Minor involvement of nitric oxide during chronic endotoxemia in anesthetized pigs | DISCOUNT CODE !

septic shock, which remains a leading cause of death in intensive care units, is characterized by hypotension, vascular hyporeactivity to vasoactive agents, myocardial dysfunction, and changes in regional blood flow (4, 25). Multiple organ dysfunction syndrome, defined as a simultaneous failure of two or more organs, occurs frequently and is associated with a high mortality rate. Endotoxins, included in the wall of Gram-negative bacteria, are responsible for most of the abnormalities seen in sepsis and are commonly used to mimic septic shock in experimental models.

The liver is crucial in severe sepsis because it contains most of the body's macrophages (Kupffer cells) capable of eliminating endotoxins and bacteria that may stimulate the systemic inflammatory response. In addition, hepatocytes synthesize the proteins and acute phase enzymes needed to modulate the inflammatory response. Finally, since hepatic blood flow accounts for 25% of cardiac output (CO), changes in hepatic perfusion can greatly impair systemic hemodynamics.

Nitric Oxide (NO) is one of the leading mediators inducing cardiovascular abnormalities during septic shock. Under physiological conditions, the constitutive endothelial isoform of NO synthase (eNOS or NOS 3) produces low levels of NO and regulates vascular tone as well as numerous cellular functions (24). In contrast, during sepsis, the inducible form of NOS (iNOS or NOS 2) produces large amounts of NO (14,33). However, there is evidence that humans produce less NO than laboratory animals during septic shock (28). The expression of NOS 2 was observed mainly in rodents that received intraperitoneal endotoxin and few studies were devoted to the determination of protein expression in large animals. Since the induction of NOS 2 requires the de novo synthesis of the enzyme by the proteins, studies of more than 4 hours are required to accurately assess the role of NO from NOS 2 in the pathogenesis of endotoxemia.

To investigate the involvement of hepatic NO over time in endotoxemia in a species different from rodents, we measured plasma concentrations of

NO2

, no3 and cGMP, tested the effect of ACh on mean arterial pressure (MAP) and hepatic blood flow over time, and assessed the expression of NOS 2 in Liver biopsies in anesthetized pigs perfused with endotoxin. Escherichia coli(160 ng ⋅ kg-1 ⋅ min-1) over a period of 18 hours.

Animals and anesthesia.

Male or female minipigs (23 ± 1 kg, not = 20) were fasted without access to water for 24 h before the experiment, were premedicated and placed in a supine position on the operating table. After induction of anesthesia with halothane, the pigs were mechanically ventilated with air and oxygen2 concentration (

FIO2

) = 0.4) to obtain a normal arterial

PCO2

. After intubation, halothane was removed and anesthesia maintained with thiopental (5 mg kg-1 ⋅ h-1) and fentanyl (10-20 μg kg)-1 ⋅ h-1). In addition, pancuronium (0.2 mg kg-1 ⋅ h-1) has been used as a skeletal muscle relaxant. The protocol was approved by the Animal Welfare Committee of the University of Geneva and the Veterinary Office and followed the Guidelines for the care and use of laboratory animals.

Surgery.

The right carotid artery was cannulated to measure the MAP and collect blood samples. A catheter was inserted into the right external jugular vein for fluid and drug administration. A pulmonary arterial catheter (131H-7F, Baxter, Düdingen, Switzerland) was inserted into the right internal jugular vein to measure mean pulmonary arterial pressure (mmHg), central venous pressure (mmHg), pulmonary wedge pressure (mmHg) ) and CO (ml ⋅ min-1 ⋅ kg-1).

After a mid-abdominal incision, the bladder was drained. Two flow probes were positioned around the portal vein and hepatic artery (above the bifurcation of the common hepatic artery and the gastroduodenal artery) to determine the blood flow of the portal vein (PVBF, ml / min) and that of the hepatic artery (HABF, ml / min). Blood flow was measured using the ultrasonic transit time flow technique (Transonic System, Ithaca, NY). To measure portal vein pressure (PVP, mmHg) and hepatic vein pressure (HVP, mmHg), catheters were inserted into the portal vein through a lateral branch and into the hepatic vein via the external jugular vein left, respectively. The location of the ends of the catheter was confirmed by direct palpation. Once surgery was completed, the abdominal wall was closely re-approximated to minimize heat loss during the experiment. The core temperature was maintained with heat lamps.

Experimental protocol.

After a stabilization period of 2 h, the hemodynamic parameters were measured from time (t) = 0 (basic value) over 18 h. Endotoxic (Endo,not = 13) the pigs received continuous intravenous infusion of endotoxin from E. coli (160 ng ⋅ kg-1 ⋅ min-1) more than 18 hours. Control (Ctrl, not = 7) The animals received an infusion of saline solution in a similar volume. The animals were perfused with saline (12 ml kg-1 ⋅ h-1during surgery and 8 ml ⋅ kg-1 ⋅ h-1during the experimental protocol) to compensate for the loss of fluid induced by anesthesia and surgery. After the recovery period, the hemodynamic parameters were recorded every 3 hours for the next 18 hours.

Determination of systemic and hepatic O2 delivery and O2 consumption.

The exhaled flow of the ventilator has been directly connected to a metabolic measuring cart (Datex, Helsinki, Finland) for continuous measurements of2 consumption (V˙o2ml / min). O2 Carotid artery (a), portal vein (pv) and hepatic vein (hv) contents were calculated according to the equation.

