Beate Wolf

Selective activation of melatonin receptors with ramelteon improves liver function and hepatic perfusion after hemorrhagic shock in rat

Objective:Melatonin may attenuate organ damage via direct antioxidative properties, and was recently demonstrated to reduce cardiac and hepatic injury through receptor-dependent effects. However, the relevance of an isolated activation of melatonin receptors for organ protection, excluding direct antioxidant effects, has not been established yet. This study was designed to investigate whether therapy with melatonin receptor agonist and hypnotic substance ramelteon may improve liver function, hepatic perfusion, and hepatic integrity after hemorrhagic shock in rat. Design:Prospective, randomized, controlled study. Setting:University research laboratory. Subjects:Male Sprague–Dawley rats (n = 10 per group). Interventions:Animals underwent hemorrhagic shock (mean arterial pressure 35 ± 5 mm Hg for 90 mins) and were resuscitated with shed blood and Ringers lactate (2 hrs). At the end of shock, animals were treated with ramelteon (1.0 mg/kg intravenously), melatonin receptor antagonist luzindole plus ramelteon (each 1.0 mg/kg intravenously), or vehicle. Measurements and Main Results:In vitro, ramelteon displayed no relevant antioxidant capacity in an 2,2′-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) assay, compared with melatonin. In vivo, liver function was assessed by plasma disappearance rate of indocyanine green, and intravital microscopy was performed for evaluation of hepatic perfusion index, nicotinamide adenine dinucleotide phosphate autofluorescence, and hepatic integrity. Compared with vehicle controls, ramelteon therapy significantly improved plasma disappearance rate of indocyanine green (7.89 ± 2.12% vs. 13.67 ± 2.51%; p = 0.006), hepatic perfusion index (352.04 ± 111.78 pl/sec/mm vs. 848.81 ± 181.38 pl/sec/mm; p = 0.002), nicotinamide adenine dinucleotide phosphate autofluorescence and hepatocellular injury. Coadministration of luzindole abolished the protective effect of ramelteon with respect to liver function and nicotinamide adenine dinucleotide phosphate autofluorescence. Conclusions:Ramelteon therapy improves liver function, hepatic perfusion, and hepatocellular integrity after hemorrhagic shock in rat. This demonstrates that an isolated activation of melatonin receptors may be sufficient for organ protection, independent from direct antioxidant effects. The hypnotic ramelteon could thus play an interesting role in future sedation concepts for critical care patients.

Melatonin pretreatment improves liver function and hepatic perfusion after hemorrhagic shock.

Exogenous administration of pineal hormone melatonin (MEL) has been demonstrated to attenuate organ damage in models of I/R and inflammation by antioxidative effects. However, specific organ-protective effects of MEL with respect to hemorrhagic shock have not been investigated yet. In the present study, we evaluated the role of MEL pretreatment for hepatic perfusion, redox state, and function after hemorrhage and resuscitation, with emphasis on MEL receptor activation. In a model of hemorrhagic shock (MAP 35 ± 5 mmHg for 90 min) and reperfusion (2 h), we measured nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) autofluorescence, hepatic microcirculation, and hepatocellular injury by intravital microscopy, as well as plasma disappearance rate of indocyanine green (PDRICG) as a sensitive maker of liver function in rat. Pretreatment with 10 mg kg−1 MEL (i.v.) 15 min before induction of hemorrhage resulted in a significantly improved PDRICG compared with controls (MEL/shock, 15.02% min−1 ± 2.9 SD vs. vehicle/shock, 6.18 ± 4.6 SD; P = 0.001). Intravital microscopy after reperfusion revealed an improved hepatic perfusion index, redox state, and reduced hepatocellular injury in pretreated animals compared with the vehicle group. Melatonin receptor antagonist luzindole (LZN; 2.5 mg kg−1) almost completely abolished the protective effects of MEL pretreatment with respect to liver function (MEL + LZN/shock PDRICG, 7.31% min−1 ± 3.4 SD). Beneficial effects regarding hepatic perfusion, redox state, and cellular injury were not influenced by LZN, indicating that they may depend on antioxidative effects of MEL. However, liver function after hemorrhage is effectively maintained by MEL pretreatment via receptor-dependent pathways.

Hemin arginate-induced heme oxygenase 1 expression improves liver microcirculation and mediates an anti-inflammatory cytokine response after hemorrhagic shock.

