Rapid Appearance of Resolvin Precursors in Inflammatory Exudates: Novel Mechanisms in Resolution

Animals

All animals used in the present study were male FVB mice (Charles River Laboratories) that were 6–8 wk old (weighing 20–25 g). They were maintained in a temperature and light-controlled environment, and had unlimited access to food (laboratory standard rodent diet 5001 (Lab Diet)), containing 1.5% EPA, 1.9% DHA of total fatty acids, and tap water. Experiments were performed in accordance with the Harvard Medical School Standing Committee on Animals guidelines for animal care (Protocol 02570).

Chemicals

RvD1 and 17-(R/S)-methyl RvD1 were each synthesized by total organic synthesis for these experiments from starting materials of known chirality in enantiomerically and geometrically pure form (N. Petasis, J. Uddin, and C. Serhan, manuscript in submission) by the Organic Synthesis Core, National Institutes of Health P50-DE-016191. Resolvin E1 (RvE1) and the 19-p-fluorophenoxy-RvE1 methyl ester were prepared in an enantiomerically and geometrically pure form, as described previously (26) (N. Petasis, R. Yang, G. Bernasconi, J. Uddin, and C. Serhan, manuscript in submission). Physical and spectroscopic properties matching biogenic and synthetic RvD1 and RvE1were as reported (20, 27). The integrity of each synthetic resolvin and their analogues was assured by monitoring the physical properties with LC-UV-tandem mass spectrometry just before evaluating their biological activities.

Murine peritonitis

Male FVB mice were anesthetized with isoflurane (Hospira). Both 1 μg of 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic-21,21,22,22,22-d₅ acid (d₅-DHA; Cayman Chemical) and 1 μg of 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic-19,19,20,20,20-d₅ acid (d₅-EPA; Cayman Chemical) in 100 μl of 2% ethanol (v/v) in saline or vehicle alone were administered by bolus tail vein injection. Approximately 5 min later, peritonitis was initiated with i.p. administration of 1 mg of zymosan A (Sigma Aldrich). For control experiments, 1 ml of saline was injected i.p. At indicated time intervals, mice were euthanized with an overdose of isoflurane, and peritoneal exudates were collected by lavage with 5 ml of Dulbecco’s PBS without either Ca²⁺ or Mg²⁺. Exudate cells and supernatants were separated by centrifugation (10 min, 1000 rpm, 4°C), aliquots of supernatants were collected, and 4 vol of cold acetone were added to precipitate proteins. Serum albumin and total protein levels were determined (28).

For tracer experiments, mice fasted overnight (∼18 h) before 500 nCi of [1-¹⁴C]DHA (docosahexaenoic acid 4,7,10,13,16,19-[1-¹⁴C]; American Radiolabeled Chemicals) were injected via the tail vein. At indicated time intervals, exudates were collected (vide supra) and the radioactivity was determined using a scintillation counter (multipurpose scintillation counter LS6500; Beckman Coulter).

Differential counts and FACS analysis

Aliquots of lavage cells were assessed for total and differential leukocyte counts via light microscopy to identify individual cell types (i.e., neutrophil, monocyte, etc.). For flow cytometry analysis, aliquots of 0.5 × 10⁶ cells were stained with 0.25 μg of FITC-conjugated anti-mouse F4/80, 0.1 μg of PE-conjugated anti-mouse Ly-6G, and 0.1 μg of PerCP-Cy5.5-conjugated anti-mouse CD11b, or 0.1 μg of PE-conjugated rat IgG2c,κ isotype control as a background stain. Cells were washed and analyzed using FACSort (BD Biosciences) (29).