O2contents=(PO20,003)+(HbSO21.36)

The following equations were used

systDO2=COaO2contents

a dO2=HABFaO2contents

pvDO2=PVBFPVO2contents

HEPDO2=pvDO2+a dO2

HEPVO2=((aO2contentsHABF)+(PVO2contentsPVBF))(hvo2contents(HABF+PVBF))

where systDo2, a do2, pvDo2and hepDo2 are systemic, hepatic artery, portal vein, and hepatic O2delivery, respectively, and hepV˙o2 is hepaticV˙o2.

Hepatic injury.

To assess liver function, we measured bile flow every 3 h (μl min-1 G 100 g-1) and calculated the ratio of wet weight to dry weight of the liver. Hepatic cell damage was assessed by arterial concentrations of aspartate transaminase (AST) and alanine transaminase (ALT), γ-glutamyl transpeptidase (γ-GT), alkaline phosphatase and lactate dehydrogenase (LDH) (all in IU / l).

Vascular reactivity to ACh.

An intravenous injection of ACh (10 μg / kg) was performed for 20 seconds, and changes in MAP, HABF and PVBF were observed within 200 seconds after drug administration. To compare the evolution of vascular reactivity over time, three tests (A, B, and C) were performed 5, 11 and 17 h after the initial administration of endotoxin or saline.

Arterial levels of

NO2

,

NO3

, CGMP and 6-keto-PGF .

Serum samples were analyzed to determine the stable end products of NO oxidation (

NO2

and

NO3

) using a modified procedure based on the Griess reaction as recently described (23). In short, the samples were deproteinized by ultrafiltration (Centrisart, Sartorius, Göttingen, Germany) to remove the high molecular weight particles.

NO3

was then reduced enzymatically to

NO2

nitrate reductase, and the samples were analyzed to determine their

NO2

concentrations (representing

NO2

+

NO3

) with a mixture of dapsone (4,4'-diaminodiphenylsulfone), andNOT(1-naphthyl) ethylenediamine. After incubation at room temperature, light absorption was measured at 550 nm in a microplate reader (EAR 300; SLT, Crailsheim, Germany).

NO2

concentrations were calculated from a standard

NO2

curve. Plasma concentrations of cGMP and 6-keto-PGF (a stable degradation product of prostacyclin) were measured by a radioimmunoassay technique according to the instructions provided by the manufacturer (Amersham, Little Chalfont, United Kingdom).

NOS 2 synthase in the liver.

To evaluate the induction of NOS 2 at various sites of the liver, biopsies were performed on the right and left lobes of the tissues surrounding the large vessels (inferior vena cava and portal vein) 6 and 9 h after the onset of the administration of endotoxins. The biopsies were collected, frozen and stored at -70 ° C before being tested.

Analysis of NOS 2 mRNA by RT-PCR.

Total RNA was isolated and subjected to RT-PCR analysis as previously described (17). Two milligrams of total RNA were reverse transcribed cDNA at 37 ° C for 60 min in 10 μl of solution containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 4 mg of MgCl2, 1 mM each dNTP (Boehringer Mannheim), 10 mMdl-dithiothreitol, 10 U of human placenta RNAase inhibitor (GIBCO BRL, Gaithersburg, MD), oligo (dT) primers (Boehringer Mannheim) and 200 U Moloney murine leukemia virus reverse transcriptase (GIBCO). The cDNA was then used to detect NOS 2 mRNA by PCR using human primers. The NOS 2 sequences were as follows: 5'-GCCTCGCTCTGGAAAGA-3 (bases 1,425 to 1,441 sense) and 5'-TCCATGCAGACAACCTT-3 (bases 908 to 1,924 antisense), amplifying a product of 499 bp. An equal load of RNA was verified by β-actin, using specific primers, as follows: 5'-CGGGAACCGCTCATTGCC-3 (sense) and 5'-ACCCACACTGTGCCCATCTA-3 & # 39; (antisense) amplifying a PCR product of 289 bp. The PCR reactions were carried out in 50 μl of solution containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 mM each, dNTP, 1.5 mM MgCl.2and 1.25 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Thirty cycles of one minute at 96 ° C (denaturation), 2 minutes at 56 ° C (annealing) and 2 minutes at 72 ° C (extension) were performed in a thermal cycler (Perkin-Elmer Cetus).

Western blot analysis.