Microvascular failure is a major determinant for the development of hepatocellular dysfunction after hemorrhagic shock. Induction of heme oxygenase (HO) 1 may confer hepatocellular protection. Hemin arginate (HAR) induces HO-1 and protects against shock-induced organ failure. The mechanisms are not completely understood, but HO-1-mediated protective effects on the microcirculation and on the inflammatory response may contribute. Therefore, the aim of the present study was to investigate the influence of HAR pretreatment on liver microcirculation and cytokine response to assess the role of HO-1-mediated effects under these conditions. Male Sprague-Dawley rats (200-300 g; n = 8 per group) were subjected to hemorrhage (MAP, 30-40 mmHg for 1 h) 24 h after pretreatment with vehicle (Ringer solution) or HAR (5 mg kg−1), followed by 2 h of resuscitation. The microcirculation and the redox state (nicotinamide adenine dinucleotide phosphate [reduced form; NADPH] autofluorescence) of the liver were assessed using intravital microscopy. Cytokine levels (TNF-&agr; and IL-10) were quantified using an enzyme-linked immunosorbent assay. A profound induction of HO-1 was observed 24 h after pretreatment with HAR. Hemorrhage significantly reduced sinusoidal perfusion and increased NADPH autofluorescence and cytokine levels. Hemin arginate pretreatment significantly improved liver microcirculation, reduced NADPH autofluorescence, significantly increased IL-10, and tended to decrease TNF-&agr; serum levels compared with shock vehicle. Blockade of the HO pathway with tin-mesoporphyrin-IX after HAR pretreatment abolished the observed beneficial effects, whereas the additional administration of the carbon monoxide donor dichloromethane reversed the tin-mesoporphyrin-IX-mediated changes. These results suggest that HAR pretreatment improves liver microcirculation and mediates an anti-inflammatory cytokine response after hemorrhagic shock through induction of HO-1 and in part through an increased carbon monoxide release.

Melatonin receptors mediate improvements of survival in a model of polymicrobial sepsis.

Objectives:Melatonin has been demonstrated to improve survival after experimental sepsis via antioxidant effects. Yet, recent evidence suggests that this protective capacity may also rely on melatonin receptor activation. Therefore, the present study was designed to investigate whether selective melatonin receptor-agonist ramelteon may influence survival and immune response in a model of polymicrobial sepsis in rats, wild-type and melatonin receptor MT1/MT2 double knockout mice. Design:Prospective, randomized, controlled study. Setting:University research laboratory. Subjects:Male Sprague-Dawley rats (200–250 g) and male C3H/HeN wild-type and MT1/MT2 receptor knockout mice (20–22 g). Interventions:Animals underwent cecal ligation and incision and remained anesthetized for evaluation of survival for 12 hours (rats: n = 15 per group) or 15 hours (mice: n = 10 per group). Analysis of immune response by means of enzyme-linked immunosorbent assay was performed before and 5 hours after cecal ligation and incision (rats only; n = 5 per group). After induction of sepsis, animals were treated IV with vehicle, different doses of melatonin (rats: 0.01/0.1/1.0/10 mg/kg; mice: 1.0 mg/kg), ramelteon, melatonin receptor-antagonist luzindole, ramelteon + luzindole, or melatonin + luzindole (each 1.0 mg/kg). Sham controls underwent laparotomy but not cecal ligation and incision. Measurements and Main Results:Compared with vehicle, administration of ramelteon or melatonin significantly improved median survival time in rats (sepsis/melatonin [0.1 mg/kg], 554 min, [1.0 mg/kg] 570 min, [10 mg/kg] 579 min; sepsis/ramelteon, 468 min; each p < 0.001 vs sepsis/vehicle, 303 min) and wild-type mice (sepsis/melatonin, 781 min; sepsis/ramelteon, 701 min; both p < 0.001 vs sepsis/vehicle, 435 min). This effect was completely antagonized by coadministration of luzindole in all groups. Melatonin, ramelteon, or luzindole had no significant effect on survival time in knockout mice. Significantly elevated concentrations of tumor necrosis factor-&agr;, interleukin-6, and interleukin-10 were observed 5 hours after cecal ligation and incision in rats (p < 0.05 vs baseline and corresponding sham); neither ramelteon nor melatonin treatment significantly affected immune response. Conclusions:Melatonin receptors mediate improvements of survival after polymicrobial sepsis in rats and mice; this effect appears to be independent from major alterations of cytokine release.