Mass spectrometric analysis of deuterium-labeled EPA and DHA

Upon collection, 2 vol of cold methanol containing internal standard (500 ng/sample, trans-parinaric acid; Molecular Probes) were added to exudates, which were stored at −20°C for 1 h. After centrifugation, samples were diluted and applied to C18 cartridge columns (Extract-Clean EV SPE; Alltech Associates). Lipids were extracted using hexane and methyl formate, and each fraction was collected and its unesterified fatty acids converted to corresponding methyl ester using diazomethane treatment (30). The esters were suspended in hexane, and then injected in gas chromatography-mass spectrometry (GC-MS). Electron-impact GC-MS was conducted using an HP 6890 GC system with HP5973 Mass Selective Detector (Hewlett-Packard) equipped with an HP-5MS capillary column (0.25 mm ID × 30 m, 0.25 μm; Agilent) operating at a mass range from m/z 70 to 800. The ionization voltage was 70 eV, and the ion source temperature was 230°C. Chromatography was conducted using a column temperature maintained at 150°C for 2 min; programmed to increase 10°C/min up to 230°C, 5°C/min up to 280°C; and then maintained at 280°C for 10 min with a helium flow rate of 1.0 ml/min. EPA and DHA were separated, and the area beneath each chromatographic peak of their deuterium labels was obtained by integration using a Chemstation integrator Version D.02.00.275 (Agilent). For an internal standard, parinaric acid was selected because it exists in plants and its spectrum does not overlap with those of endogenous fatty acids for diagnostic ions. We selected the ω-3-derived ion ⁺CH(CH=CH)CH₂(CH=CH)CD₂CD₃ = m/z 113 for both d₅-EPA and d₅-DHA methyl ester and M⁺ (290) for methyl parinarate. Identification was conducted by examining and retention times as in Fig. 1⇓ (d₅-EPA-methyl ester: 11.7 min, methyl-parinarate; 13.1 min, d₅-DHA; 13.9 min). Calibration curves were obtained for each, as follows: d₅-EPA, y = 0.0016x + 0.0313 (r² = 0.9954, 50–500 ng/ml); d₅-DHA, y = 0.0015x − 0.0524 (r² = 0.9809, 50–500 ng/ml).

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FIGURE 1.

Circulating deuterium-labeled ω-3 fatty acids (d₅-EPA and d₅-DHA) appear in inflammatory exudates. FVB mice (6–8 wk) received d₅-EPA and d₅-DHA i.v. (1 μg/mouse) just before i.p. challenge with zymosan A (1 mg/ml/mouse). Peritoneal exudates were collected at 0, 2, 4, 12, 24, and 48 h and taken to solid-phase extraction. Eluate fractions were treated with diazomethane and taken to GC-MS. Results represent the mean ± SEM from n = 3, separate mice. Mass spectra of d₅-EPA methyl ester and d₅-DHA methyl ester. d₅-EPA and d₅-DHA were derivatized into methyl esters and injected into GC-MS. A, Mass spectrum of d₅-EPA methyl ester and selected ion monitoring chromatogram of d₅-EPA in mouse exudate. The ω-3-derived ion (⁺CH(CH=CH)CH₂(CH=CH) CD₂CD₃ = m/z 113) and α ion 180 were selected as diagnostic ions. The retention time was 11.7 min. B, Mass spectrum of d₅-DHA methyl ester and selected ion monitoring chromatogram of d₅-DHA in mouse exudate. The ω-3 ion 113 and α ion 160 were selected as diagnostic ions. The retention time was 13.9 min. C, Time course of d₅-EPA and d₅-DHA in peritoneal exudates. Upper panel, Results shown as percentage of i.v. injection: ▪, d₅-EPA; •, d₅-DHA; ▵, d₅-EPA in control (absence of zymosan A); ○, d₅-DHA in control (absence of zymosan A). Results represent the mean ± SEM from n = 3. ∗, Significantly different from 24 h, p < 0.05. Control: each two separate experiments. Middle panel, Time course of PMN infiltration. ▪, Zymosan A treatment; •, zymosan A + d₅-EPA and d₅-DHA. Results represent the mean ± SEM from n = 3. Lower panel: ▴, Increase of total protein amount in peritoneal exudates without d₅-EPA and d₅-DHA. Results represent the mean ± SEM from n = 7∼20 (35 ). ∗, Significantly different from 0 h, p < 0.05. ‡, Significantly different from 4 h, p < 0.05. •, Increase of total protein amount in peritoneal exudates of control mice. Mice were injected i.v. with d₅-DHA and d₅-EPA, and 5 min later 1 ml of saline was administered i.p. Time zero = total protein was 1.67 mg with zymosan A, and in the absence of zymosan A was 1.17 mg. Control experiment: each two separate experiments.