The tissues were homogenized in 20 mM TES, 2 mMdldithiothreitol, 10% glycerol, 25 mg / ml antipain, 25 mg / ml aprotinin, 25 mg / ml leupeptin, 25 mg / ml chymostatin, 50 mM phenanthroline, 10 mg / ml pepstatin A and 100 mM phenylmethylsulfonyl fluoride and centrifuged at 100,000 g (crude cytosol) as previously described (20). Equal amounts of protein (60 μg) obtained from biopsies were separated by 7.5% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes using a transbutter ( Pase, Lübeck, Germany). Nonspecific binding to the membrane was blocked with 5% skimmed milk powder in PBS-Tween 20 overnight at 4 ° C. The blots were washed twice in PBS and then incubated with an antiserum antibody. affinity purified polyclonal IgG rabbit mice (1/1000, Transduction Laboratories, Lexington, KY). This antibody was chosen because it cross-reacts with the porcine isoform NOS 2 (13). As a positive control, we used isolated porcine hepatocytes exposed for 9 h to interferon-rh (γ-rh, IFN-γ, 200 U / ml), tumor necrosis factor-α (rh-TNF-α 1000 U / ml). rh-interleukin-1β (rh-IL-1β, 15 U / ml), endotoxin (10 μg / ml) and 1 mM l-arginine. All culture additives were purchased from Sigma (Deideshoffen, Germany). After incubation with the first antibody, the membranes were washed (5 times) with PBS-Tween-20 and incubated with the second antibody (1: 1000; goat and rabbit IgG, conjugated with horseradish peroxidase, Amersham) for 1 hour in the room. Temperature. After incubation, the membranes were washed with PBS-Tween-20, developed with 10 ml of a 1: 1 mixture of solutions 1 and 2 of the ECL detection system (Amersham) and exposed to a movie.

Statistical analyzes.

The data are expressed in means ± SE. The data were analyzed using unidirectional and bidirectional ANOVA for repeated measurements with the Bonferroni test for intra-group comparison over time, as appropriate. The meaning has been established P <0.05.

Hemodynamic parameters in Ctrl and Endo pigs.

All animals survived in the Ctrl group, while three pigs died in the Endo group after 3, 9, and 12 h of endotoxemia, respectively. Data from these three pigs were not included in the Endo group (Table 1). In the Ctrl group, MAP decreased after t = 12 h, while the CO remained stable over time. On the other hand, in the Endo group, the MAP decreased rapidly t = 3 h and remained low until the end of the experimental protocol, whereas the only significant decrease in CO was observed at t = 6 h. Heart rate increased by t = 3 h to t = 18 h in the Endo group but remains stable in the Ctrl group. HABF and PVBF have not been changed over time in both groups.

Table 1. Systemic and hepatic hemodynamic parameters in control and endotoxic pigs

Groups Endotoxin or saline infusion, h P
0 3 6 9 12 15 18
Mean arterial pressure, mmHg Ctrl 93 ± 3 82 ± 4 83 ± 5 83 ± 5 78 ± 5 * 79 ± 6 * 77 ± 4 *
Endo 104 ± 5 70 ± 4 * 64 ± 4 * 62 ± 3 * 59 ± 4 * 59 ± 5 * 50 ± 4 * 0,001
Cardiac rate, ml ⋅ min-1 ⋅ kg-1 Ctrl 78 ± 5 78 ± 5 84 ± 7 73 ± 6 75 ± 7 75 ± 5 76 ± 4
Endo 76 ± 4 72 ± 6 56 ± 5 * 58 ± 5 64 ± 5 67 ± 6 66 ± 11 0.489
Heart rate, beats / min Ctrl 85 ± 6 87 ± 6 92 ± 5 83 ± 5 95 ± 5 95 ± 5 100 ± 6
Endo 87 ± 5 139 ± 8 * 126 ± 7 * 130 ± 9 * 126 ± 8 * 127 ± 7 * 133 ± 6 * 0,029
HABF, ml ⋅ min-1 G 100 g-1 Ctrl 14 ± 3 14 ± 2 15 ± 3 14 ± 2 18 ± 3 22 ± 4 27 ± 5
Endo 12 ± 3 8 ± 1 5 ± 1 7 ± 1 11 ± 2 12 ± 2 12 ± 2 0.354
PVBF, ml ⋅ min-1 G 100 g-1 Ctrl 47 ± 5 54 ± 5 64 ± 6 51 ± 5 54 ± 5 57 ± 5 58 ± 8
Endo 64 ± 4 54 ± 4 62 ± 8 72 ± 8 77 ± 8 87 ± 12 83 ± 15 0.331
THBF, ml ⋅ min-1 G 100 g+1 Ctrl 61 ± 5 68 ± 5 79 ± 8 65 ± 4 72 ± 5 79 ± 3 85 ± 8
Endo 79 ± 4 62 ± 4 67 ± 7 79 ± 8 88 ± 8 99 ± 12 94 ± 14 0.297

Systemic and hepatic parameters of oxygenation in Ctrl and Endo pigs.

sysDo2 and Vo2 remained stable over time in both groups (Table 2). In pigs Ctrl, pvDo2 and aDo2 has not changed over time. The endotoxin infusion did not change the pvDo2 and aDo2. However, hepDo2/ sysDo2 increased to t = 18 h in Ctrl pigs and did not change in Endo pigs.