Melatonin receptor antagonist luzindole is a powerful radical scavenger in vitro

To the Editor The radical scavenging potential of melatonin is well documented [1]. Consequentially, exogenous administration of melatonin has proven useful in different models of stress in vivo, such as in sepsis [2], after ischemia/reperfusion [3, 4], and in a variety of toxic challenges [5, 6]. Some of these beneficial effects of melatonin are mediated via receptor dependent processes [7]. To identify the relevance of melatonin receptors, the receptor antagonist luzindole was introduced. This substance displays an affinity to all melatonin receptor (MT) subtypes, MT1, MT2, and MT3 (quinone reductase 2) [8]. Despite the extensive use of luzindole, many pharmacological aspects of this substance remain unknown. Possible dose-dependent variations in the response to luzidole have been reported [9], partial agonist behavior was suggested in human leukemia cells [10], and only limited data are available with respect to precise agonist–antagonist relationships [11]. The failure of luzindole to antagonize melatonin s effect on hepatocellular redox state in a recent study on hemorrhagic shock [12] prompted the question on possible antioxidant properties of luzindole itself. Therefore, this study was designed to evaluate the radical scavenging properties of melatonin receptor antagonist luzindole in vitro. Radical scavenging was evaluated using a spectrophotometrical scavenger competition assay based on the formation of 2,2¢-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation [13]. All measurements were carried out at room temperature and performed in triplicates. Total antioxidant capacity was determined for luzindole, melatonin, ascorbic acid, and vehicle dimethyl sulfoxide (DMSO). ABTS was diluted in water to a concentration of 7.0 mm and incubated with potassium persulfate (2.45 mm final concentration) for 12 hr at room temperature, protected from light, for formation of ABTS radical cation (ABTS). ABTS was diluted with 5 mm phosphate buffered saline (PBS), pH 7.2, to an absorbance value of 0.7 at 734 nm in a spectrophotometer (Ultrospect2000; Pharmacia Biotech, Piscataway, NJ, USA). Luzindole, melatonin, and ascorbic acid were freshly dissolved and added to ABTS (final concentrations: 5.0 lm, 10.0 lm, and 50.0 lm for each substance). Total antioxidant capacity is expressed as the percentage of inhibition of the absorbance of ABTS at room temperature at 734 nm as a function of time. A strong inhibition of ABTS radical was achieved for melatonin, luzindole, and ascorbic acid within 30 min, using the concentration of 5.0 lm (Fig. 1A) and 10.0 lm (Fig. 1B). No significant inhibition of ABTSwas detected for DMSO. At a concentration of 50 lm, melatonin, luzindole, and ascorbic acid reached an inhibition of ABTS >90%within seconds, while DMSO did not affect the absorption of ABTS for 30 min (data not shown). These data demonstrate that luzindole is a potent antioxidant itself, exhibiting an inhibition of ABTS radical of almost 80% at a concentration of 10 lm after 30 min in vitro. The ABTS assay was previously shown to be a reliable method to estimate the antioxidant capacity of a variety of substances, including food products and indoles [13, 14]. The results obtained for melatonin and ascorbic

Melatonin receptors mediate improvements of liver function but not of hepatic perfusion and integrity after hemorrhagic shock in rats.