Ischemia-reperfusion-induced second-organ injury

Mice were anesthetized by i.p. injection of pentobarbital (80 mg kg⁻¹, Nembutal sodium solution NDC 0074-3778-04). Hind limb ischemia was initiated using tourniquets consisting of a rubber band placed on each hind limb (as in Ref. 31). Mice were subjected to hind limb ischemia for 1 h, after which the tourniquets were removed to initiate reperfusion. Resolvins and their analogues were each administered at 1 μg/mouse (i.e., DHA, RvD1, RvD1 methyl ester, 17-(R/S)-methyl-RvD1 methyl ester, RvE1, or 19-p-fluorophenoxy-RvE1 methyl ester) in vehicle (5 μl of ethanol in 120 μl of sterile saline) and compared with vehicle alone. They were administered i.v. to the tail vein ∼5 min before the start of the reperfusion period. For postreperfusion challenge, at 60 and 90 min after tourniquet release, mice were administered 100 ng of 17-(R/S)-methyl-RvD1-methyl ester (prepared as in Ref. 20) or vehicle alone i.v. At the end of this reperfusion period (2 h), the mice were euthanized with an overdose of anesthetic and the lungs were quickly harvested, frozen in liquid nitrogen, and stored at −80°C. The right lungs were homogenized from individual mice and centrifuged, and the tissue levels of myeloperoxidase (MPO) in the resulting supernatants were determined using a mouse MPO ELISA (Hycult Biotechnology; Cell Sciences).

Microfluidic chambers and leukocyte motility

A new microfluidic chamber was designed for testing the actions of putative novel lipid mediators on the chemotactic function(s) of neutrophils. These analyses were performed immediately after neutrophils were separated from components of whole blood within the chamber (vide infra), which routinely averaged less than 5 min, and lipid mediator (test compounds) adsorption to the chamber surfaces was prevented through the use of polyethylene glycol (PEG) coatings. The microfluidic chamber with microstructured valves was fabricated in elastomeric materials using standard microfabrication technologies (32). Briefly, two layers of poly(dimethylsiloxane) (PDMS), molded on two silicon wafers, were bonded together and on top of a glass slide. A network of channels was formed between the glass and the first layer, and control channels for the microstructured valves were formed between the first and second layers of PDMS. For details of the fabrication process, see Supplementary Materials.⁵

After assembly, different sections of the chamber were isolated through the actuation of the valves, and each section was chemically modified for specific functionalities. The surfaces of the network channels in the gradient generator section were modified with PEG to prevent absorption of the lipid mediators to the PDMS surfaces (see Results; Fig. 3⇓ illustration). Briefly, the PDMS surface of the gradient generation networks was treated with 5% (v/v) 3-(trimethoxysilyl)-propyl acrylate (92%; Aldrich) in acetone and followed by rinsing the chamber with acetone. Next, PEG diacrylate (Aldrich) with 1% photo initiator (2,2-dimethoxy-2-phenyl-acetophenone; Aldrich) was introduced to the chamber. After 15 min, the chamber was washed with 5% H₂O in acetone and with pure acetone subsequently, and then dried with air. After this surface modification procedure, the chambers were stored in the refrigerator for up to 1 wk. The surfaces of the chemotaxis chambers, where neutrophils were captured from whole blood, were modified by physical absorption of P-selectin (10 ng/ml for 30 min; R&D Systems), followed by blocking the entire channel network with 2% human serum albumin (Sigma-Aldrich) in HBSS (Sigma-Aldrich) immediately before use. The separation between the different sections of the chamber during the surface modification was achieved by keeping the valves between the main channels and the gradient generators closed.

For neutrophil chemotaxis experiments, the chambers were purged with HBSS containing 0.1% human serum albumin, with care to remove all bubbles. Four syringes, two with buffer, one with IL-8 chemoattractant (R&D Systems), and one with the lipid mediator, i.e., RvD1 or other related compounds tested in HBSS were placed in syringes (1 ml), with a syringe pump set for 0.1 μl/min. The flow was allowed to stabilize for 3 min, with the microscale valves between the chemotaxis chamber and the two gradient generators closed (see Fig. 3). Two steady-state gradients of IL-8 (0–10 nM) were formed in the two gradient generators, one alone and one with an overlaying uniform concentration of the lipid mediator, and were ready to be delivered to the chemotaxis chambers. Approximately 5–10 μl of capillary blood was collected from healthy volunteers by finger prick using a BD genie lancet (BD Biosciences) using Protocol 1999-P-001297 approved by the Partners Human Research Committee. The whole blood was then quickly mixed with heparin (10 μl) in a syringe tip (30G; Small Parts). After opening the cell inlet valve, the suspension was slowly pushed through the tubing (Tygon; Small Parts) into the microfluidic chamber. The blood stayed in the main channel for 3 min, and then the valve was opened for the two chemokine gradient generators. The flow removed the majority of RBC and other cells that were not tethered with the chamber-attached P-selectin, thus allowing direct observation of the neutrophils captured on the surface of the chamber. After 10–15 min, the gradient was switched to the gradient containing the chemokine alone or either native DHA or RvD1. The migration of neutrophils in the chemokine gradient and their individual response(s) to addition of RvD1 or DHA were recorded with a video and/or digital camera mounted on the microscope, and cell migration was analyzed using the cell tracking function in Metamorph (Molecular Devices). At least a dozen cells per condition were tracked and analyzed for displacement in the direction of the gradient and along the flow.