Table 2. Systemic and hepatic parameters of oxygenation in Ctrl and Endo pigs

Groups Endotoxin or saline infusion, h P
0 3 6 9 12 15 18
systDo2, ml ⋅ min-1 ⋅ kg-1 Ctrl 11 ± 1 11 ± 1 12 ± 1 11 ± 1 11 ± 1 11 ± 1 11 ± 1
Endo 11 ± 1 14 ± 1 10 ± 1 10 ± 1 10 ± 1 10 ± 1 9 ± 1 0.269
systVo2, ml ⋅ min-1 ⋅ kg-1 Ctrl 4 ± 1 4 ± 1 4 ± 1 4 ± 1 4 ± 1 4 ± 1 4 ± 1
Endo 4 ± 1 4 ± 1 4 ± 1 4 ± 1 4 ± 1 4 ± 1 4 ± 1 0.822
systo2ER% Ctrl 38 ± 2 36 ± 2 36 ± 3 37 ± 4 38 ± 4 41 ± 5 40 ± 4
Endo 36 ± 6 33 ± 2 40 ± 3 42 ± 3 43 ± 2 44 ± 3 47 ± 5 0.660
hado2, ml / min Ctrl 7 ± 1 7 ± 1 7 ± 1 8 ± 1 10 ± 2 11 ± 2 14 ± 3
Endo 7 ± 2 6 ± 1 4 ± 1 5 ± 1 7 ± 1 7 ± 2 7 ± 2 0.284
pvDo2, ml / min Ctrl 17 ± 2 20 ± 3 26 ± 5 20 ± 3 21 ± 3 22 ± 3 22 ± 4
Endo 27 ± 3 26 ± 3 34 ± 4 37 ± 6 34 ± 4 39 ± 5 33 ± 6 0.753
HEPDo2/ systDo2,% Ctrl 13 ± 1 15 ± 1 17 ± 2 16 ± 2 17 ± 2 19 ± 1 21 ± 2 *
Endo 15 ± 1 12 ± 1 19 ± 2 24 ± 4 22 ± 2 23 ± 2 21 ± 3 0.326
HEPVo2, ml / min Ctrl 4 ± 1 5 ± 1 5 ± 1 5 ± 1 8 ± 3 7 ± 1 7 ± 1
Endo 8 ± 2 9 ± 2 9 ± 2 12 ± 2 12 ± 2 11 ± 2 13 ± 2 0.973
HEPVo2/ systVo2,% Ctrl 5 ± 1 8 ± 2 7 ± 1 7 ± 2 9 ± 1 10 ± 1 10 ± 2
Endo 10 ± 1 10 ± 1 11 ± 2 12 ± 3 13 ± 2 12 ± 2 15 ± 2 0.983

Biological parameters in Ctrl and Endo pigs.

Metabolic acidosis induced by endotoxin infusion began at t= 3 pm and peaked at t = 18 h (Table 3). In addition, we observed a significant increase in arterial lactate concentration after 18 hours. In Ctrl pigs, the arterial pH and

HCO3

remained in normal concentrations, while arterial lactate concentrations decreased over time. Hemoconcentration was observed only from t= 3 h to t = 9 h in the Endo group. The platelet count decreased over time in both groups, but the decrease was greater in the Endo group than in the Ctrl group. The leukocyte count remained stable in the Ctrl group, but the leukopenia was severe in the Endo group at t = 6 h, with a partial recovery in time. the

PennsylvaniaO2

-at-

FIO2

the ratio was unchanged in the Ctrl group over time. In Endo pigs, the ratio decreased slightly t = 6 h after the start of the endotoxin infusion and remained low until the end of the protocol. The temperature remained stable in both groups over time.

Table 3. Biological parameters in the arterial plasma of Ctrl and Endo pigs

Groups Endotoxin or saline infusion, h P
0 3 6 9 12 15 18
pH Ctrl 7.43 ± 0.01 7.43 ± 0.01 7.44 ± 0.02 7.43 ± 0.01 7.41 ± 0.01 7.42 ± 0.01 7.38 ± 0.02
Endo 7.44 ± 0.01 7.33 ± 0.023-150 7.34 ± 0.013-150 7.35 ± 0.013-150 7.31 ± 0.013-150 7.29 ± 0.023-150 7.22 ± 0.043-150 0,001
Pco2, mmHg Ctrl 5.3 ± 0.2 5.2 ± 0.1 4.9 ± 0.2 4.9 ± 0.1 5.1 ± 0.2 4.9 ± 0.2 5.5 ± 0.2
Endo 4.9 ± 0.2 5.8 ± 0.3 5.2 ± 0.2 5.1 ± 0.2 5.1 ± 0.1 5.1 ± 0.2 5.5 ± 0.3 0.309
PennsylvaniaO2/ FIO2 Ctrl 486 ± 13 489 ± 8 481 ± 15 470 ± 16 463 ± 20 457 ± 20 443 ± 23
Endo 496 ± 15 382 ± 28 332 ± 343-150 320 ± 363-150 319 ± 373-150 327 ± 373-150 301 ± 413-150 0.058

