Carla Bazzani

Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock.

ABSTRACT The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited I&kgr;B&agr; liver loss, blunted the augmented nuclear factor-&kgr;B activity, decreased hepatic tumor necrosis factor (TNF)-&agr; mRNA (VGX + SAO + Sham STIM = 1.0 ± 1.9 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 ± 0.2 TNF-&agr;/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-&agr; (VGX + SAO + Sham STIM = 118 ± 19 pg/mL; VGX + SAO + STIM = 39 ± 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 ± 1 U/g tissue; VGX + SAO + STIM = 3.1 ± 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 ± 1.0 U/g tissue; VGX + SAO + STIM = 3.2 ± 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor-&kgr;B and TNF-&agr; in SAO shock.

Oxidative stress causes nuclear factor-κB activation in acute hypovolemic hemorrhagic shock

Nuclear Factor kappaB (NFkappaB) is an ubiquitous rapid response transcription factor involved in inflammatory reactions and exerts its action by expressing cytokines, chemokines, and cell adhesion molecules. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock. Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Hemorrhagic shocked rats died in 26.3 +/- 2.1 min following the discontinuance of bleeding, experienced a marked hypotension (mean arterial blood pressure = 20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (200 +/- 15 pg/ml, 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE; 1nM to 10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding) and enhanced plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA; 6 +/- 2.2 microm), 2,3-dihydroxybenzoic acid (2,3-DHBA; 13 +/- 2.1 microm), both studied to evaluate OH(*) production. Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm became decreased at 5 min after the end of bleeding. IRFI-042, a vitamin E analogue (20 mg/kg intraperitoneally 2 min after the end of bleeding), inhibited the loss of IkappaBalpha protein from the cytoplasm and blunted the increase in NF-kappaB binding activity. Furthermore IRFI-042 increased survival time (117.8 +/- 6.51 min; p <.01) and survival rate (vehicle = 0% and IRFI-042 = 80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (21 +/- 4.3 pg/ml), and restored to control values the hypo-reactivity to PE. Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB, likely through an increased production of reactive oxygen species. This experiment indicates that NF-kappaB-triggered inflammatory cascade becomes early activated during acute hemorrhage even in the absence of resuscitation procedures.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

In circulatory shock, melanocortins have life‐saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists.

Resuscitating effect of melanocortin peptides after prolonged respiratory arrest

The resuscitating activity of melanocortin peptides (MSH‐ACTH peptides) was tested in an experimental model of prolonged respiratory arrest. Anaesthetized, endotracheally intubated rats subjected to a 5 min period of ventilation interruption, invariably died from cardiac arrest within 6–9 min of resumption of ventilation. When resumption of ventilation was associated with the simultaneous intravenous (i.v.) injection of a melanocortin peptide (α‐MSH or ACTH‐(1–24)) (160 μg kg−1) there was an almost immediate (within 1 min), impressive increase in cardiac output, heart rate, mean arterial pressure (+560% of the before‐treatment value) and pulse pressure (+356% of the before‐treatment value), with full recovery of electroencephalogram after 30–45 min. Blood gases and pH were normalized within 15–60 min after treatment, and all treated animals eventually recovered completely and survived indefinitely (=more than 15 days). The same response was observed in adrenalectomized animals, as well as in animals pretreated with a β1‐adrenoceptor blocking agent (atenolol, 3 mg kg−1, i.v.), or with an α1‐adrenoceptor blocking agent (prazosin, 0.1 mg kg−1, i.v.), or with an adrenergic neurone blocking agent (guanethidine, 10 mg kg−1, intraperitoneally). An effect quite similar to that produced by melanocortins was obtained with ouabain (0.1 mg kg−1, i.v.); the antioxidant drug, glutathione (75 mg kg−1, i.v.) also produced 100% resuscitation, but the effect was slower in onset. On the other hand, adrenaline (0.005 mg kg−1, i.v.) was able to resuscitate only 1 out of 8 rats and dobutamine (0.02 mg kg−1, i.v.) resuscitated 4 out of 8 rats; moreover, the effect of both catecholamines was much slower in onset than that of melanocortins and the initial, impressive stimulation of cardiovascular function was absent. These results show that melanocortin peptides have a resuscitating effect in a pre‐terminal condition produced in rats by prolonged asphyxia. This effect seems primarily due to the restoration of cardiac function, not mediated by catecholamines. These data also suggest that these peptides may have potential therapeutic value in conditions of transient cardiac hypoxia and re‐oxygenation such as occur in coronary artery disease.

Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC(3) receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH(2) (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC(3) receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC(3)/MC(4) receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(1-24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC(3) receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC(3) receptors, the C-terminal Arg-Phe structure being relevant.

Tumour necrosis factor‐α as a target of melanocortins in haemorrhagic shock, in the anaesthetized rat

The cytokine tumour necrosis factor‐α (TNF‐α) is involved (mostly through the activation of inducible nitric oxide synthase) in the pathogenesis of circulatory shock. On the other hand, melanocortin peptides are potent and effective in reversing haemorrhagic shock, both in animals (rat, dog) and in humans. This prompted us to study the influence of the melanocortin peptide ACTH‐(1–24) on the blood levels of TNF‐α in haemorrhage‐shocked rats and on the in vitro production of TNF‐α by lipopolysaccharide (LPS)‐activated macrophages. Plasma levels of TNF‐α were undetectable before starting bleeding and greatly increased 20 min after bleeding termination in saline‐treated rats. In rats treated with ACTH‐(1–24) the almost complete restoration of cardiovascular function was associated with markedly reduced levels of TNF‐α 20 min after bleeding termination. On the other hand, ACTH‐(1–24) did not influence TNF‐α plasma levels in sham‐operated, unbled rats. In vitro, ACTH‐(1–24) (25–100 nM) dose‐dependently reduced the LPS‐stimulated production of TNF‐α by peritoneal macrophages harvested from untreated, unbled rats. These results indicate that inhibition of TNF‐α overproduction may be an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock.

Early treatment with ACTH-(1-24) in a rat model of hemorrhagic shock prolongs survival and extends the time-limit for blood reinfusion to be effective.

The ability of ACTH-(1-24) to prolong survival and to extend the deadline for effective blood reinfusion has been studied in a model of lethal hypovolemic shock in the rat. Anesthetized rats were bled to a mean arterial pressure of 18 to 25 mm Hg and then subjected to one of the following iv treatments: a) saline; b) ACTH-(1-24), 160 micrograms/kg; c) blood reinfusion; d) ACTH-(1-24), 160 micrograms/kg; c) blood reinfusion; d) ACTH-(1-24), with saline 5 min after bleeding died within 0.05 h. On the other hand, the treatment with ACTH-(1-24) induced an almost complete and sustained recovery of cardiovascular and respiratory functions associated with a survival time of 44 +/- 18 h, while four of six rats reinfused with the withdrawn blood were still alive 15 days later. The time-lapse between bleeding and treatment was of crucial importance, and neither ACTH-(1-24) injection nor blood reinfusion had any effect if performed 25 min after bleeding. However, treatment with ACTH-(1-24) shortly after bleeding (5 min) greatly improved the effect of a later blood reinfusion. These data indicate that ACTH-(1-24) can prolong survival and permit the time-lapse between blood loss and blood reinfusion to be extended.

Adrenocorticotropin reverses vascular dysfunction and protects against splanchnic artery occlusion shock

Tumour necrosis factor (TNF‐α) is involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. On the other hand, inhibition of TNF‐α is an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock. We therefore investigated the effects of the melanocortin peptide ACTH‐(1–24) (adrenocorticotropin fragment 1–24) on the vascular failure induced by SAO shock. SAO‐shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham‐shocked rats survived for more than 4 h), enhanced serum TNF‐α concentrations (755±81 U ml−1), decreased mean arterial blood pressure, leukopenia, and increased ileal leukocyte accumulation, as revealed by means of myeloperoxidase activity (MPO=9.4±1 U g−1 tissue). Moreover, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM–10 μM) (Emax and ED50 in shocked rats=7.16 mN mg−1 tissue and 120 nM, respectively; Emax and ED50 in sham‐shocked rats=16.31 mN mg−1 tissue and 100 nM, respectively), reduced responsiveness to acetylcholine (ACh, 10 nM‐10 μM) (Emax and ED50 in shocked rats=30% relaxation and 520 nM, respectively; Emax and ED50 in sham‐shocked rats=82% relaxation and 510 nM, respectively) and increased staining for intercellular adhesion molecule‐1 (ICAM‐1). ACTH‐(1–24) [160 μg kg−1 intravenously (i.v.), 5 min after SAO] increased survival rate [SAO+ACTH‐(1–24)=80% at 4 h of reperfusion], reversed hypotension, reduced serum TNF‐α (55±13 U ml−1), ameliorated leukopenia, reduced ileal MPO (1.2±0.2 U g−1 tissue), restored the reactivity to PE, improved the responsiveness to ACh and blunted the enhanced immunostaining for ICAM‐1 in the aorta. Adrenalectomy only in part–but not significantly–reduced the ACTH‐induced shock reversal, the survival rate of SAO+ACTH‐(1–24) adrenalectomized rats being 60% at 4 h of reperfusion; and methylprednisolone (80 mg−1 i.v., 5 min after SAO) had a non‐significant effect (10% survival) at 4 h of reperfusion. The present data show that melanocortins are effective also in SAO shock, their effect being, at least in part, mediated by reduced production of TNF‐α. Furthermore, they demonstrate, for the first time, that this inhibition is responsible for the adrenocorticotropin‐induced reversal of vascular failure and leukocyte accumulation.