Statistical analysis

Results from both in vitro and in vivo experiments were analyzed by Student’s t test with p values ≤0.05 taken as statistically significant. The migration of neutrophils in the microfluidic chambers is presented as average and SEM displacement from the original position at the time of the gradient switching.

From circulation to inflammatory exudates

To determine whether circulating ω-3 fatty acids in their unesterified form are available to evolving inflammatory exudates, we administered i.v. deuterium-labeled ω-3 fatty acids, e.g., d₅-EPA and d₅-DHA, to mice simultaneously experiencing localized inflammatory responses, namely peritonitis. The magnitude of the inflammatory insult was selected to be a self-limited spontaneous resolving peritonitis (33) to monitor the potential presence of deuterium-labeled fatty acids in the inflammatory exudates (Fig. 1⇑, A and B). Both deuterium-labeled EPA and DHA were identified within the local inflammatory exudates within 2 h. As shown in Fig. 1⇑C (upper panel), both d₅-EPA and d₅-DHA rapidly appeared in exudates during the time course of initiation of inflammation. The levels of both unesterified d₅-EPA and d₅-DHA gradually declined at 24 h. In this spontaneous resolution system, maximum inflammation as defined by maximum PMN infiltrates within the exudates was at ∼4 h, and the resolution phase ranged between 12 and 24 h.

As depicted in Fig. 1⇑C (middle panel), PMN infiltration into the exudates was monitored. By definition, these exudates contain serum proteins (34) that were determined within the peritoneal exudates throughout the time course (Fig. 1⇑C, lower panel; cf Ref. 35). Of interest, the time course of both d₅-EPA and d₅-DHA paralleled the initial appearance of edema and increases in protein levels in these inflammatory exudates. Their presence also coincided with PMN infiltration (Fig. 1⇑C). At 48 h, both d₅-EPA and d₅-DHA levels were significantly greater than at 24 h. It is noteworthy that, without inflammation (i.e., peritonitis), neither d₅-EPA nor d₅-DHA appeared in peritoneal lavages (Fig. 1⇑). Thus, EPA and DHA, the precursors for resolvins and protectins, rapidly appeared within these developing inflammatory exudates from peripheral circulation and are delivered to exudates during both the initiation and resolution phases.

Next, we used a second approach to verify that DHA, i.e., a representative ω-3 fatty acid, indeed appears in exudates directly from peripheral circulation (Fig. 2⇓). To this end, radiolabeled ω-3 fatty acid tracer ¹⁴C-DHA was administered i.v., and we monitored recovery of ¹⁴C from DHA and the total protein amount in murine exudates. To reduce potential and immediate influences of diet, these experiments were performed with mice that fasted overnight before receiving i.v. ¹⁴C-DHA. Exudates were collected at 1, 2, 4, and 12 h. At 1 h, ¹⁴C label had already reached maximal exudate levels, and protein levels appeared to parallel the ¹⁴C radioactivity profile. These results suggest that ¹⁴C-DHA and its products rapidly appear coincident with increases in protein levels and increases in PMN infiltration within the exudate site of inflammation. Thus, they confirm that tracer levels of ¹⁴C-DHA and its initial transformation products rapidly appeared within the exudates during early initiation of the inflammatory response in vivo (Fig. 2⇓). Of note, these tracer levels of DHA did not reduce leukocyte trafficking as monitored by FACS analysis of the exudates (see Supplement⁵ Fig. 2).

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FIGURE 2.

Rapid exudate appearance of ¹⁴C-DHA from circulation. Each exudate was mixed with scintillation fluid, and radioactivity was counted with a scintillation counter. Extracellular protein levels were determined by the Bradford method and expressed as the total amount in each sample. Three different groups of animals and experiments were performed.