HCO3

, mmol / l

Ctrl 25.2 ± 0.5 24.9 ± 0.6 23.9 ± 0.6 23.2 ± 0.6 23.2 ± 0.6 23.3 ± 0.8 23.8 ± 0.9
Endo 24.0 ± 0.4 21.6 ± 0.5 20.1 ± 0.33-150 19.8 ± 0.43-150 18.5 ± 0.43-150 18.0 ± 0.83-150 16.3 ± 1.33-150 0,001
Hematocrit,% Ctrl 30.5 ± 1.1 30.9 ± 1.5 30.7 ± 1.1 32.1 ± 2.3 31.0 ± 2.2 31.2 ± 2.5 30.9 ± 2.4
Endo 32.9 ± 1.3 44.0 ± 1.73-150 42.3 ± 1.43-150 39.6 ± 2.0 35.4 ± 1.6 35.0 ± 2.2 32.1 ± 2.4 0,010
Lactate, mmol / l Ctrl 1.4 ± 0.1 0.6 ± 0.13-150 0.7 ± 0.13-150 0.6 ± 0.23-150
Endo 1.4 ± 0.2 1.3 ± 0.1 1.5 ± 0.2 3.5 ± 1.13-150 0.012
Number of platelets, cells / ml (× 103) Ctrl 301 ± 19 257 ± 11 203 ± 153-150 179 ± 173-150
Endo 230 ± 23 102 ± 73-150 75 ± 63-150 52 ± 83-150 0.014
Number of leukocytes, cells / ml Ctrl 3,600 ± 760 3,160 ± 360 2,650 ± 300 2,870 ± 520
Endo 3,830 ± 600 500 ± 803-150 1200 ± 2903-150 1,730 ± 5303-150 0.031
Temperature, ° C Ctrl 38.4 ± 0.1 38.3 ± 0.3 38.3 ± 0.3 38.4 ± 0.2 38.3 ± 0.2 38.3 ± 0.3 38.5 ± 0.2
Endo 38.8 ± 0.2 38.2 ± 0.2 38.1 ± 0.3 38.1 ± 0.2 38.4 ± 0.3 38.0 ± 0.4 38.7 ± 0.7 0.722

Liver function in Ctrl and Endo pigs.

Bile flow decreased similarly in groups over time (Table 4). The ratio of dry and wet liver weights was also similar in the Ctrl (3.61 ± 0.14) and Endo (3.88 ± 0.09) groups. ALT and alkaline phosphatase remained stable in the Ctrl group, while γ-GT and LDH levels decreased and ASAT increased. In the Endo group, ASAT also increased and the increase was higher in this group compared to the Ctrl group. LDH and alkaline phosphatase were doubled over time, whereas, as in the Ctrl group, γ-GT decreased.

Table 4 Liver function in Ctrl and Endo pigs

Groups Endotoxin or saline infusion, h P
0 6 12 18
Alanine transaminase, IU / l Ctrl 61 ± 17 48 ± 13 45 ± 11 43 ± 8
Endo 52 ± 8 39 ± 6 41 ± 5 53 ± 5 0.685
Aspartate transaminase, IU / l Ctrl 96 ± 14 130 ± 18 195 ± 214-150 245 ± 224-150
Endo 95 ± 5 162 ± 20 273 ± 314-150 413 ± 514-150 0,023
γ-glutamyl transpeptidase, IU / l Ctrl 47 ± 7 28 ± 34-150 21 ± 24-150 19 ± 34-150
Endo 42 ± 6 36 ± 6 20 ± 14-150 19 ± 24-150 0.551
Lactate dehydrogenase, IU / l Ctrl 1,356 ± 272 1 090 ± 2454-150 912 ± 1314-150 842 ± 984-150
Endo 1 113 ± 83 1,326 ± 121 1.659 ± 86 2 163 ± 3114-150 0.012
Alkaline phosphatase, IU / l Ctrl 68 ± 5 70 ± 4 71 ± 6 76 ± 6
Endo 86 ± 12 117 ± 17 152 ± 19 204 ± 274-150 0,002
Biliary flow, μl min-1 G 100 g-1 Ctrl 4.0 ± 0.7 3.5 ± 0.3 2.7 ± 0.2 2.4 ± 0.3
Endo 4.8 ± 0.5 2.9 ± 0.54-150 2.4 ± 0.34-150 2.3 ± 0.34-150 0.774

Plasma concentrations of

NO2

,

NO3

, CGMP and 6-keto-PGF.

We found that

NO2

+

NO3

and cGMP did not change over time in Ctrl and Endo pigs (Table 5). In contrast, the 6-keto-PGF remained stable in Ctrl pigs and increased significantly in Endo pigs over time.

Table 5

NO2

+

NO3

, CGMP and 6-keto-PGF in the arterial plasma of Ctrl and Endo pigs

Endotoxin or saline infusion, h P
0 6 12 18

NO2

+

NO3

μmol / l

Ctrl 51.2 ± 10.7 77.6 ± 10.5 61.2 ± 14.1 74.7 ± 16.2
Endo 70.1 ± 17.1 87.9 ± 24.8 96.3 ± 25.7 109.0 ± 27.5 0.920
CGMP, pmol / l Ctrl 145.0 ± 27.9 132.9 ± 13.8 139.7 ± 7.8 133.1 ± 8.1
Endo 142.1 ± 10.4 195.3 ± 22.7 178.6 ± 24.3 162.5 ± 21.5 0.402
6-keto-PGF, ng / ml Ctrl 60.4 ± 18.3 46.0 ± 10.3 42.3 ± 10.8 44.0 ± 11.4
Endo 198.6 ± 74.1 602.5 ± 123.1 604.0 ± 193.7 1 875.5 ± 656.35-150 0,015

MAP and hepatic blood flow change after ACh injection.

To test for NO-dependent vascular reactivity, we compared MAP response to ACh in Ctrl and Endo 5 pigs (test A), 11 (test B) and 17 (C test) h after the initial administration of endotoxin or saline (Fig 1). The MAP decreased after ACh injection and this decrease remained constant in both groups. However, the decrease was significantly lower in the Endo group (A: -14 ± 2 mmHg; B: -17 ± 3 mmHg;C: -12 ± 2 mmHg) than in the Ctrl group (A: -31 ± 3 mmHg; B: -31 ± 4 mmHg; C: -26 ± 3 mmHg). HABF increased after ACh injection. The increase in HABF decreased with time, but the altered vascular response was similar in both groups. In contrast, the decrease in PVBF after ACh was constant over time in Ctrl pigs but decreased significantly in Endo pigs 5 h after the start of endotoxin infusion.