Objective:Melatonin has been demonstrated to attenuate organ damage in models of ischemia and reperfusion. Melatonin treatment before hemorrhagic shock has been shown to improve liver function and hepatic perfusion. Proposed mechanisms of the pineal hormone involve direct inactivation of reactive oxygen species and induction of antioxidative enzymes. However, recent evidence suggests a strong influence of melatonin receptor activation for these effects. Specific protection of organ function by melatonin after hemorrhage has not been investigated yet. In this study, we evaluated whether melatonin therapy after hemorrhagic shock improves liver function and hepatic perfusion, with emphasis on melatonin receptor activation. Design:Prospective, randomized, controlled study. Setting:University research laboratory. Subjects:Male Sprague-Dawley rats, 200–300 g (n = 10 per group). Interventions:Animals underwent hemorrhagic shock (mean arterial pressure, 35 ± 5 mm Hg for 90 mins) and were resuscitated with shed blood and Ringers solution. At the end of shock, animals were treated with either melatonin (10 mg/kg, intravenously), melatonin receptor antagonist luzindole (2.5 mg/kg, intravenously) plus melatonin (10 mg/kg, intravenously), luzindole alone (2.5 mg/kg, intravenously), or vehicle. Measurements and Main Results:After 2 hrs of reperfusion, either liver function was assessed by plasma disappearance rate of indocyanine green or intravital microscopy of the liver was performed for evaluation of hepatic perfusion, hepatocellular redox state, and hepatic integrity. Compared with vehicle controls, melatonin therapy after hemorrhagic shock significantly improved plasma disappearance rate of indocyanine green, hepatic redox state, hepatocellular injury, and hepatic perfusion index. Coadministration of luzindole completely abolished the protective effect with respect to liver function only, and improvements regarding hepatic redox state, perfusion, and integrity were comparable with melatonin treatment alone. Conclusions:Melatonin therapy after hemorrhagic shock improves liver function, hepatic perfusion, redox state, and hepatic integrity. With respect to liver function, beneficial effects of the pineal hormone seem to be dependent on melatonin receptor activation.

Dobutamine pretreatment improves survival, liver function, and hepatic microcirculation after polymicrobial sepsis in rat.

ABSTRACT Dobutamine is recommended for the treatment of sepsis-related circulatory failure in international guidelines. Furthermore, dobutamine has been demonstrated to improve liver function and hepatic perfusion after experimental hemorrhagic shock. Yet, it is unknown whether dobutamine may also induce hepatoprotective effects in sepsis. This study was designed to investigate the effect of dobutamine on survival, hepatic function, and microcirculation after polymicrobial sepsis in rat. Under general anesthesia, male Sprague-Dawley rats (n = 25/group) underwent pretreatment with dobutamine (10 &mgr;g/kg per minute) in the presence or absence of &bgr;1-receptor antagonist esmolol (500 &mgr;g/kg per minute), esmolol alone, or vehicle for 6 h, before induction of sepsis (cecal ligation and incision [CLI]). Sham-operated animals were treated likewise but underwent no CLI. Five hours after CLI, either liver function was assessed by plasma disappearance rate of indocyanine green (n = 5/group), or intravital microscopy was performed (n = 5/group) for evaluation of hepatic perfusion index and hepatic integrity (as propidium iodide–stained cells per field). Alternatively, survival time after induction of CLI was monitored under general anesthesia (n = 15/group). Compared with controls, dobutamine pretreatment significantly improved plasma disappearance rate of indocyanine green (13.8% ± 4.1% vs. 20.6% ± 4.6%; P = 0.029), hepatic perfusion index (275.0 ± 126.1 vs. 703.5 ± 177.4 pL/s per mm; P < 0.001), hepatocellular injury (22.2 ± 6.7 vs. 6.4 ± 3.1 cells per field; P < 0.001), and survival time (326 ± 20 vs. 603 ± 41 min; P < 0.001). Coadministration of esmolol abolished the protective effect of dobutamine completely. Our results indicate that pretreatment with dobutamine may improve survival, liver function, and hepatic microcirculation after polymicrobial sepsis in rat via &bgr;1-adrenoceptor activation. Dobutamine could therefore play a relevant role for hepatoprotection under septic conditions.

Volatile Organic Compounds during Inflammation and Sepsis in Rats: A Potential Breath Test Using Ion-mobility Spectrometry