Single-cell monitoring of real-time responses demonstrates the direct impact of RvD1, but not its precursor DHA

The original isolation and structure elucidation of RvD1 demonstrated its presence and potent anti-inflammatory and proresolution actions in spontaneous resolving exudates and disease models in vivo (19, 20). In this study, we questioned whether RvD1 or its precursor DHA has direct actions with human neutrophils, and specifically whether RvD1 can alter the directed movements of single cells along chemotactic gradients. This is a central point because excessive infiltration of PMN and release of proinflammatory mediators is a well-appreciated key event in causing tissue damage and uncontrolled inflammation. To address this, we used a 1-μl microfluidic chamber that was engineered with microstructure membranes (see Fig. 3⇓). The chamber permitted the isolation of individual PMNs from one drop of whole blood (see Materials and Methods and Fig. 3⇓C) following a simple finger prick. After isolation, the microstructured valves in the chamber (Fig. 3⇓B) were used to control the timing of exposure of the captured PMNs to the two distinct chemotactic gradients (Fig. 3⇓A) (see Materials and Methods). Images of the PMN before and during exposure to the two gradients were continuously monitored using a camera and recorded (Fig. 4⇓A).

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FIGURE 3.

Microfluidic chamber for PMN chemotaxis from whole blood. A, Overview of the microfluidic chamber for isolating neutrophils from whole blood and successive exposure of the cells to preformed chemokine gradients and lipid mediators. B, Rapid switching between chemotaxis conditions was achieved by using microfluidic valves integrated on the chip, and pneumatically controlled from the outside. C, One drop of blood from a finger stick illustrates the loading of the chamber with whole blood.

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FIGURE 4.

RvD1 stops neutrophil chemotaxis. A, Neutrophils having polarized morphology during exposure to IL-8 gradient (−1 min) quickly become rounded (+1 min) and were unable to regain their polarized morphology (+5 min) after exposure to RvD1. B, Population analysis shows that normal neutrophil migration in an IL-8 gradient is immediately and effectively blocked after exposure to RvD1. Results represent the mean ± SEM, n = 12.

The PMN trafficked along the IL-8 gradient (see Supplement⁵ Fig. 3) and displayed the typical shape change and morphology of PMN during chemotaxis in a linear gradient (cf Ref. 36 and Fig. 4⇑A, left panel). Between 0 and 8 min, RvD1 at 10 nM was uniformly infused in the microfluidic chamber. Before exposure to RvD1, individual PMN movements and distances were proportional to time and totaled ∼30–40 μm. Almost immediately on exposure to RvD1, PMN dramatically changed shape (see Fig. 4⇑A, middle panel) and ceased directed chemotactic movements (Fig. 4⇑, A and B, and see Supplementary Video online).⁵ Fig. 4⇑B shows the average displacement, demonstrating a highly reproducible stopping of PMN migration when exposed to RvD1 (n = 12). In sharp contrast, DHA, the biosynthetic precursor to RvD1, at equimolar concentrations did not stop PMN migration (Fig. 5⇓). Thus, by tracing the displacement of single cells in the direction of an IL-8 chemotactic gradient before and after RvD1 was introduced into the chamber, we were able to record the direct actions of RvD1 on PMN. Thus, these results establish the ability of RvD1 to essentially completely stop PMN movements, almost immediately upon exposure, as well as the ability of RvD1 to stimulate rapid shape changes of PMN. These single-cell-recorded PMN responses were not shared by DHA, the RvD1 metabolic precursor in exudates, when introduced at equal molar concentrations in these microfluidic chambers.

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FIGURE 5.

DHA, precursor of RvD1, did not stop neutrophil chemotaxis. Direct comparisons of DHA and RvD1 in an IL-8 gradient with PMN isolated from a drop of whole blood. •, RvD1; ▵, DHA. Results represent the mean ± SEM, n = 12, for each.

Resolvins are organ protective in vivo

Ischemia followed by reperfusion is a well-appreciated pathophysiologic mechanism of organ injury and local tissue damage that can be initiated by excessively activated PMN. This system in mice (Fig. 6⇓) models the second organ injury and tissue damage observed in humans following tourniquet release of vessels in surgery involving, for example, extremities (37, 38). The occlusion of blood vessels creates local ischemia and remote organ injury that has many features of rapid, acute inflammation and tissue damage (31). On release of the tourniquet occlusion, reflow is initiated and aberrantly activated PMN rapidly perfuse and infiltrate secondary organs, causing local damage (31). In this study, we evaluated RvD1 in a remote organ injury system, i.e., hind limb ischemia-reperfusion, to assess whether the ability of RvD1 to stop PMN migration in microchambers can in turn reduce PMN-mediated tissue and organ damage in vivo. Using the time line illustrated in Fig. 6⇓ (upper panel), PMN accumulation in the lung was assessed by monitoring increases in both tissue histology and tissue MPO, a leukocyte marker enzyme. The extent of lung tissue injury is associated with PMN activation and organ infiltration (31).

