Involvement of the histaminergic system in the resuscitating effect of centrally acting leptin in haemorrhagic shock in rats.

Leptin, acting centrally as a neuromodulator, induces the activation of the sympathetic nervous system, which may lead to a pressor action in normotensive animals. In haemorrhagic shock, leptin administered intracerebroventricularly (icv.) evokes the resuscitating effect, with long-lasting rises in mean arterial pressure (MAP) and heart rate (HR), subsequent increase in peripheral blood flows, and a 100% survival at 2 h. Since leptin is able to activate histaminergic neurons, and centrally acting histamine also induces the resuscitating effect with the activation of the sympathetic nervous system, in the present study, we investigated an involvement of the histaminergic system in leptin-evoked cardiovascular effects in haemorrhagic shock. The model of irreversible haemorrhagic shock, with MAP decreased to and stabilised at 20 - 25 mmHg, has been used. Leptin (20 µg) given icv. at 5 min of critical hypotension evoked 181.5% increase in extracellular hypothalamic histamine concentration during the first 10 min after injection. Rises in MAP, HR and renal, mesenteric and hindquarters blood flows induced by leptin were inhibited by icv. pre-treatment with histamine H1 receptor antagonist chlorpheniramine (50 nmol). In contrast, there was no effect of H2, H3 and H4 receptor antagonists ranitidine (25 nmol), VUF 5681 (25 nmol) and JNJ 10191584 (25 nmol), respectively. In conclusion, the histaminergic system is involved in centrally-acting leptin-induced resuscitating effect in haemorrhagic shock in rats.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP--MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. KEY RESULTS: All shocked rats treated with saline died within 30-35 min. Treatment with NDP--MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. CONCLUSIONS AND IMPLICATIONS: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

Nuclear factor-kappaB as a target of cyclosporin in acute hypovolemic hemorrhagic shock.

BACKGROUND: Cyclosporin is an immunosuppressive drug that blocks Nuclear Factor kappaB (NF-kappaB) activation. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock and the effects of cyclosporin in this model of experimental shock. METHODS: 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. Two minutes after bleeding cessation, animals received intravenously cyclosporin (1 mg kg(-1)) or its vehicle. Survival rate and survival time were evaluated for 120 min after bleeding was discontinued. Plasma TNF-alpha levels were investigated at different time points after bleeding cessation. Moreover we investigated levels of TNF-alpha mRNA in the liver, vascular reactivity, liver NF-kappaB binding activity and levels of the inhibitory protein IkappaBalpha in the cytoplasm. RESULTS: Hemorrhagic shocked rats died in 27+/-6 min following the cessation of bleeding, experienced a marked hypotension (mean arterial blood pressure=20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (208+/-22 pg ml(-1), 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: 1 nM-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). 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 protein IkappaBalpha in the cytoplasm decreased at 5 min after the end of bleeding. Cyclosporin inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Furthermore, cyclosporin increased survival time (118+/-7 min; P<0.01) and survival rate (vehicle=0% and cyclosporin=80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (28+/-7 pg ml(-1)), and restored to control values the hypo-reactivity to PE. CONCLUSIONS: 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 which triggers an inflammatory cascade leading to a fatal outcome. Cyclosporin blocks NF-kappaB activation and protects against hypovolemic hemorrhagic shock.

Survival rate after early treatment for acute type-A aortic dissection with ACTH-(1-24).

Haemorrhagic shock, usually as a consequence of major trauma, is the most frequent cause of death among people younger than 40 years. Reports indicate that melanocortin peptides are effective in reversing haemorrhagic shock. We found that in patients with aortic-dissection-induced haemorrhagic shock, the addition of an early intravenous bolus injection of the melanocortin andrenocorticotrophic hormone (ACTH)-(1-24) to standard treatment significantly improved cardiovascular function and increased survival rate. Because administration of ACTH-(1-24) is simple, and because melanocortin peptides have no acute toxicity, their use in the early critical care of patients in shock should be more extensively assessed.

Protective effect of melanocortin peptides in rat myocardial ischemia.