Fig. 1.

Fig. 1.Blood flow in the hepatic arteries (HABF; A) and the blood flow of the portal vein (PVBF; B) after injection of ACh (10 μg / kg). The tests were carried out 5 h (test A), 11 am (test B) and 5 pm (C test) after the initial administration of endotoxin or saline. Endotoxic pigs (filled bars, not = 10) were perfused with endotoxin (160 ng kg)-1 ⋅ min-1) and control pigs (open bars, not = 7) were infused with saline. * † P <0.05 vs control pigs during test A.

NOS 2 expression of mRNA and proteins in liver biopsies.

After the injection of endotoxin, the mRNA of NOS 2 is expressed between 2 and 6 h (20). No NOS 2 mRNA was detected in Ctrl pigs at t = 6 h (Fig. 2). In contrast, mRNA was positive in the right and left lobes of the liver, as well as in the tissues surrounding the inferior vena cava and portal vein. Similar results were observed at t = 9 h.

Fig. 2

Fig. 2The expression of nitric oxide synthase (NOS) 2 mRNA in liver biopsies collected from controls (lanes 1 and 2) and endotoxic (lanes 3 to 9) pigs. Biopsies were collected around the portal vein (lane 3), the inferior vena cava (lane 4), right upper lobe (lanes 5 and 6), left upper lobe (lanes 7 and 8) and the right lower lobe (way 9). Biopsies were collected 6 h after the start of saline or endotoxin infusion. MW, molecular weight.

AT t = 9 h, NOS 2 protein was not detected in Ctrl pigs (Fig. 3). In an endo pig, the protein was expressed only in tissues surrounding the portal vein and inferior vena cava, whereas in other liver regions no protein could be detected. The porcine isoform NOS 2 also gave a positive result in hepatocytes exposed to cytokines and endotoxin. Similar results were found in two other Endo pigs t = 9 pm, while at t = 6 h, the expression of NOS 2 was much lower (Table 6). Thus, although mRNA was found in all hepatic regions, the protein was expressed only in regions surrounding large vessels.

Fig. 3

Fig. 3Expression of NOS 2 protein in liver biopsies collected in 1 Ctrl pig and 1 Endo pig. Positive control (PC) shows protein expression in porcine hepatocytes incubated with endotoxin and cytokines (interferon-γ rh (200 U / ml), tumor necrosis factor-α (1000 U / ml) rh-interleukin-1β (15 U / ml)) over 9 h. Biopsies were collected around the portal vein (lane 3), the inferior vena cava (lane 4), right upper lobe (lanes 5 and 6), left upper lobe (lanes 7 and 8) and the right lower lobe (way 9) 9 h after the start of the administration of endotoxins (lipopolysaccharide (LPS)) or a saline infusion (Ctrl).

Table 6 Expression of NOS 2 protein in porcine liver biopsies

Groups Time, h The portal vein Vena Cava Right upper lobe Upper left lobe Right lower lobe
Sample 1 Sample 2 Sample 1 Sample 2
Ctrl 9 North Dakota. North Dakota. North Dakota. North Dakota. North Dakota.
Ctrl 9 North Dakota. North Dakota. North Dakota. North Dakota. North Dakota.
Endo 6 + + ±
Endo 6 ++ +
Endo 6 + ++
Endo 9 +++ +++ ±
Endo 9 +++ ++
Endo 9 ++ +++ +

In anesthetized pigs, continuous infusion of endotoxin for 18 h caused hypotensive and normokinetic shock in association with minor changes in hepatic blood flow and blood pressure.2 delivery to the liver. Metabolic acidosis and pulmonary dysfunction progressively developed in Endo pigs. Minor liver injury was observed in both groups. Endotoxin infusion did not significantly increase plasma concentrations of

NO2

+

NO3

and cGMP. In addition, the decrease in MAP after ACh injection was lower in Endo pigs than in Ctrl pigs. The increase in HABF after ACh gradually decreased over time, while the decrease in PVBF remained constant in both groups. Endotoxins did not affect hepatic responses to ACh except for t = 5 h, when the decrease in PVBF was lower in Endo pigs than in Ctrl pigs. The mRNA of NOS 2 was not detected in liver biopsies taken from Ctrl pigs. Although mRNA of NOS 2 is present in various liver segments collected in endo pigs, NOS 2 protein expression was only detected in the tissues surrounding the portal vein and vein lower cellar. In contrast, plasma concentrations of prostacyclin increased significantly over time. Thus, although endotoxemia induces the expression of NOS 2 in the liver, the involvement of NO remains minor in this pattern of sepsis.

Endotoxic model.