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FIGURE 6.

RvD1 protects from second organ lung injury following ischemia-reperfusion. A, Time course of experiment. B, RvD1 protects lung from ischemia/reperfusion injury. Hind limb ischemia was induced in mice by creating tourniquets using a rubber band on each hind limb. After 1 h, the tourniquets were removed and reperfusion ensued. Test compounds (1 μg per mouse of DHA, RvD1, RvE1) in vehicle were administered i.v. 5 min before the start of the reperfusion period. At the end of the reperfusion period (2 h), lungs were collected and MPO levels were determined. Results indicate percentage of reduction of control, mean ± SEM (n = 4∼6, control; n = 12). RvD1, ▪ Values for DHA and RvE1 were not significant changes. ∗, Significantly different from values obtained with vehicle, p < 0.02. †, Significantly different from values obtained with DHA, p < 0.01. ‡, Significantly different from values obtained with RvE1, p < 0.002.

In this system, we also directly compared the actions of RvD1 with its biosynthetic precursor DHA as well as with another resolvin, namely, RvE1, which is a potent product of EPA that is anti-inflammatory in both oral inflammation (39) and colitis (reviewed in Ref. 1). RvD1, but neither RvE1 nor DHA, was able to protect the lung tissues from excessive leukocyte infiltration (Fig. 6⇑). The histology and reduction in leukocyte infiltration (data not shown) were confirmed by the reduction in the lung-associated MPO values. At 1 μg/mouse, RvD1 sharply reduced ∼50% leukocyte infiltration in the reperfusion tissues. In contrast, at equal doses, neither DHA nor native RvE1 gave significant reduction in PMN tissue infiltration. Because RvD1 undergoes local metabolic inactivation (20), as does RvE1 (26, 40), blocking of their respective metabolic sites of inactivation within each molecule de novo was undertaken using metabolically stable analog mimetics. To this end, both 17-(R/S)-methyl RvD1 and RvD1 were prepared by total organic synthesis for in vivo administration. Both RvD1 and the related analogues (structures shown in Fig. 7⇓) sharply reduced MPO levels in lung tissues. Of interest, the metabolically stable analog of RvE1, namely 19-p-fluorophenoxy RvE1 at 1 μg/mouse, also significantly reduced leukocyte infiltration, whereas native RvE1 was unable to protect lung from PMN-induced damage in this system. It is noteworthy that mouse lung tissue enzymatically converts RvE1 to 18-oxo-RvE1 (26), which is devoid of bioactivity. The 19-p-fluorophenoxy RvE1 prevents this metabolic inactivation (Fig. 7⇓), and as documented in this study, displayed potent protective actions dramatically reducing PMN infiltration and second organ injury of the lung. The actions of resolvins during reperfusion were investigated. Ninety minutes after tourniquet release, 100 ng of 17-(R/S)-methyl-RvD1 methyl ester was administered. The RvD1 analog lowered the increases in MPO levels in lung, whereas at 60 min after ischemia-reperfusion, administration of this RvD1 analog (100 ng i.v.) did not protect from second organ injury. Thus, by preventing the local metabolic inactivation of resolvins, their ability to reduce PMN infiltration into distal organs was sustained for an enhanced beneficial action when given before reflow and at 90 min after reflow compared with controls.

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FIGURE 7.

Resolvin and related analogues are protective in vivo. A, Structures and actions of compounds used in this experiment. Test compounds (1 μg of RvD1 carboxymethyl ester (RvD1-Me), 17-(R/S)-methyl-RvD1 carboxymethyl ester (17-(R/S)-methyl-RvD1-Me), and 19-p-fluorophenoxy-RvE1 methyl ester (19-p-fluorophenoxy-RvE1-Me)) in vehicle were administered i.v. Results indicate percentage of reduction compared with control, mean ± SEM (n = 3∼5, control; n = 12). RvD1-Me, ▪; 17-(R/S)-methyl-RvD1-Me, □; 19-p-fluorophenoxy-RvE1-Me, ▧. ∗, Significantly different from values obtained with vehicle, p < 0.02. †, Significantly different from values obtained with DHA; p < 0.01. B, Postreperfusion challenge with RvD1. At 60 and 90 min after tourniquet release, 100 ng of 17-(R/S)-methyl-RvD1-Me or vehicle was administered i.v. Results indicate percentage of reduction compared with parallel controls, mean ± SEM (n = 3). ∗, Significantly different from values obtained with vehicle; p < 0.02.