Influence of ACTH‐(1–24) on free radical levels in the blood of haemorrhage‐shocked rats: direct ex vivo detection by electron spin resonance spectrometry

1 The influence of ACTH‐(1–24) on the blood levels of highly reactive free radicals in haemorrhagic shock was studied in rats. 2 Volume‐controlled haemorrhagic shock was produced in adult rats under general anaesthesia (urethane, 1.25 g kg−1 intraperitoneally) by stepwise bleeding until mean arterial pressure stabilized at 20–23 mmHg. Rats were intravenously (i.v.) treated with either ACTH‐(1–24) (160 μg kg−1 in a volume of 1 ml kg−1) or equivolume saline. Free radicals were measured in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin‐trapping agent (α‐phenyl‐N‐tert‐butylnitrone). 3 Blood levels of free radicals were 6490 ± 273 [arbitrary units (a.u.) ml−1 whole blood, before starting bleeding, and 30762 ± 2650 after bleeding termination (means ± s.e.mean of the values obtained in all experimental groups). All rats treated with saline died within 30 min, their blood levels of free radicals being 35450 ± 5450 a.u. ml−1 blood, 15 min after treatment. Treatment with ACTH‐(1–24) produced a rapid and sustained restoration of arterial pressure, pulse pressure, heart rate and respiratory function, with 100% survival at the end of the observation period (2 h); this was associated with an impressive reduction in the blood levels of free radicals, that were 12807 ± 2995, 10462 ± 2850, 12294 ± 4120, and 10360 ± 2080 a.u. ml−1 blood, 15, 30, 60 and 120 min after ACTH‐(1–24) administration, respectively. 4 These results provide a direct demonstration that (i) in haemorrhagic shock there is a rapid and massive production of highly reactive free radicals, and that (ii) the sustained restoration of cardiovascular and respiratory functions induced by the i.v. injection of ACTH‐(1–24) is associated with a substantial reduction of free radical blood levels. It is suggested that ACTH‐(1–24) prevents the burst of free radical generation during blood mobilisation and subsequent tissue reperfusion, and this may be an important component of its mechanism of action in effectively preventing death for haemorrhagic shock.

Adrenocorticotropin normalizes the blood levels of nitric oxide in hemorrhage-shocked rats.

Anesthetized rats were subjected to volume-controlled hemorrhagic shock by stepwise bleeding. Besides cardiovascular and respiratory functions, nitric oxide (NO)-hemoglobin formation in arterial blood was directly evaluated by means of electron spin resonance spectroscopy. During hemorrhagic shock there was a massive increase in NO-hemoglobin, associated with a fall in mean arterial pressure, pulse pressure, respiratory rate and heart rate, and there was a further increase in NO-hemoglobin 15 min after intravenous (i.v.) treatment with saline. All rats died within 30 min. The reversal of the shock condition induced by the i.v. injection of the adrenocorticotropin (ACTH) fragment 1-24 (160 microg/kg, 5 min after bleeding termination) was associated with a prompt disappearance of NO-hemoglobin. Also S-methylisothiourea (3 mg/kg i.v.), a selective inhibitor of inducible NO synthase, provoked a disappearance of NO-hemoglobin and reversal of the shock condition. The present results provide a direct demonstration that volume-controlled hemorrhagic shock is associated with highly increased blood levels of NO, as indicated by increased NO-hemoglobin, and indicate that ACTH-induced reversal of the shock condition is associated with the normalization of NO blood levels, and a parallel improvement of cardiovascular and respiratory functions. This occurs probably through the inhibition of inducible NO synthase, as suggested by the fact that S-methylisothiourea, a selective inhibitor of this NO synthase isoform, produced the same results.