The influence of the melanocortin peptide ACTH-(1-24) (adrenocorticotropin) on the consequences of short-term coronary ischemia (5 min) followed by reperfusion, and the effect of the long-acting melanocortin [Nle(4),D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-MSH) on the damage induced by a permanent coronary occlusion, were investigated in anesthetized rats. Ischemia was produced by ligature of the left anterior descending coronary artery. Reperfusion-induced arrhythmias [ventricular tachycardia (VT), ventricular fibrillation (VF)] and survival rate within the 5 min following reperfusion, blood levels of free radicals detected 2 min after reperfusion by electron spin resonance spectrometry, and amount of healthy myocardial tissue, measured 72 h after permanent coronary occlusion on immunohistologically stained serial sections, were evaluated. Postischemic reperfusion induced VT in all saline-treated rats, and VF and death in a high percentage of animals (87%). In rats treated i.v. (2.5 min after coronary occlusion) with ACTH-(1-24) (0.16-0.48 mg/kg) there was a significantly dose-dependent reduction in the incidence of arrhythmias and lethality. Ischemia/reperfusion caused a large increase in free radical blood levels; treatment with ACTH-(1-24) (0.48 mg/kg i.v.) almost completely prevented this increase. In rats subjected to permanent coronary occlusion, the amount of healthy myocardial tissue was much reduced in saline-treated rats, while in rats treated s.c. with NDP-MSH (0.27 mg/kg every 12 h) it was significantly higher. The present data demonstrate, for the first time, an unforeseen property of melanocortin peptides, i.e., their ability to significantly reduce both heart ischemia/reperfusion injury and size of the ischemic area induced by permanent coronary occlusion.

Oxidative stress causes nuclear factor-kappaB 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.

Adrenocorticotropin inhibits nitric oxide synthase II mRNA expression in rat macrophages.

During hemorrhagic shock there is a massive overproduction of nitric oxide (NO). In such conditions, the intravenous (i.v.) injection of melanocortin peptides in nanomolar amounts produces a long-lasting restoration of cardiovascular and respiratory functions associated with the normalization of NO blood levels. To clarify the mechanism of such melanocortin-induced inhibition of NO overproduction, the influence of the adrenocorticotropin fragment 1-24 [ACTH-(1-24)] on the NO synthesizing activity of rat macrophages was studied in vitro. Nitrite production, an indicator of NO synthesis, was measured in the supernatant of rat macrophages whose inducible NO synthase (NOS II, iNOS) had been stimulated by the addition of S. enteritidis lipopolysaccharide (LPS, 50 microg/ml). ACTH-(1-24) (25, 50 and 100 nM) inhibited nitrite production when incubated together with LPS, but had no effect when applied 6 h after LPS. Further, the effect of ACTH-(1-24) on the expression of iNOS mRNA in rat macrophages activated with LPS was studied by means of a reverse transcriptase-polymerase chain reaction assay. ACTH-(1-24) (25, 50 and 100 nM), applied together with LPS, dose-dependently suppressed iNOS gene activation. The present data suggest that the melanocortin-induced normalization of NO blood levels during hemorrhagic shock is due, at least in part, to a direct inhibition of iNOS induction, at the level of mRNA transcription.

Evidence that melanocortin 4 receptor mediates hemorrhagic shock reversal caused by melanocortin peptides.

Melanocortin peptides are known to be extremely potent in causing the sustained reversal of different shock conditions, both in experimental animals and humans; the mechanism of action includes an essential brain loop. Three melanocortin receptor subtypes are expressed in brain tissue: MC(3), MC(4,) and MC(5) receptors. In a volume-controlled model of hemorrhagic shock in anesthetized rats, invariably causing the death of control animals within 30 min after saline injection, the i.v. bolus administration of the adrenocorticotropin fragment 1-24 (agonist at MC(4) and MC(5) receptors) at a dose of 160 microg/kg i.v. (54 nmol/kg) produced an almost complete and sustained restoration of cardiovascular and respiratory functions. An equimolar dose of gamma(1)-melanocyte stimulating hormone (selective agonist at MC(3) receptors) was completely ineffective. The selective antagonist at MC(4) receptors, HS014, although having no influence on cardiovascular and respiratory functions per se, dose-dependently prevented the antishock activity of adrenocorticotropin fragment 1-24, with the effect being complete either at the i.v. dose of 200 microg/kg or at the i.c.v. dose of 5 microg/rat (17-20 microg/kg). We concluded that the effect of melanocortin peptides in hemorrhagic shock is mediated by the MC(4) receptors in the brain.

