Maria-Michela Cainazzo

Efferent Vagal Fibre Stimulation Blunts Nuclear Factor-κB Activation and Protects Against Hypovolemic Hemorrhagic Shock

Background—We investigated whether electrical stimulation (STIM) of efferent vagus nerves may suppress nuclear factor (NF)-&kgr;B activation and the inflammatory cascade in hemorrhagic (Hem) shock. Methods and Results—Rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Hem shock was induced by intermittent withdrawing of blood until mean arterial pressure stabilized within the range of 35 to 40 mm Hg. Application of constant voltage pulses to the caudal vagus ends (STIM; 5 V, 2 ms, 1 Hz for 12 minutes, 5 minutes after mean arterial pressure stabilization) increased survival time (VGX+Hem+Sham STIM=38±3 minutes; VGX+Hem+STIM >180 minutes), reverted the marked hypotension (VGX+Hem+Sham STIM=33±3 mm Hg; VGX+Hem+STIM=66±5 mm Hg), inhibited I&kgr;B&agr; liver loss, and blunted the augmented NF-&kgr;B activity, decreased hepatic tumor necrosis factor (TNF)-&agr; mRNA (VGX+Hem+Sham STIM=1.42±0.5 amount of TNF-&agr; m-RNA; VGX+Hem+STIM=0.51±0.2 amount of TNF-&agr; mRNA), and reduced plasma TNF-&agr; (VGX+Hem+Sham STIM=190±24 pg/mL; VGX+Hem+STIM=87±15 pg/mL). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Conclusions—Our results show a parasympathetic inhibition of NF-&kgr;B by which the brain opposes NF-&kgr;B activation in the liver and modulates the inflammatory response during acute hypovolemic hemorrhagic shock.

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.

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.

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 µg/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 µM), 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 30–40 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.