Many models of porcine endotoxemia have been described in the literature, but the dose and duration of infusion vary from one study to the next. For example, Hasibeder et al. (15) infused 2 μg / kg of endotoxin in 20 minutes, Cohn et al. (7) infused 250 μg / kg over 20 min, Breslow et al. (5) infused at 5 μg / kg in 40 minutes and Klemm et al. (18) infused 15 μg / kg in 3 h. In all studies, except for the one published by Weingand et al. (36), the pigs were anesthetized. The mortality rate in these experimental models ranged from 38% to 58% and was primarily a consequence of severe pulmonary hypertension. In our study, the mortality rate was lower because 13 pigs had received endotoxin and only 3 pigs had died before the end of the experiment. As observed in this study, most porcine sepsis models have described hypotensive shock and normal or hypokinetic shock with pulmonary hypertension (30, 35). This model was also characterized by hemoconcentration, decreased platelet count, leukopenia, and metabolic acidosis. the

PennsylvaniaO2

-at-

FIO2

The ratio decreased significantly over time in Endo pigs, but remained stable in Ctrl pigs. Interestingly, surgery and long-term anesthesia also altered hemodynamic and biological parameters in Ctrl pigs: MAP decreased while CO remained constant and platelet count decreased, while leukocyte count decreased. #39;hématocrite, le pH et le lactate plasmatique ne changeaient pas avec le temps. Contrairement aux rongeurs, chez lesquels l&#39;endotoxine est principalement injectée par voie intrapéritonéale, les grands animaux ont reçu de l&#39;endotoxine en bolus ou en perfusion continue. Étant donné que les macrophages extra-hépatiques peuvent détoxifier les endotoxines lorsqu’elles sont injectées par voie intraveineuse, la quantité totale d’endotoxines atteignant le foie peut être plus faible chez les porcs que chez les rongeurs chez lesquels des endotoxines sont injectées par voie intrapéritonéale. Ce fait pourrait expliquer la différence d&#39;expression de NOS 2 entre porcs et rongeurs au cours de l&#39;endotoxémie. De plus, la translocation bactérienne pouvant survenir au cours d&#39;une endotoxémie était plus susceptible d&#39;atteindre la circulation systémique par le ganglion mésentérique que par la veine porte (22, 34).

Flux hépatique et fonction pendant l&#39;endotoxémie.

Dans la présente étude, la perfusion hépatique n&#39;a pas été modifiée de manière significative au fil du temps dans les deux groupes. Récemment, chez des porcs anesthésiés, Saetre et al. (30) ont mis en évidence une hypoperfusion hépatique grave dans les 3 heures suivant la perfusion d&#39;endotoxine, qui a été inversée par l&#39;aminoéthyl-isothiourée, un inhibiteur sélectif de l&#39;iNOS. Concomitamment, hépatique Do2 diminué, alors que hepaticV˙o2 a été maintenu constant par le hépatique O2 augmentation de l&#39;extraction (30). Des hypoperfusions hépatiques mineures et transitoires ont également été observées après une injection unique d&#39;endotoxine en bolus par Maeda et al. (21). Dans un modèle de péritonite porcine, Rasmussen et al. (29) ont constaté que hepaticV˙o2 est limitée par une diminution de la D hépatiqueo2 associée à une acidose lactique et à une libération de lactate hépatique, ce qui suggère que l’ischémie hépatique s’est produite dans ce modèle.

La diminution du débit biliaire et le rapport entre le poids humide et le poids sec du foie étaient similaires dans ces groupes. De plus, des lésions hépatiques mineures ont également été observées dans les groupes Endo et Ctrl. La modification des tests hépatiques après une intervention chirurgicale chez des porcs anesthésiés perfusés avec une solution saline a déjà été décrite (21).

NO2

and

NO3

concentrations dans le plasma artériel.

Parmi les nombreuses méthodes disponibles pour estimer les rejets de NO, la détermination des concentrations plasmatiques des produits finaux stables contenant du NO, à savoir:

NO2

and

NO3

(ou NOx) était couramment utilisé. Les concentrations plasmatiques de NOx chez les grands animaux témoins étaient <5 µM (11, 18, 38), mais Santak et al. (32) ont mesuré des concentrations de NOx plus élevées que celles observées dans la présente étude. Après une injection d’endotoxine en bolus, une augmentation minimale de NOx artériel (<12 μM) a été mesurée (11, 18, 38) et cette augmentation était de loin inférieure à celle mesurée chez les rongeurs (20). Chez les humains en bonne santé, les concentrations plasmatiques de NOx étaient comprises entre 20 et 30 µM et atteignaient jusqu&#39;à 150 µM lors du choc septique (23, 28). De plus, les hépatocytes humains (26) et les macrophages (2) ont produit moins de NO que les mêmes types de cellules isolés de rongeurs. En conséquence, il a été prouvé que, dans les conditions septiques, les concentrations plasmatiques de NOx étaient plus élevées chez les rongeurs que chez l’homme, tandis que les concentrations de NOx étaient beaucoup plus faibles chez les grands animaux que chez les rongeurs. De plus, les concentrations plasmatiques de NOx observées chez les porcs et les chiens anesthésiés pourraient ne pas refléter de manière fiable la production globale de NO. Chez les porcs endotoxiques, Santak et al. (32) ont montré que l&#39;absence d&#39;augmentation des NOx ne reflétait pas l&#39;augmentation de la production de NO mesurée par15NaNO3 méthode cinétique. De manière similaire à notre étude, les concentrations plasmatiques initiales de NOx ne différaient pas 9 h après un bol d’endotoxines, alors que la mesure directe de la production de NO augmentait de manière significative avec le temps. De plus, les mesures de NOx plasmatiques auraient pu conduire à des erreurs d&#39;estimation de la quantité réelle de formation de NO lorsque la fonction rénale et / ou le volume extracellulaire étaient altérés (37). Ces résultats ont rendu difficile la corrélation entre les changements hémodynamiques au cours de l&#39;endotoxémie et les concentrations plasmatiques de NOx (11). Enfin, comme le facteur natriurétique auriculaire agissait également sur la guanylate cyclase et que la concentration sérique de l&#39;ANF était augmentée par l&#39;endotoxine, les mesures du GMPc ne reflétaient pas uniquement la production de NO, mais pourraient aussi être la conséquence d&#39;une production accrue d&#39;ANF (1 ).