Adrenocorticotropin reverses vascular dysfunction and protects against splanchnic artery occlusion shock.

1. Tumour necrosis factor (TNF-alpha) is involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. On the other hand, inhibition of TNF-alpha 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. 2. 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-alpha 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 microM) (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 microM) (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). 3. ACTH-(1 - 24) [160 microg 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-alpha (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. 4. 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. 5. 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-alpha. Furthermore, they demonstrate, for the first time, that this inhibition is responsible for the adrenocorticotropin-induced reversal of vascular failure and leukocyte accumulation.

High blood levels of nitric oxide in rats subjected to prolonged respiratory arrest and their modulation during adrenocorticotropin-induced resuscitation.

Anaesthetized rats, endotracheally intubated and mechanically ventilated with room air, were subjected to a 5-min period of asphyxia by turning off the ventilator. The ventilator was then turned back on and, simultaneously, the animals were treated with either the adrenocorticotropin fragment 1-24 [ACTH-(1-24), 160 microg/kg in a volume of 1 ml/kg i.v.] or an equivalent volume of saline. Nitric oxide (NO)-haemoglobin formation was detected ex vivo in arterial blood by electron spin resonance spectrometry; arterial blood pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for a 60-min observation period, or until prior death. During asphyxia, there was massive formation of NO (red cell concentrations 40-80 microM), associated with a dramatic fall in mean arterial pressure and pulse pressure, marked bradycardia and ECG signs of ischaemic damage, as well as an isoelectric EEG. Treatment with ACTH-(1-24) produced a prompt (within 15 min) and long-lasting drop in NO blood levels, associated with an almost immediate (within 1 min) restoration of cardiovascular function and with a more gradual recovery of EEG, which became normal after 3040 min; all parameters remained stable throughout the 60-min observation period. In saline-treated rats, on the other hand, there was a further increase in NO blood levels, as detected 3 min after treatment, and all died within 5-8 min. Moreover, pretreatment and treatment with S-methylisothiourea sulphate (SMT, 3 mg/kg i.v.), a relatively specific inhibitor of inducible NO synthase, inhibited NO formation, but did not affect the mortality rate (100% within 5-8 min). The present results provide the first evidence that prolonged asphyxia is associated with high blood concentrations of NO, and that the life-saving effect of melanocortin peptides in severe hypoxic conditions is associated with a complete normalization of NO blood levels. However, the lack of SMT protection in this experimental model seems to rule out the possibility that the ACTH-(1-24)-induced resuscitation is due to an effect on NO overproduction.

Tumour necrosis factor-alpha as a target of melanocortins in haemorrhagic shock, in the anaesthetized rat.

The cytokine tumour necrosis factor-alpha (TNF-alpha) 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-alpha in haemorrhage-shocked rats and on the in vitro production of TNF-alpha by lipopolysaccharide (LPS)-activated macrophages. Plasma levels of TNF-alpha 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-alpha 20 min after bleeding termination. On the other hand, ACTH-(1-24) did not influence TNF-alpha plasma levels in sham-operated, unbled rats. In vitro, ACTH-(1-24) (25-100 nM) dose-dependently reduced the LPS-stimulated production of TNF-alpha by peritoneal macrophages harvested from untreated, unbled rats. These results indicate that inhibition of TNF-alpha overproduction may be an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock.

Adrenocorticotropin counteracts the increase in free radical blood levels, detected by electron spin resonance spectrometry, in rats subjected to prolonged asphyxia.

