (Fig. 8d
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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described a patient with occlusion of the superior mesenteric artery resulting in acute renal failure, intestinal ischemia, necrosis of both legs, and multiple organ failure leading to death. Several animal models have been described in order to understand the pathophysiologic mechanism. Among them, SAO is a severe form of circulatory shock produced by ischemia and reperfusion of the splanchnic organs. This type of shock is characterized by a decrease in systemic blood pressure upon release of the splanchnic arteries, which leads to a fatal outcome (8
, 27)
. Other important characteristics of the SAO shock are local splanchnic release of lysosomal hydrolases, enhanced proteolysis, hemoconcentration, intestinal injury, and the production of cardiotoxic substances (27
, 28)
. Our data demonstrate that genetic or pharmacological PARG inactivation exerts a protective effect against SAO shock. Specifically, the present study provides evidence that PARG activity is responsible for 1) the fall of blood pressure, 2) the development of SAO-induced shock, 3) morphological injury and neutrophil infiltration, and 4) the up-regulation of P-selectin and ICAM-1. The PARG gene disruption and enzymatic inhibition of PARG reversed the above symptoms. What, then, is the mechanism by which PARG inhibition protects the ileum against injury and dysfunction?
PARG inhibitors have been evaluated in vitro against necrotic cell death. For example, PARG inhibitors gallotannin and nobotannin reduced the death rate of the astrocytes of neurons treated with hydrogen peroxide, N-methyl-N-nitro-N-nitroguanidine, or N-methyl-D-aspartate (19
, 20)
. New PARG inhibitors, including GPI 16552 and GPI 18214, have recently been developed (21
, 28
, 30)
. In one in vivo study, GPI 16552 exerted significant neuroprotection in a cerebral focal ischemia model (8)
. In one study, we demonstrated that another PARG inhibitor, GPI 18214, significantly reduced multiple organ failure induced by zymosan in rats (22)
.
PARP-1 and PARG inhibitors both reduce SAO-induced intestinal injury at the same level and in the same time window for treatment (4
5
6
7
, 22
, 31)
. These results are consistent with the conjecture that PARG inhibition may be as effective as PARP-1 inhibition for suppressing NAD/ATP depletion. Alternatively, auto-poly(ADP-ribosylation) is known to inhibit PARP-1 (32
, 33)
and PARG inactivation simply enhances the autoinhibition of PARP-1 (17)
. In either scenario, interruption the poly(ADP-ribose) pathway that can be achieved by either PARG or PARP-1 inhibitors may represent a common mechanism for anti-inflammatory effects. Recent studies clearly demonstrate that PARP-1 inhibitors attenuate intestinal injury during SAO shock (5
6
7)
. Like most biological pathways that offer multiple targets for drug intervention, the current study shows that PARG may be another key target for modulating pADPR metabolism.
What are the secondary biochemical changes subsequent to PARG activation that may contribute to SAO injuries? There is evidence that proinflammatory cytokines (e.g., TNF-) help propagate the extension of ischemia and reperfusion shock (25)
. We confirm here that SAO shock leads to a substantial increase in plasma levels of TNF-. However, levels of this proinflammatory cytokine are significantly lower in plasma obtained from PARG110KO and WT mice or rats treated with PARG inhibitors. These findings suggest that the degree of SAO shock, and hence the formation of TNF-, depend at least partially on PARG activity. Various studies have clearly shown that adhesion molecules play an important role in the regulation of the process of neutrophil chemoattraction, adhesion, and emigration from the vasculature to tissue (34
, 35)
. In accordance with previous findings (25)
, we observed that SAO shock-induced the appearance of P-selectin on the endothelial vascular wall and up-regulated surface expression of ICAM-1 on endothelial cells in an intestinal section from WT mice or vehicle-treated rats. Genetic inactivation (in PARG110 KO mice) or pharmacological inhibition of PARG (by two new PARG inhibitors) abolished expression of P-selectin and the up-regulation of ICAM-1, but did not affect the constitutive expression of ICAM-1 on endothelial cells. These results suggest that inhibition of PARG activation may interfere with the interaction of neutrophils and endothelial cells at both the early rolling phase mediated by P-selectin and the late firm adhesion phase mediated by ICAM-1. The absence of an increased expression of the adhesion molecules in intestinal tissue of SAO-shocked PARG110KO and WT mice or rats treated with PARG inhibitors correlated with the reduction of leukocyte infiltration, as assessed by the specific granulocyte enzyme myeloperoxidase, and with moderation of the tissue damage as evaluated by histological examination.
PARG
2-3/2-3 mutant mice are hypersensitive to LPS-induced septic shock (17). Since PARP-1 can still be activated in PARG110 KO mutant mice but with a low degree of automodification, DNA damage-induced cell death pathway may become prominent in this model. However, we cannot rule out the possibility that deficient poly(ADP-ribosylation) of PARP-1 can also alter NF-
B-mediated anti-inflammatory pathways since PARP-1 is proposed to be a coactivator for NF-B-mediated transcription (36)
.
Involvement of the pADPR pathway in pathological conditions is well documented by numerous studies that use PARP-1 inhibition and PARP-1 gene deletion (33)
. The data presented in the present study, together with other reports (27
, 28)
, demonstrate that homeostasis of pADPR modulated by PARP-1/PARG directly or indirectly regulates the inflammation response. The mechanism of these actions clearly requires further investigation. Additional experiments with multiple approaches can be expected to unlock the full potential for intervening in the pADPR pathway for therapeutic purposes through PARP-1 and/or PARG inhibition. Thus, we propose the following cycle: ischemia/reperfusion -> reactive oxygen species production -> PARP-1/PARG-mediated endothelial injury -> PMN infiltration -> cytokines release -> organ damage. Inhibition of PARG would intercept this cycle before endothelial injury. Confirmation of this proposed feedback cycle, however, requires further investigation. In conclusion, we have demonstrated for the first time in vivo that genetic and pharmacological inhibition of PARG attenuates intestinal injury associated with ischemia and reperfusion. Therefore, our results provide experimental evidence that PARG may be a novel target by therapeutic applications for treating shock and inflammation.
Received for publication October 6, 2004. Accepted for publication December 2, 2004.
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This article has been cited by other articles:
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  E. Esposito, E. Mazzon, C. Muia, R. Meli, E. Sessa, and S. Cuzzocrea Splanchnic ischemia and reperfusion injury is reduced by genetic or pharmacological inhibition of TNF-{alpha} J. Leukoc. Biol., April 1, 2007; 81(4): 1032 - 1043. [Abstract] [Full Text] [PDF]
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 S. Cuzzocrea, T. Genovese, E. Mazzon, C. Crisafulli, W. Min, R. Di Paola, C. Muia, J.-H. Li, E. Esposito, P. Bramanti, et al. Poly(ADP-Ribose) Glycohydrolase Activity Mediates Post-Traumatic Inflammatory Reaction after Experimental Spinal Cord Trauma J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 127 - 138. [Abstract] [Full Text] [PDF]
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