PAS de libération après l&#39;injection d&#39;ACh.

Une autre façon de détecter la production globale de NO consiste à étudier la réponse vasculaire à l&#39;injection d&#39;ACh, qui induit une relaxation de l&#39;endothélium par la libération de NO. En conséquence, l&#39;injection d&#39;ACh a diminué la MAP dans les deux groupes expérimentaux. Cet effet pourrait être totalement inversé par les inhibiteurs de NOS (8). Chez les porcs Endo, la diminution de la MAP après l&#39;injection d&#39;ACh était inférieure à celle observée chez les porcs Ctrl, ce qui montre que la libération de NO par l&#39;endothélium était altérée pendant l&#39;endotoxémie. Fait intéressant, la réponse du MAP à l&#39;ACh est restée stable chez les porcs Ctrl et Endo au fil du temps et les réponses du HABF à l&#39;ACh ont diminué dans les deux groupes. In contrast, the PVBF decrease (which reflects decreased blood flow coming from splanchnic organs) did not change over time. Moreover, the PVBF response during test A was lower in Endo pigs than in Ctrl pigs.

NOS 2 detection in hepatic biopsies.

Finally, detection of NOS in these models of endotoxic shock is an additional way to determine the involvement of NO. Three different isoforms of NOS have been characterized. Two of them are constitutive (NOS 1 and NOS 3) and produce low levels of NO. NO released from the constitutive NOS isoforms functions as a cell signaling molecule in different physiological processes, such as neurotransmission, regulation of vascular tone, and platelet aggregation. The iNOS isoform (NOS 2) produces large amounts of NO and is detected during various inflammatory events. Induction of NOS 2 in vivo in response to endotoxin was observed in numerous tissues including the liver (6, 9,19, 20, 31). In the liver, NOS 2 was induced in hepatocytes, Kupffer cells, endothelial cells, and stellate cells during endotoxemia (20). Although numerous studies found NOS 2 expression in various tissues collected from rodents injected with endotoxin, the protein has not yet been detected in large animals infused with endotoxin. The present study clearly provides evidence for NOS 2 mRNA expression in various hepatic locations of the liver, whereas NOS 2 protein expression was found only in the hepatic tissues surrounding the great vessels. The difference between NOS 2 mRNA and protein expression might indicate some problems with regard to the specificity of the antibody used to detect NOS 2 protein. In the present study we used an affinity-purified IgG polyclonal rabbit-anti-mouse antibody that scored positively with NOS 2 protein (Fig. 3) and has been demonstrated to cross-react with porcine NOS 2 (13). Although we tried to precipitate the NOS 2 protein from the sample that scored negatively, we were unable to detect any NOS 2 protein.

Because NOS 2 has not been cloned in pigs, we used human-specific NOS 2 primers. This approach was successful in porcine platelets (3). This result was not surprising because, across species, each isoform is well conserved (>80%) (12). However, although a homology of >80% was observed among the different NOS 2 sequences, other differences, such as the partial calcium dependence of human NOS 2 or the cofactor dependence between human and murine NOS 2, have been described (26). To what extent these differences are important in the detection of porcine NOS 2 is unknown. Finally, the fact that NOS 2 mRNA was expressed in some hepatic locations where NOS 2 protein was undetectable has already been demonstrated in porcine platelets (3).

6-keto-PGF concentrations in arterial plasma.

In contrast to plasma NOx concentrations, which did not increase significantly over time, the plasma concentrations of 6-keto-PGF (stable breakdown product of prostacyclin) increased in Endo pigs and remained steady in Ctrl pigs. Prostacyclin is a mediator of inflammation synthesized from arachidonic acid through the cyclooxygenase pathway. Increased release of 6-keto-PGF was also observed in numerous experimental models of sepsis (10, 16, 27).

In summary, infusion of endotoxin resulted in hypotensive and normokinetic shock in association with minor modifications of hepatic perfusion over a 18-h experimental period. The ACh-dependent decrease of MAP was reduced in Endo pigs, whereas a minor difference was observed between Ctrl and Endo pigs for ACh-dependent modification of hepatic perfusion. Endotoxin did not modify the arterial concentrations of NOx and cGMP. However, in hepatic biopsies, NOS 2 protein was expressed 6 and 9 h after the start of endotoxin injection and localized around the great vessels. Thus, although endotoxemia induces NOS 2 expression in the liver, NO involvement remains minor in this model of sepsis.

We thank Elisabeth Bernouilli, Jennifer Hantson, Manuel Jorge-Costa, Sylvie Roulet, and Aiguo Wang for excellent technical assistance.

FOOTNOTES

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