We investigated the influence of the adrenocorticotropic fragment 1-24 [ACTH-(1-24)] on the blood levels of highly-reactive free radicals in a rat model of prolonged asphyxia. Anesthetized animals were endotracheally intubated and mechanically ventilated with room air; after a 10 min stabilization period, the ventilator was turned off to induce asphyxia for 5 min; then, the ventilator was turned back on, and, simultaneously, the rats were intravenously treated with either ACTH-(1-24) (160 microg/kg in a volume of 1 ml/kg) or equivolume saline. Free radicals were detected in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent employed (alpha-phenyl-N-tert-butylnitrone). Arterial pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for the 60 min observation period, or until prior death. At the end of the 5 min period of respiratory arrest, blood levels of free radicals were about four times higher than those of the basal, pre-asphyxia condition, arterial pressure had dramatically decreased, ECG showed marked bradycardia and signs of ischemic damage and the EEG had become isoelectric. Treatment with ACTH-(1-24) produced an immediate normalization of the blood levels of free radicals, associated with a restoration of cardiovascular function and full recovery of EEG within 30-45 min; all the saline-treated rats, on the other hand, died within 6.89 +/- 0.96 min. These results provide direct evidence that in a severe condition of prolonged asphyxia there is a rapid and massive production of highly-reactive free radicals and suggest that the resuscitating effect of adrenocorticotropin fragments in severe hypoxic conditions may be largely due to the inhibition of free radical overproduction during tissue reoxygenation.

Physostigmine has a life-saving effect in rats subjected to prolonged respiratory arrest.

We have previously reported that centrally-acting cholinomimetic drugs have a prompt and sustained resuscitating effect in pre-terminal conditions of hemorrhagic shock in rats. Here we have studied the effect of physostigmine in another experimental condition of hypoxia in anesthetized rats, which were endotracheally intubated and subjected to prolonged (5 min) interruption of ventilation. This led to a dramatic fall in mean arterial pressure (MAP), pulse pressure (PP), heart rate (HR), pH, PO2, SO2 and base excess, while PCO2 increased; the electroencephalogram (EEG) became isoelectric, and the electrocardiogram (ECG) showed marked bradycardia, P-wave inversion, partial atrio-ventricular block and S-T segment elevation; all saline-treated rats died of cardiac arrest within 7.01 +/- 0.85 min of ventilation being resumed. When ventilation resumption was associated with the simultaneous intravenous (i.v.) injection of physostigmine (70 microg/kg) there was an almost immediate and impressive increase in MAP, PP and HR, with normalization of ECG within 4 min and full recovery of EEG after 30-50 min. This was associated with a normalization of blood gases and pH. Fifteen days later 40% of treated animals were still alive and apparently in normal health, the mean survival time of the remaining 60% animals being 22.67 +/- 10.19 h. Pretreatment with atropine sulfate or hemicholinium-3 did not modify the response to physostigmine, which, however, was strongly antagonized by the intracerebroventricular injection of mecamylamine. These results suggest that centrally-acting cholinomimetic agents may have a resuscitating effect in pre-terminal conditions produced by prolonged asphyxia, probably through the direct activation of nicotinic receptors in the central nervous system.

Resuscitating effect of melanocortin peptides after prolonged respiratory arrest.

1. The resuscitating activity of melanocortin peptides (MSH-ACTH peptides) was tested in an experimental model of prolonged respiratory arrest. 2. 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. 3. When resumption of ventilation was associated with the simultaneous intravenous (i.v.) injection of a melanocortin peptide (alpha-MSH or ACTH-(1-24)) (160 microg 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). 4. The same response was observed in adrenalectomized animals, as well as in animals pretreated with a beta1-adrenoceptor blocking agent (atenolol, 3 mg kg(-1), i.v.), or with an alpha1-adrenoceptor blocking agent (prazosin, 0.1 mg kg(-1), i.v.), or with an adrenergic neurone blocking agent (guanethidine, 10 mg kg(-1), intraperitoneally). 5. 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. 6. 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.

Inhibition of nitric oxide synthases enhances the effect of ACTH in hemorrhagic shock.

In a model of volume-controlled hemorrhagic shock in rats, invariably leading to death within 30 min of bleeding termination, the intravenous (i.v.) bolus injection of ACTH-(1-24) at the dose of 0.16 mg/kg restored cardiovascular and respiratory functions and greatly prolonged survival. I.v. or intracerebroventricular (i.c.v.) treatment with NG-nitro-L-arginine methylester (L-NAME), a non-isoform-selective inhibitor of nitric oxide synthases (NOSs), at the doses of 2.5-10 mg/kg i.v. or 0.015-0.135 mg/kg i.c.v., as well as i.v. treatment with S-methylisothiourea (SMT), a selective inhibitor of the inducible isoform of NOS, at the doses of 0.001-3 mg/kg, dose-dependently improved cardiovascular and respiratory functions and potentiated the effect of a subthreshold dose (0.02 mg/kg) of ACTH-(1-24). On the other hand, either intraperitoneal or i.c.v. pretreatment with L-arginine, the substrate of NOSs, prevented the effect of ACTH-(1-24). These data suggest that inhibition of NO overproduction is involved in the mechanism of action of ACTH-(1-24) in shock reversal.