Background:Multicapillary column ion-mobility spectrometry (MCC-IMS) may identify volatile components in exhaled gas. The authors therefore used MCC-IMS to evaluate exhaled gas in a rat model of sepsis, inflammation, and hemorrhagic shock. Methods:Male Sprague–Dawley rats were anesthetized and ventilated via tracheostomy for 10 h or until death. Sepsis was induced by cecal ligation and incision in 10 rats; a sham operation was performed in 10 others. In 10 other rats, endotoxemia was induced by intravenous administration of 10 mg/kg lipopolysaccharide. In a final 10 rats, hemorrhagic shock was induced to a mean arterial pressure of 35 ± 5 mmHg. Exhaled gas was analyzed with MCC-IMS, and volatile compounds were identified using the BS-MCC/IMS-analytes database (Version 1209; B&S Analytik, Dortmund, Germany). Results:All sham animals survived the observation period, whereas mean survival time was 7.9 h in the septic animals, 9.1 h in endotoxemic animals, and 2.5 h in hemorrhagic shock. Volatile compounds showed statistically significant differences in septic and endotoxemic rats compared with sham rats for 3-pentanone and acetone. Endotoxic rats differed significantly from sham for 1-propanol, butanal, acetophenone, 1,2-butandiol, and 2-hexanone. Statistically significant differences were observed between septic and endotoxemic rats for butanal, 3-pentanone, and 2-hexanone. 2-Hexanone differed from all other groups in the rats with shock. Conclusions:Breath analysis of expired organic compounds differed significantly in septic, inflammation, and sham rats. MCC-IMS of exhaled breath deserves additional study as a noninvasive approach for distinguishing sepsis from inflammation.

Inhibition of glycogen synthase kinase (GSK)-3-β improves liver microcirculation and hepatocellular function after hemorrhagic shock.

Ischemia and reperfusion may cause liver injury and are characterized by hepatic microperfusion failure and a decreased hepatocellular function. Inhibition of glycogen synthase kinase (GSK)-3β, a serine-threonine kinase that has recently emerged as a key regulator in the modulation of the inflammatory response after stress events, may be protective in conditions like sepsis, inflammation and shock. Therefore, aim of the study was to assess the role of GSK-3β in liver microcirculation and hepatocellular function after hemorrhagic shock and resuscitation (H/R). Anesthetized male Sprague-Dawley rats underwent pretreatment with Ringer´s solution, vehicle (DMSO) or TDZD-8 (1 mg/kg), a selective GSK-3β inhibitor, 30 min before induction of hemorrhagic shock (mean arterial pressure 35±5 mmHg for 90 min) and were resuscitated with shed blood and Ringer´s solution (2h). 5h after resuscitation hepatic microcirculation was assessed by intravital microscopy. Propidium iodide (PI) positive cells, liver enzymes and alpha-GST were measured as indicators of hepatic injury. Liver function was estimated by assessment of indocyanine green plasma disappearance rate. H/R led to a significant decrease in sinusoidal diameters and impairment of liver function compared to sham operation. Furthermore, the number of PI positive cells in the liver as well as serum activities of liver enzymes and alpha-GST increased significantly after H/R. Pretreatment with TDZD-8 prevented the changes in liver microcirculation, hepatocellular injury and liver function after H/R. A significant rise in the plasma level of IL-10 was observed. Thus, inhibition of GSK-3β before hemorrhagic shock modulates the inflammatory response and improves hepatic microcirculation and hepatocellular function.

Melatonin modifies cellular stress in the liver of septic mice by reducing reactive oxygen species and increasing the unfolded protein response

BACKGROUND & AIMS Melatonins hepatoprotective actions have numerously been demonstrated in the past but the underlying molecular mechanisms are widely unknown. For a better understanding of melatonins effects on hepatic stress response this study aimed to elucidate alterations in oxidative stress, unfolded protein response and acute phase response in septic mice. METHODS Male C3H/HeN mice underwent sham operation or cecal ligation and incision and remained anesthetized for 5h. Production of reactive oxygen species was determined by electron spin resonance spectroscopy. Protein and mRNA expression levels were determined by western blot analysis and quantitative real-time PCR, respectively. RESULTS Production of reactive oxygen species was strongly increased in the aorta and liver after 5h of polymicrobial sepsis which was entirely inhibited by treatment with melatonin. SOD-1 levels did not differ between the groups. Sepsis also induced the upregulation of VCAM-1 and ICAM-1 independent of melatonin treatment but probably regulated via ERK1/2 signaling. Melatonin triggered the transcriptional upregulation of PERK in septic animals which seems to be independent on ERK1/2 signaling and NR4A1 activation. Melatonin therapy also engendered an increased expression of CHOP, but apoptosis was not initiated. Furthermore, sepsis reduced the expression of the transcription factor CREBH which was entirely suppressed by melatonin. CONCLUSIONS This study gives new insight into the mechanisms by which melatonin might confer its hepatoprotective actions during polymicrobial sepsis. The results clearly show the melatonin-mediated amelioration of oxidative stress as well as alterations in the cellular stress mechanisms via the unfolded protein response and the acute phase response.