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.

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 micrograms 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 (alpha-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.

Antithrombotic activity of a 2-kDa heparin fragment in an experimental model of carotid artery thrombosis in rats.

The antithrombotic activity of a 2-kDa heparin fragment was studied in a rat model of common carotid artery thrombosis that causes a completely occlusive thrombus with cessation of the blood flow within 10-15 min. The compound reduced thrombus formation in a dose-dependent manner, starting from an intravenous dose of 5 mg kg-1. A dose of 20 mg kg-1 completely prevented thrombus formation and apparently induced the almost complete lysis of the already formed occlusive thrombus. At none of the doses used did the compound cause increased bleeding or the formation of haematomas. The present results indicate that low molecular weight heparins, which have an established, highly beneficial effect in venous thromboembolism, are also highly effective in an animal model of arterial thrombosis.

Serotonin is involved in the ACTH-induced reversal of hemorrhagic shock in anesthetized rats.

In a rat model of volume-controlled hemorrhagic shock (mean arterial pressure = 20-24 mm Hg) causing the death of all saline-treated animals within 30 min, the i.v. bolus injection of ACTH-(1-24) (160 micrograms/kg) produced an almost complete and sustained reversal of the shock condition, with recovery of arterial blood pressure, pulse pressure and respiratory rate, and with 100% survival at the end of the experiment (2 h). The serotonin-depleting agent p-chlorophenylalanine (316 mg/kg i.p., administered 66-70 h before hemorrhage) almost completely prevented the effect of ACTH. The 5-HT1/5-HT2 receptor antagonist, methysergide, prevented the effect of ACTH completely when injected i.v. (5 mg/kg), but only in part when injected into a brain ventricle (i.c.v.) (15 micrograms/rat); the 5-HT2 antagonist, ketanserin, prevented the effect of ACTH completely when injected i.c.v. (1.5 micrograms/rat), but only in part when injected i.v. (0.5 mg/kg); the 5-HT3 antagonist, MDL 72222, largely prevented the effect of ACTH when injected i.c.v. (10 micrograms/rat), but had no influence at all when injected i.v. (3 mg/kg); finally, the 5-HT4 antagonist, GR 125487, had no effect when injected i.v. (5 micrograms/kg) or when injected i.c.v. (30 ng/rat). Overall, these data indicate that both CNS and peripheral serotonin play an important role in the complex mechanism of the ACTH-induced hemorrhagic shock reversal.

A highly reproducible model of arterial thrombosis in rats.

The objective of this investigation was to develop a reproducible and reliable method of arterial thrombosis in a small laboratory animal. Rats were anesthetized with urethane, and a common carotid artery was exposed. A completely occlusive thrombus was produced by applying an electric current to the arterial wall (2 mA for 5 min) while simultaneously constricting the artery with a hemostatic clamp placed immediately downstream from the electrodes. A complete and persistent cessation of blood flow was obtained in all the control rats starting 10-15 min after the thrombogenic lesion. Histological examination revealed a picture of mixed white and red thrombus, stratified and rich in platelets aggregates and fibrin, with piles of red cells trapped in the fibrin network. On the other hand, stasis alone (clamping) was ineffective at all, whereas electric current application alone caused non-occlusive thrombosis only in 30% of animals. An antithrombotic dose of heparin (3 mg/kg i.v., 30 min before thrombus induction) prevented the formation of a persistent thrombus, blood flow being progressively restored (up to 59% of basal value within 45 min). Similarly, a thrombolytic dose of urokinase-type plasminogen activator (2 mg/kg, for 120 min, starting 15 min after thrombus induction) caused a rapid and progressive resumption of blood flow (up to 80% of basal value). This method gives highly consistent and reproducible results and may be suitable for the study or the screening of antithrombotic as well as thrombolytic agents.